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The eight human “Canonical” Ribonucleases: Molecular diversity, catalytic properties, and special biological actions of the enzyme proteins
FEBS Letters
Abstract
Human ribonucleases (RNases) are members of a large superfamily of rapidly evolving homologous proteins. Upon completion of the human genome, eight catalytically active RNases (numbered 1-8) were identified. These structurally distinct RNases, characterized by their various catalytic differences on different RNA substrates, constitute a gene family that appears to be the sole vertebrate-specific enzyme family. Apart from digestion of dietary RNA, a wide variety of biological actions, including neurotoxicity, angiogenesis, immunosuppressivity, and anti-pathogen activity, have been recently reported for almost all members of the family. Recent evolutionary studies suggest that RNases started off in vertebrates as host defence or angiogenic proteins.
... Interestingly, according to Hornung and co-workers, the release of U > p ends by RNase2 would participate in the activation of TLR8 at the endolysosomal compartment and will contribute to sense the presence of pathogen RNA [52]. To note, we find a good agreement between RNase2 substrate specificity identified in the present cell assay study on tRNAs and the previously reported for synthetic single stranded oligonucleotides [53,54] (see Table 2). However, some differences are evidenced at the miRNAs cleavage and in particular at the B2 site specificity, which does not fully match the reported on synthetic substrates. ...
... This discrepancy is also evident for the other two RNaseA family members described to release specific tRFs [55][56][57][58], i.e., RNase5/Ang and Onconase, an RNase purified from Rana pipiens with antitumoral properties (Table S5). Previous kinetic studies on RNaseA family cleavage preference using single stranded RNA substrates revealed a specificity for pyrimidines at the main B1 site and preference for purines at B2 [53,54]. Among the family members, we observe distinct preferences for U vs C and A vs G at B1 and B2 sites, respectively. ...
... Among the family members, we observe distinct preferences for U vs C and A vs G at B1 and B2 sites, respectively. Interestingly, RNase 2 shows a marked preference for U at B1 and A at B2 on synthetic oligonucleotides [53,59], which mostly corresponds to the observed preference identified by cP-RNAseq for tRNA in this study (Fig. 5). Nevertheless, our analysis on tRNA cleavage sites would suggest a U/C ratio for B1 a bit lower than the estimated for some synthetic substrates ( Table 2). ...
... Thus, nucleases function as biological scissors that possess a shared catalytic capability, but exhibit a large variation in structure, function and substrate selectivity [1]. Nucleases play crucial roles in nature, including survival, homeostasis [2][3][4][5][6] and immune defense [3,4,[7][8][9][10]. They are involved in genome stability [11], genome editing and proof-reading [12], DNA repair [13] and cell apoptosis [14][15][16][17][18]. Nucleases are also key elements in host defense against pathogens [10,19] and innate immunity modulation during infection and cancer [20,21]. ...
... Thus, nucleases function as biological scissors that possess a shared catalytic capability, but exhibit a large variation in structure, function and substrate selectivity [1]. Nucleases play crucial roles in nature, including survival, homeostasis [2][3][4][5][6] and immune defense [3,4,[7][8][9][10]. They are involved in genome stability [11], genome editing and proof-reading [12], DNA repair [13] and cell apoptosis [14][15][16][17][18]. Nucleases are also key elements in host defense against pathogens [10,19] and innate immunity modulation during infection and cancer [20,21]. ...
... They are involved in genome stability [11], genome editing and proof-reading [12], DNA repair [13] and cell apoptosis [14][15][16][17][18]. Nucleases are also key elements in host defense against pathogens [10,19] and innate immunity modulation during infection and cancer [20,21]. Some nucleases exert neurotoxicity and angiogenesis [3,4]. ...
... Upon the completion of the human genome project, eight canonical RNase A genes were found on chromosome 14q11.2 (Cho et al., 2005;Goo and Cho, 2013;Sorrentino, 2010). These include RNase 1 (pancreatic RNase), RNase 2 (eosinophil-derived neurotoxin, EDN), RNase 3 (eosinophil cationic protein, ECP), RNase 4, RNase 5 (angiogenin, ANG), RNase 6 (RNase k6), RNase 7, and RNase 8 Sorrentino, 2010). ...
... (Cho et al., 2005;Goo and Cho, 2013;Sorrentino, 2010). These include RNase 1 (pancreatic RNase), RNase 2 (eosinophil-derived neurotoxin, EDN), RNase 3 (eosinophil cationic protein, ECP), RNase 4, RNase 5 (angiogenin, ANG), RNase 6 (RNase k6), RNase 7, and RNase 8 Sorrentino, 2010). This chromosome location also includes several homologous genes (RNases 9-13), which are related to the RNase A family based on amino acid sequence homology; however, these genes lack one or more elements necessary for enzymatic activity (Goo and Cho, 2013). ...
... However, their ribonucleolytic activity varies in magnitude. Compared with RNase 1, RNase 2 and 4 have similar activities, RNase 3, 6, 7, and 8 exhibits 8-100-fold lower activity, whereas RNase 5 has the lowest activity (approximately 10 -5 -fold) (Sorrentino, 2010;Wang et al., 2018b;Zhang et al., 2003). Accumulating evidence indicates that these proteins can be internalized into cells through multiple pathways, including membrane receptors-dependent endocytosis or dynamin-independent penetrating (Chao and Raines, 2011;Ferguson and Subramanian, 2018;Yu et al., 2017), and then regulate cellular processes in a ribonucleolytic activity-dependent or -independent manner. ...
The ribonuclease A (RNase A) family is one of the best-characterized vertebrate-specific proteins. In humans, eight catalytically active RNases (numbered 1-8) have been identified and have unique tissue distributions. Apart from the digestion of dietary RNA, a broad range of biological actions, including regulation of intra- or extra-cellular RNA metabolism as well as antiviral, antibacterial, and antifungal activities, neurotoxicity, promotion of cell proliferation, anti-apoptosis, and immunomodulatory abilities, have been recently reported for the members of this family. Based on multiple biological roles, RNases are found to participate in the pathogenic processes of many diseases, such as infection, immune dysfunction, neurodegeneration, cancer, and cardiovascular disorders. This review summarizes the available data on the human RNase A family and illustrates the significant roles of the eight canonical RNases in health and disease, for stimulating further basic research and development of ideas on the potential solutions for disease diagnosis and treatment.
... Interestingly, according to Hornung and co-workers, the release of U > p ends by RNase2 would participate in the activation of TLR8 at the endolysosomal compartment and will contribute to sense the presence of pathogen RNA [52]. To note, we find a good agreement between RNase2 substrate specificity identified in the present cell assay study on tRNAs and the previously reported for synthetic single stranded oligonucleotides [53,54] (see Table 2). However, some differences are evidenced at the miRNAs cleavage and in particular at the B2 site specificity, which does not fully match the reported on synthetic substrates. ...
... This discrepancy is also evident for the other two RNaseA family members described to release specific tRFs [55][56][57][58], i.e., RNase5/Ang and Onconase, an RNase purified from Rana pipiens with antitumoral properties (Table S5). Previous kinetic studies on RNaseA family cleavage preference using single stranded RNA substrates revealed a specificity for pyrimidines at the main B1 site and preference for purines at B2 [53,54]. Among the family members, we observe distinct preferences for U vs C and A vs G at B1 and B2 sites, respectively. ...
... Among the family members, we observe distinct preferences for U vs C and A vs G at B1 and B2 sites, respectively. Interestingly, RNase 2 shows a marked preference for U at B1 and A at B2 on synthetic oligonucleotides [53,59], which mostly corresponds to the observed preference identified by cP-RNAseq for tRNA in this study (Fig. 5). Nevertheless, our analysis on tRNA cleavage sites would suggest a U/C ratio for B1 a bit lower than the estimated for some synthetic substrates ( Table 2). ...
RNase2 is the member of the RNaseA family most abundant in macrophages. Here, we knocked out RNase2 in THP-1 cells and analysed the response to Respiratory Syncytial Virus (RSV). RSV induced RNase2 expression, which significantly enhanced cell survival. Next, by cP-RNAseq sequencing, which amplifies the cyclic-phosphate endonuclease products, we analysed the ncRNA population. Among the ncRNAs accumulated in WT vs KO cells, we found mostly tRNA-derived fragments (tRFs) and second miRNAs. Differential sequence coverage identified tRFs from only few parental tRNAs, revealing a predominant cleavage at anticodon and d -loops at U/C (B1) and A (B2) sites. Selective tRNA cleavage was confirmed in vitro using the recombinant protein. Likewise, only few miRNAs were significantly more abundant in WT vs RNase2-KO cells. Complementarily, by screening of a tRF & tiRNA array, we identified an enriched population associated to RNase2 expression and RSV exposure. The results confirm the protein antiviral action and provide the first evidence of its cleavage selectivity on ncRNAs.
Graphical abstract
... Humans express genes encoding 13 such enzymes, clustered near one another on chromosome 14 (Zhang et al., 2002). Eight of these ptRNases are classified as "canonical" (Sorrentino, 2010). The remaining five exhibit more unusual features, such as the absence of one or more catalytic residues or the lack of cationic character (Cho et al., 2005). ...
... The appreciation of other biological roles has been growing for decades (Benner and Allemann, 1989;D'Alessio, 1993;Wang et al., 2018a;Lee et al., 2019). Antimicrobial activity has been described for a cluster of closely related ptRNases, including ribonucleases 2 and 3, as well as 6, 7, and 8 (Sorrentino, 2010), which are expressed in eosinophil granules, placenta, and skin and exhibit relatively broad-spectrum cytotoxicity (Ackerman et al., 1985;Molina et al., 1988;Lehrer et al., 1989;Venge et al., 1999;Harder and Schroder, 2002;Boix and Nogués, 2007;Rosenberg, 2008;Attery and Batra, 2017) and antiviral activity (Domachowske et al., 1998a;Domachowske et al., 1998b;Ilinskaya and Mahmud, 2014;Li and Boix, 2021). Angiogenins have well-documented angiogenic activity, but some subfamily members have also been reported to exhibit immune activity. ...
... This hypothesis is supported by the presence of enzymes in fish that are more similar to human angiogenin than to RNase 1 or other ptRNases (Kazakou et al., 2008). Additionally, angiogenin has three intramolecular disulfide bonds in contrast to the four in other ptRNases (Sorrentino, 2010) (Figures 2A and 2B). That fourth disulfide bond is reduced more readily than the other three (Zhou and Strydom, 1993), suggesting that it is less important to enzymic structure and function and was likely a later addition (Strydom, 1998). ...
Pancreatic-type ribonucleases (ptRNases) are a large family of vertebrate-specific secretory endoribonucleases. These enzymes catalyze the degradation of many RNA substrates and thereby mediate a variety of biological functions. Though the homology of ptRNases has informed biochemical characterization and evolutionary analyses, the understanding of their biological roles is incomplete. Here, we review the functions of two ptRNases: RNase 1 and angiogenin. RNase 1, which is an abundant ptRNase with high catalytic activity, has newly discovered roles in inflammation and blood coagulation. Angiogenin, which promotes neovascularization, is now known to play roles in the progression of cancer and amyotrophic lateral sclerosis, as well as in the cellular stress response. Ongoing work is illuminating the biology of these and other ptRNases.
... Interestingly, according to Hornung and co-workers, the release of U>p ends by RNase2 would participate in the activation of TLR8 at the endolysosomal compartment and will contribute to sense the presence of pathogen RNA [48]. To note, we find a good agreement between RNase2 substrate specificity identified in the present cell assay study on tRNAs and the previously reported for synthetic single stranded oligonucleotides [49,50] (see Table 2). However, some differences are evidenced at the miRNAs cleavage and in particular at the B2 site specificity, which does not fully match the reported on synthetic substrates. ...
... RNase5/Ang and Onconase, an RNase purified from Rana pipiens with antitumoral properties (Table S5). Previous kinetic studies on RNaseA family cleavage preference using single stranded RNA substrates revealed a specificity for pyrimidines at the main B1 site and preference for purines at B2 [49,50]. Among the family members, we observe distinct preferences for U vs C and A vs G at B1 and B2 sites respectively. ...
... Among the family members, we observe distinct preferences for U vs C and A vs G at B1 and B2 sites respectively. Interestingly, RNase 2 shows a marked preference for U at B1 and A at B2 on synthetic oligonucleotides [49,55], which mostly corresponds to the observed preference identified by Cp-RNAseq for tRNA in this study ( Fig 5). Nevertheless, our analysis on tRNA cleavage sites would suggest a U/C ratio for B1 a bit lower than the estimated for some synthetic substrates ( Table 2). ...
RNase2, also named the Eosinophil derived Neurotoxin (EDN), is one of the main proteins secreted by the eosinophil secondary granules. RNase2 is also expressed in other leukocyte cells and is the member of the human ribonuclease A family most abundant in macrophages. The protein is endowed with a high ribonucleolytic activity and participates in the host antiviral activity. Although RNase2 displays a broad antiviral activity, it is mostly associated to the targeting of single stranded RNA viruses. To explore RNase2 mechanism of action in antiviral host defence we knocked out RNase2 expression in the THP1 monocyte cell line and characterized the cell response to human Respiratory Syncytial Virus (RSV). We observed that RSV infection induced the RNase2 expression and protein secretion in THP1 macrophage-derived cells, whereas the knockout (KO) of RNase2 resulted in higher RSV burden and reduced cell viability. Next, by means of the cP-RNAseq methodology, which uniquely amplifies the RNA 2'3'cyclic-phosphate-end products released by an endonuclease cleavage, we compared the ncRNA population in native and RNase2-KO cell lines. Among the ncRNAs accumulated in WT versus KO cells, we found mostly tRNA-derived fragments and secondly miRNAs. Analysis of the differential sequence coverage of tRNAs molecules in native and KO cells identified fragments derived from only few parental tRNAs, revealing a predominant cleavage at anticodon loops and secondarily at D-loops. Inspection of cleavage region identified U/C and A, at 5' and 3' sides of cleavage sites respectively (namely RNase B1 and B2 base binding subsites). Likewise, only few selected miRNAs were significantly more abundant in WT versus RNase2-KO cells, with cleavage sites located at the end of stem regions with predominance for pyrimidines at B1 but following an overall less defined nucleotide specificity. Complementarily, by screening of a tRF/tiRNA PCR array we identified an enriched population of tRNA-derived fragments associated to RNase2 expression and RSV infection. The present results confirm the contribution of the protein in macrophage response against virus infection and provide the first evidence of its cleavage selectivity against ncRNA population. A better understanding of the mechanism of action of RNase2 recognition of cellular RNA during the antiviral host defence should pave the basis for the design of novel antiviral drugs.
... Angiogenin has also been found to cleave within the TC-loop, however this site is less frequently used. Ribonuclease (RNase) 1 is also a member of the vertebrate secreted ribonuclease family which shows high activity against double-stranded RNA (dsRNA) 38 , where it digests RNAs to single nucleotide products. RNase 1 is ubiquitously expressed but has been found to be upregulated during oxidative stress, and has been reported to cleave tRNA indicating it may also function to generate stress-induced tRFs 39,40 . ...
... This also suggests that tRFs generated in neural cells do not appear to be substrates for de novo methylation, although this was a short experimental time frame 52 . Cleavage by vertebrate secreted ribonucleases generates 2'3' cyclic phosphates at the 3' end of the 5'tRF, and a 5' hydroxyl group on the 3'tRF 38 . ...
Transfer RNAs play a crucial role in protein translation where they bring amino acids to the ribosome to be incorporated into nascent polypeptide chains. During stress conditions tRNAs can be cleaved to generate tRNA-derived fragments. Several ribonucleases have been identified that cleave tRNA, however mutations in the stress-induced ribonuclease Angiogenin have been identified in a range of neurological disorders including Amyotrophic Lateral Sclerosis, Parkinson’s Disease, and Alzheimer’s Disease, suggesting that tRNA cleavage may be dysregulated in neurological disease. tRNA fragments have been detected in biofluids indicating they may be of use as biomarkers for neurological diseases. There is considerable variability in the methods used to quantify tRFs from size selection, adapter ligation, removal of RNA modifications, and sequence analysis approaches which can make it difficult to reconcile multiple studies. Here we review the biology of transfer RNAs and the biogenesis of tRNA-derived fragments, with a focus on the methods used to identify and quantify tRNA fragments and how different methodological approaches can influence tRNA fragment detection. We provide an overview of current literature on the identification of tRNA fragments in neurological disease models and patient samples, with a focus on circulating tRNA fragments as potential biomarkers of neurological diseases.
... RNase can be divided into two main classes, endoribonucleases and exoribonucleases [3][4][5]. Endoribonuclease RNase1 belongs to the pancreatic RNase A superfamily and can be detected in human serum/plasma [6,7]. The studies of RNase in the last 50-60 years discovered numerous RNase family [8,9], however, the biological function of RNase1 have not yet been completely defined. ...
... The surfaces of the canonical RNases 1-8 contain continuous basic patches, except of RNase5, which exhibits extremely weak RNase enzymatic activity [41]. On the other hand, non-canonical members of the human RNases 9-13 harbor neither long basic patch nor RNase activities [6], indicating that both of the surface basic patch and the active site residues are important for enzymatic activity. Because RNase1, RNase5 and RNase7 can specifically bind to target RTKs (i.e. ...
It has been shown that several ribonuclease (RNase) A superfamily proteins serve as ligands of receptor tyrosine kinases (RTKs), representing a new concept for ligand/receptor interaction. Moreover, recent studies indicate high clinical values for this type of ligand/RTK interactions. However, there is no structural report for this new family of ligand/receptor. In an attempt to understand how RNase and RTK may interact, we focused on the RNase1/ephrin type-A receptor 4 (EphA4) complex and predicted their structure by using the state-of-the-art machine learning method, AlphaFold and its derivative method, AF2Complex. In this model, electrostatic force plays an essential role for the specific ligand/receptor interaction. We found the R39 of RNase1 is the key residue for EphA4-binding and activation. Mutation on this residue causes disruption of an essential basic patch, resulting in weaker ligand-receptor association and leading to the loss of activation. By comparing the surface charge distribution of the RNase A superfamily, we found the positively charged residues on the RNase1 surface is more accessible for EphA4 forming salt bridges than other RNases. Furthermore, RNase1 binds to the ligand-binding domain (LBD) of EphA4, which is responsible for the traditional ligand ephrin-binding. Our model reveals the location of RNase1 on EphA4 partially overlaps with that of ephrin-A5, a traditional ligand of EphA4, suggesting steric hindrance as the basis by which the ephrin-A5 precludes interactions of RNase1 with EphA4. Together, our discovery of RNase1/EphA4 interface provides a potential treatment strategy by blocking the RNase1-EphA4 axis.
... The tiRNA products are about 31-40 nt-long derivatives, differentiated in 5 tiRNA or 3 tiRNA, as a function of the availability of the 3 or 5 end at the anticodon cleavage site [18]. Angiogenin (ANG), a ribonuclease (RNase 5) [19,20] secreted by stressed cells [21], can generate tiRNA products as a result of paracrine signaling [22]. Since ANG normally elicits prosurvival signaling, many resulting tiRNAs facilitate the cellular response to stress by reprogramming translation, hence inhibiting apoptosis and degrading mRNAs [22]. ...
... Many reviews described the properties of the secretory "pancreatic-type" (pt), or RNase A-like, RNases [20,[41][42][43][44][45], so called because they display structural and functional similarities with the well-known bovine pancreatic RNase A [46]. Among the most known present in humans, and numbered as RNase 1-8, human pancreatic (HP)-RNase (RNase 1), angiogenin (RNase 5) are noteworthy. ...
The majority of transcribed RNAs do not codify for proteins, nevertheless they display crucial regulatory functions by affecting the cellular protein expression profile. MicroRNAs (miRNAs) and transfer RNA-derived small RNAs (tsRNAs) are effectors of interfering mechanisms, so that their biogenesis is a tightly regulated process. Onconase (ONC) is an amphibian ribonuclease known for cytotoxicity against tumors and antiviral activity. Additionally, ONC administration in patients resulted in clinical effectiveness and in a well-tolerated feature, at least for lung carcinoma and malignant mesothelioma. Moreover, the ONC therapeutic effects are actually potentiated by cotreatment with many conventional antitumor drugs. This review not only aims to describe the ONC activity occurring either in different tumors or in viral infections but also to analyze the molecular mechanisms underlying ONC pleiotropic and cellular-specific effects. In cancer, data suggest that ONC affects malignant phenotypes by generating tRNA fragments and miRNAs able to downregulate oncogenes expression and upregulate tumor-suppressor proteins. In cells infected by viruses, ONC hampers viral spread by digesting the primer tRNAs necessary for viral DNA replication. In this scenario, new therapeutic tools might be developed by exploiting the action of ONC-elicited RNA derivatives.
... Interestingly, evolutionary studies suggest that the RNase A family started off with a host-defense role [2][3][4]. Antimicrobial and immune-regulatory properties were reported for several family members and their presence in a variety of biological fluids is associated to a protective physiological role [5][6][7][8][9][10]. ...
... In particular, we observe a shift from cytidine to uridine preference at B1 in the last two variants, which could be attributed to a gradual predominance of RNase 3 traits. Previous kinetic data highlighted the uridine versus cytidine preference of RNase 3 in contrast to RNase 1 [9,46,47]. Further work is currently in progress to analyse the chimera structural peculiarities that explain their differential substrate preferences (Fernández-Millán et al., in preparation). ...
Bacterial resistance to antibiotics urges the development of alternative therapies. Based on the structure-function of antimicrobial members of the RNase A superfamily, we have developed a hybrid enzyme. Within this family, RNase 1 exhibits the highest catalytic activity and the lowest cytotoxicity; in contrast, RNase 3 shows the highest bactericidal action, alas with a reduced catalytic activity. Starting from both parental proteins, we designed a first RNase 3/1-v1 chimera. The construct had a catalytic activity much higher than RNase 3, unfortunately without reaching an equivalent antimicrobial activity. Thus, two new versions were created with improved antimicrobial properties. Both of these versions (RNase 3/1-v2 and -v3) incorporated an antimicrobial loop characteristic of RNase 3, while a flexible RNase 1-specific loop was removed in the latest construct. RNase 3/1-v3 acquired both higher antimicrobial and catalytic activities than previous versions, while retaining the structural determinants for interaction with the RNase inhibitor and displaying non-significant cytotoxicity. Following, we tested the constructs’ ability to eradicate macrophage intracellular infection and observed an enhanced ability in both RNase 3/1-v2 and v3. Interestingly, the inhibition of intracellular infection correlates with the variants’ capacity to induce autophagy. We propose RNase 3/1-v3 chimera as a promising lead for applied therapeutics.
... 5OH ends may be generated by many endogenous nucleases (36,38) but may also reflect exogenous RNA fragmentation. The latter may be due to contamination by RNases secreted by our epidermis and mucosal surfaces (36,39,40), or by commercial RNAses (such as RNAse A, RNAse T1, RNAse I). All these RNAses use acid-base catalysis, taking advantage of the 2 OH group of RNA as a reactive species and generate 5OH termini (36,40). ...
... The latter may be due to contamination by RNases secreted by our epidermis and mucosal surfaces (36,39,40), or by commercial RNAses (such as RNAse A, RNAse T1, RNAse I). All these RNAses use acid-base catalysis, taking advantage of the 2 OH group of RNA as a reactive species and generate 5OH termini (36,40). Another source of exogenous degradation is due to the inherent instability of RNA caused be spontaneous cleavage of phosphodiester bonds by intramolecular, in-line nucleophilic attacks that also generate 5OH termini (41,42). ...
Direct sequencing of single, native RNA molecules through nanopores has a strong potential to transform research in all aspects of RNA biology and clinical diagnostics. The existing platform from Oxford Nanopore Technologies is unable to sequence the very 5′ ends of RNAs and is limited to polyadenylated molecules. Here, we develop True End-to-end RNA Sequencing (TERA-Seq), a platform that addresses these limitations, permitting more thorough transcriptome characterization. TERA-Seq describes both poly- and non-polyadenylated RNA molecules and accurately identifies their native 5′ and 3′ ends by ligating uniquely designed adapters that are sequenced along with the transcript. We find that capped, full-length mRNAs in human cells show marked variation of poly(A) tail lengths at the single molecule level. We report prevalent capping downstream of canonical transcriptional start sites in otherwise fully spliced and polyadenylated molecules. We reveal RNA processing and decay at single molecule level and find that mRNAs decay cotranslationally, often from their 5′ ends, while frequently retaining poly(A) tails. TERA-Seq will prove useful in many applications where true end-to-end direct sequencing of single, native RNA molecules and their isoforms is desirable.
... T he pancreatic hRNase A superfamily is comprised of eight canonical hRNases, including human ribonuclease 1 (hRNase 1), and five noncanonical hRNases, all of which can be detected in human plasma/serum 1,2 . Secretory hRNase 1 is found in various organs 3,4 , and its biochemical properties and post-translational modifications, such as glycosylation, have been extensively investigated [5][6][7] . ...
... Secretory hRNase 1 is found in various organs 3,4 , and its biochemical properties and post-translational modifications, such as glycosylation, have been extensively investigated [5][6][7] . Nonetheless, the biological function of hRNase 1 has not yet been completely defined. Studies on bovine homolog RNase A indicated that its human homolog hRNase 1 also plays a role in the digestive system by degrading dietary RNAs 4,8 . ...
p>The pancreatic human ribonuclease (hRNase) A superfamily is comprised of eight canonical hRNases, including hRNase 1, all of which can be detected in various body fluids, e.g., serum and plasma. Secretory hRNase 1 exerts its ribonucleolytic activity to function in extracellular RNA clearance. Moreover, hRNase 1 regulates hemostasis, inflammation, and innate immunity, indicating that hRNase 1 possesses multiple functions in addition to RNA clearance. Studies on hRNase 1 have been extensively investigated the biochemical properties and post-translational modifications, such as glycosylation. Nonetheless, the biological function of hRNase1 and whether it plays a role in cancer have not yet been completely defined. Recent studies indicated that hRNase 5 serves as a ligand for the receptor tyrosine kinase (RTK) epidermal growth factor receptor and plexin-B2 receptor in solid and hematopoietic cancers. Those findings reveal a role of the hRNase A superfamily in tumor progression and an unconventional ligand-receptor relationship between RNase and RTK families. Here, we demonstrate that hRNase 1, independently its ribonucleolytic activity, enriches the stem-like cell population and enhances the tumor-initiating ability of breast cancer cells. Specifically, secretory hRNase 1 binds to and activates the RTK Eph receptor A4 (EphA4) signaling to promote breast tumor initiation in an autocrine/paracrine manner, which is distinct from the classical ligand-receptor ephrin-EphA4 juxtacrine signaling through contact-dependent cell-cell communication. In addition, analysis of human breast tumor tissue microarrays reveals a positive correlation between hRNase 1, EphA4 activation, and stem cell marker CD133. Notably, high hRNase 1 level in plasma samples is positively associated with EphA4 activation in tumor tissues from the paired breast cancer patients, highlighting the pathological relevance of the hRNase 1-EphA4 axis in breast cancer. The discovery of hRNase 1 as a secretory ligand of EphA4 to enhance breast cancer stemness suggests a potential treatment strategy for breast cancer by inactivating the hRNase 1-EphA4 axis.
Citation Format: Ying-Nai Wang, Heng-Huan Lee, Wen-Hao Yang, Gabriel N. Hortobagyi, Dihua Yu, Mien-Chie Hung. Secretory human ribonuclease 1 functions as Eph receptor A4 ligand to promote breast tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3081.</p
... T he pancreatic hRNase A superfamily is comprised of eight canonical hRNases, including human ribonuclease 1 (hRNase 1), and five noncanonical hRNases, all of which can be detected in human plasma/serum 1,2 . Secretory hRNase 1 is found in various organs 3,4 , and its biochemical properties and post-translational modifications, such as glycosylation, have been extensively investigated [5][6][7] . ...
... Secretory hRNase 1 is found in various organs 3,4 , and its biochemical properties and post-translational modifications, such as glycosylation, have been extensively investigated [5][6][7] . Nonetheless, the biological function of hRNase 1 has not yet been completely defined. Studies on bovine homolog RNase A indicated that its human homolog hRNase 1 also plays a role in the digestive system by degrading dietary RNAs 4,8 . ...
Human ribonuclease 1 (hRNase 1) is critical to extracellular RNA clearance and innate immunity to achieve homeostasis and host defense; however, whether it plays a role in cancer remains elusive. Here, we demonstrate that hRNase 1, independently of its ribonucleolytic activity, enriches the stem-like cell population and enhances the tumor-initiating ability of breast cancer cells. Specifically, secretory hRNase 1 binds to and activates the tyrosine kinase receptor ephrin A4 (EphA4) signaling to promote breast tumor initiation in an autocrine/paracrine manner, which is distinct from the classical EphA4-ephrin juxtacrine signaling through contact-dependent cell-cell communication. In addition, analysis of human breast tumor tissue microarrays reveals a positive correlation between hRNase 1, EphA4 activation, and stem cell marker CD133. Notably, high hRNase 1 level in plasma samples is positively associated with EphA4 activation in tumor tissues from breast cancer patients, highlighting the pathological relevance of the hRNase 1-EphA4 axis in breast cancer. The discovery of hRNase 1 as a secretory ligand of EphA4 that enhances breast cancer stemness suggests a potential treatment strategy by inactivating the hRNase 1-EphA4 axis.
... doi: bioRxiv preprint controlling RNA expression (Griesemer et al., 2021). This di-nucleotide specificity may partially be attributed to the sequence preference of a human ribonuclease superfamily in which eight catalytically-active RNases (numbered 1-8) all share homology with bovine pancreatic ribonuclease A. RNase A cleaves 3'-5' phosphodiester bonds with a specificity for pyrimidines (U/C) at the main anchoring site and purines (A/G) at the secondary site (Sorrentino, 2010). ...
UTRs contain crucial regulatory elements for RNA stability, translation and localization, so their integrity is indispensable for gene expression. It has been estimated that ∼3.7% of disease-associated genetic variants are located in UTRs. However, functional interpretation of UTR variants is largely incomplete because efficient means of experimental or computational assessment are lacking. To systematically evaluate the effects of UTR variants on RNA stability, we established a massively parallel reporter assay on 6,555 UTR variants reported in human disease databases. We examined the RNA degradation patterns mediated by the UTR library in multiple cell lines, and then applied LASSO regression to model the influential regulators of RNA stability. We found that TA dinucleotides are the most prominent destabilizing element. Gain of TA dinucleotide outlined mutant UTRs with reduced stability. Studies on endogenous transcripts indicate that high TA-dinucleotide ratios in UTRs promote RNA degradation. Conversely, elevated GC content and protein binding on TA dinucleotides protect high-TA RNA from degradation. Further analysis reveals polarized roles of TA-dinucleotide-binding proteins in RNA protection and degradation. Furthermore, the TA-dinucleotide ratio of both UTRs is a common characteristic of genes in innate immune response pathways, implying that the global transcriptomic regulon involves stability coordination via UTRs. We also demonstrate that stability-altering UTRs are associated with changes in biobank-based health indices, providing evidence that UTR-mediated RNA stability contributes to establishing robust gene networks and potentially enabling disease-associated UTR variants to be classified for precision medicine.
... These active small noncoding RNAs are also involved in the regulation of mRNA stability, interaction with cytochrome C to modulate apoptosis and the SGs assembly [18]. The release of tiRNAs is mediated by angiogenin (ANG), a member of the Vertebrate RNase Superfamily [19,20], widely expressed in most tissues and associated to angiogenesis, hematopoiesis, oncogenesis, neurodegenerative diseases [21,22], inflammation, and immunity [23,24]. ANG is actively secreted and captured by cells both in an autocrine and in a paracrine way [25]. ...
Human angiogenin (hANG) is the most studied stress-induced ribonuclease (RNase). In physiological conditions it performs its main functions in nucleoli, promoting cell proliferation by rDNA transcription, whereas it is strongly limited by its inhibitor (RNH1) throughout the rest of the cell. In stressed cells hANG dissociates from RNH1 and thickens in the cytoplasm where it manages the translational arrest and the recruitment of stress granules, thanks to its propensity to cleave tRNAs and to induce the release of active halves. Since it exists a clear connection between hANG roles and its intracellular routing, starting from our recent findings on heterologous ANG (ANG) properties in human keratinocytes (HaCaT cells), here we designed a variant unable to translocate into the nucleus with the aim of thoroughly verifying its potentialities under stress. This variant, widely characterized for its structural features and biological attitudes, shows more pronounced aid properties than unmodified protein. The collected evidence thus fully prove that ANG stress-induced skills in assisting cellular homeostasis are strictly due to its cytosolic localization. This study opens an interesting scenario for future studies regarding both the strengthening of skin defences and in understanding the mechanism of action of these special enzymes potentially suitable for any cell type.
... The ligation-based methods were originally tailored for capturing intracellular miRNAs that are 5′-phosphorylated, and until recently, the biases associated with standard ligation-based commercial kits have not been considered. However, extracellular RNAs are exposed to RNases and as a result are converted to 5′-hydroxyl and 3′-(cyclo) phosphate entities (Sorrentino, 2010;Giraldez et al., 2019;Nechooshtan et al., 2020). Because ssRNA adapters cannot be ligated to such RNAs, the latter will not be included into the final library and sequenced without prior end-repair. ...
Since its original discovery over a decade ago, extracellular RNA (exRNA) has been found in all biological fluids. Furthermore, extracellular microRNA has been shown to be involved in communication between various cell types. Importantly, the exRNA is protected from RNases degradation by certain carriers including membrane vesicles and non-vesicular protein nanoparticles. Each type of carrier has its unique exRNA profile, which may vary depending on cell type and physiological conditions. To clarify putative mechanisms of intercellular communication mediated by exRNA, the RNA profile of each carrier has to be characterized. While current methods of biofluids fractionation are continuously improving, they fail to completely separate exRNA carriers. Likewise, most popular library preparation approaches for RNA sequencing do not allow obtaining exhaustive and unbiased data on exRNA transcriptome. In this mini review we discuss ongoing progress in the field of exRNA, with the focus on exRNA carriers, analyze the key methodological challenges and provide recommendations on how the latter could be overcome.
... Since then, other homologous RNases with structural conservation have been discovered, forming the RNase A superfamily that is unique to vertebrates. In this superfamily, there are eight canonical members present in humans (now named RNASE1-8) [18]; all of these are encoded on chromosome 14 [19]. Each of this superfamily of RNases contain a signal peptide that leads to them being secreted [20,21]. ...
Simple Summary
The rate by which the ribosome decodes an mRNA determines the final protein output of the mRNA. This can vary greatly between different mRNAs. Methods which allow accurate determination of translation rates of specific mRNAs require conditions that preserve native mRNA–ribosome interactions and thus cannot be denaturing-based. Complex tissues sometimes contain RNases that can breakdown the RNA. Here, we optimise methods in non-denaturing conditions enabling the application of methods that examine translational control to primary mouse tissue in an accurate and reproducible manner.
Abstract
The protein output of different mRNAs can vary by two orders of magnitude; therefore, it is critical to understand the processes that control gene expression operating at the level of translation. Translatome-wide techniques, such as polysome profiling and ribosome profiling, are key methods for determining the translation rates occurring on specific mRNAs. These techniques are now widely used in cell lines; however, they are underutilised in tissues and cancer models. Ribonuclease (RNase) expression is often found to be higher in complex primary tissues in comparison to cell lines. Methods used to preserve RNA during lysis often use denaturing conditions, which need to be avoided when maintaining the interaction and position of the ribosome with the mRNA is required. Here, we detail the cell lysis conditions that produce high-quality RNA from several different tissues covering a range of endogenous RNase expression levels. We highlight the importance of RNA integrity for accurate determination of the global translation status of the cell as determined by polysome gradients and discuss key aspects to optimise for accurate assessment of the translatome from primary mouse tissue.
... Ribonuclease (RNase) 1 belongs to the family of pancreatic-type endoribonucleases called the human RNase A superfamily, which includes 13 members closely located on chromosome 14 and divided into two subgroups, canonical RNases 1-8 and noncanonical RNases 9-13 [11,12]. All the RNase genes encode secretory proteins containing an N-terminal hydrophobic signal peptide; among them, RNase1 is the most broadly expressed in various tissues and secreted abundantly in the circulatory system such as serum and plasma [13,14]. ...
The secretory enzyme human ribonuclease 1 (RNase1) is involved in innate immunity and anti-inflammation, achieving host defense and anti-cancer effects; however, whether RNase1 contributes to adaptive immune response in the tumor microenvironment (TME) remains unclear. Here, we established a syngeneic immunocompetent mouse model in breast cancer and demonstrated that ectopic RNase1 expression significantly inhibited tumor progression. Overall changes in immunological profiles in the mouse tumors were analyzed by mass cytometry and showed that the RNase1-expressing tumor cells significantly induced CD4+ Th1 and Th17 cells and natural killer cells and reduced granulocytic myeloid-derived suppressor cells, supporting that RNase1 favors an antitumor TME. Specifically, RNase1 increased expression of T cell activation marker CD69 in a CD4+ T cell subset. Notably, analysis of cancer-killing potential revealed that T cell-mediated antitumor immunity was enhanced by RNase1, which further collaborated with an EGFR-CD3 bispecific antibody to protect against breast cancer cells across molecular subtypes. Our results uncover a tumor-suppressive role of RNase1 through adaptive immune response in breast cancer in vivo and in vitro, providing a potential treatment strategy of combining RNase1 with cancer immunotherapies for immunocompetent patients.
... DDX60 L and PARP12 are related to immune and inflammatory responses that contribute to the development of atherosclerosis 44 . The endothelial extracellular endonuclease ribonuclease 1 (RNase1) belongs to the ribonuclease A superfamily 45 , which is mainly expressed in various vascular endothelial cells 46 . There are many Weibel-Palade Bodies (WPBs) in the vascular endothelium, which store inflammatory mediators, including RNase1, IL-8, and chemokines 47 . ...
RNA-binding proteins (RBPs) are involved in the regulation of RNA splicing, stability, and localization. How RBPs control the development of atherosclerosis, is not fully understood. To explore the relevant RNA-binding proteins (RBPs) and alternative splicing events (ASEs) in atherosclerosis. We made a comprehensive work to integrate analyses of differentially expressed genes, including differential RBPs, and variable splicing characteristics related to different stages of atherosclerosis in dataset GSE104140. A total of 3712 differentially expressed genes (DEGs) were identified, including 2921 upregulated genes and 791 downregulated genes. Further analysis screened out 54 RBP genes, and 434 AS genes overlapped DEGs. We selected high expression ten RBP genes (SAMHD1, DDX60 L, TLR7, RBM47, MYEF2, RNASE6, PARP12, APOBEC3G, SMAD9, and RNASE1) for co-expression analysis. Meanwhile, we found seven regulated alternative splicing genes (RASGs) (ABI1, FXR1, CHID1, PLEC, PRKACB, BNIP2, PPP3CB) that could be regulated by RBPs. The co-expression network was used to further elucidate the regulatory and interaction relationship between RBPs and AS genes. Apoptotic process and innate immune response, revealed by the functional enrichment analysis of RASGs regulated by RBPs were closely related to atherosclerosis. In addition, 26 of the 344 alternative splicing genes regulated by the above 10 RBPs were transcription factors (TFs), We selected high expression nine TFs (TFDP1, RBBP7, STAT2, CREB5, ERG, ELF1, HMGN3, BCLAF1, and ZEB2) for co-expression analysis. The target genes of these TFs were mainly enriched in inflammatory and immune response pathways that were associated with atherosclerosis. indicating that AS abnormalities of these TFs may have a function in atherosclerosis. Furthermore, the expression of differentially expressed RBPs and the alternative splicing events of AS genes was validated by qRT-PCR in umbilical vein endothelial cells (HUVEC). The results showed that RBM47 were remarkedly difference in HUVEC treated with ox-LDL and the splicing ratio of AS in BCLAF1which is regulated by RBM47 significantly changed. In conclusion, the differentially expressed RBPs identified in our analysis may play important roles in the development of atherosclerosis by regulating the AS of these TF genes.
... However, the involvement of nonvesicular exRNAs (nv-exRNAs) in intercellular communication pathways faces an important conceptual challenge. As a consequence of the strong ribonuclease activity that characterizes the extracellular space (36), nv-exRNAs are expected to be rapidly degraded unless protected by RNA-binding proteins. How, then, do these RNAs resist degradation, diffuse to recipient cells, and trigger downstream effects, or even remain measurable and thus serve as potential disease biomarkers? ...
Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function, and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine, and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of several hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.
... However, the involvement of nonvesicular exRNAs (nv-exRNAs) in intercellular communication pathways faces an important conceptual challenge. As a consequence of the strong ribonuclease activity that characterizes the extracellular space (Sorrentino, 2010), nv-exRNAs are expected to be rapidly degraded unless protected by RNA-binding proteins. How, then, do these RNAs resist degradation, diffuse to recipient cells and trigger downstream effects, or even remain measurable and thus serve as potential disease biomarkers? ...
Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of up to three hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.
... However, the experiments presented here may offer a glimpse into a more realistic in vivo scenario, in which multiple antimicrobial agents work in concert against infection. Eight RNases are encoded by the human genome, many of which have potent antimicrobial activity, such as RNase 7 expressed in epithelial cells (Sorrentino 2010). Bovine pancreatic RNase A, on the other hand, has a digestive function degrading RNA and an antimicrobial function has not normally been ascribed to it. ...
Background: HNP1, LL-37, and HBD1 are antimicrobial against Escherichia coli ATCC 25922 at the standard inoculum but less active at higher inocula.
Methods: The virtual colony count (VCC) microbiological assay was adapted for high inocula and the addition of yeast tRNA and bovine pancreatic ribonuclease A (RNase). 96-well plates were read for 12 hours in a Tecan Infinite M1000 plate reader and photographed under 10x magnification.
Results: Adding tRNA 1:1 to HNP1 at the standard inoculum almost completely abrogated activity. Adding RNase 1:1 to HNP1 at the standard inoculum of 5x105 CFU/mL did not enhance activity. Increasing the inoculum to 6.25x107 CFU/mL almost abrogated HNP1 activity. However, adding RNase 25:1 to HNP1 enhanced activity. Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present. HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA. At the high inoculum, LL-37 activity was enhanced by RNase. HBD1 activity was not enhanced by RNase. RNase was not antimicrobial in the absence of antimicrobial peptides. Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides and at the standard inoculum in the presence of HNP1+tRNA.
Conclusions: Antimicrobial peptide-ribonuclease combinations have the potential to be active against high cell concentrations and biofilms, conditions where the antimicrobial agent alone is relatively ineffective.
... Banerjee et al. reported that the 2Cs of PV, HRV and HAV all bind to the 3 -UTR of the negative-strand viral RNA; thus, they postulated that 2C could immobilize negative-strand viral RNA to the membrane-anchored replication complex where it serves as the template for synthesizing plus-strand RNAs. Here, we provide evidence that HAV 2C harbors an unusual ssRNA-specific ribonuclease activity with a preference for poly (U) regions, similar to vertebrate ribonuclease RNase 4 (43). The substrate specificity and structural basis for the nuclease activity of other 2C proteins will require detailed investigations. ...
The HAV nonstructural protein 2C is essential for virus replication; however, its precise function remains elusive. Although HAV 2C shares 24–27% sequence identity with other 2Cs, key motifs are conserved. Here, we demonstrate that HAV 2C is an ATPase but lacking helicase activity. We identified an ATPase-independent nuclease activity of HAV 2C with a preference for polyuridylic single-stranded RNAs. We determined the crystal structure of an HAV 2C fragment to 2.2 Å resolution, containing an ATPase domain, a region equivalent to enterovirus 2C zinc-finger (ZFER) and a C-terminal amphipathic helix (PBD). The PBD of HAV 2C occupies a hydrophobic pocket (Pocket) in the adjacent 2C, and we show the PBD–Pocket interaction is vital for 2C functions. We identified acidic residues that are essential for the ribonuclease activity and demonstrated mutations at these sites abrogate virus replication. We built a hexameric-ring model of HAV 2C, revealing the ribonuclease-essential residues clustering around the central pore of the ring, whereas the ATPase active sites line up at the gaps between adjacent 2Cs. Finally, we show the ribonuclease activity is shared by other picornavirus 2Cs. Our findings identified a previously unfound activity of picornavirus 2C, providing novel insights into the mechanisms of virus replication.
... First, an exogeneous nucleic acid medication must pass through the skin's tight junctions and then the cell membrane or nucleus. Then it needs to resist degradation by endogenous enzymes such liver metabolism and nucleases until its internalization into the cell (Sorrentino 2010). As a result, it relies on a drug delivery system that can protect against nuclease and protein binding while allowing for enhanced membrane penetration (McCaffrey et al. 2015;Pardi et al. 2018;Hu et al. 2020). ...
Background
Nucleic acid-based gene therapy is a promising technology that has been used in various applications such as novel vaccination platforms for infectious/cancer diseases and cellular reprogramming because of its fast, specific, and effective properties. Despite its potential, the parenteral nucleic acid drug formulation exhibits instability and low efficacy due to various barriers, such as stability concerns related to its liquid state formulation, skin barriers, and endogenous nuclease degradation. As promising alternatives, many attempts have been made to perform nucleic acid delivery using a microneedle system. With its minimal invasiveness, microneedle can deliver nucleic acid drugs with enhanced efficacy and improved stability.Area coveredThis review describes nucleic acid medicines' current state and features and their delivery systems utilizing non-viral vectors and physical delivery systems. In addition, different types of microneedle delivery systems and their properties are briefly reviewed. Furthermore, recent advances of microneedle-based nucleic acid drugs, including featured vaccination applications, are described.Expert opinionNucleic acid drugs have shown significant potential beyond the limitation of conventional small molecules, and the current COVID-19 pandemic highlights the importance of nucleic acid therapies as a novel vaccination platform. Microneedle-mediated nucleic acid drug delivery is a potential platform for less invasive nucleic acid drug delivery. Microneedle system can show enhanced efficacy, stability, and improved patient convenience through self-administration with less pain.
... RNases are members of a family of important enzymes that catalyze the scission of RNA into smaller molecules. 35 RNase A is a prototype of this family. 36 It has been recently pointed out that several therapies based on small molecules or oligonucleotides present the general aim of targeting RNA biology. ...
The structure, stability, and enzymatic activity of the adduct formed upon the reaction of the V–picolinato (pic) complex [VIVO(pic)2(H2O)], with an octahedral geometry and the water ligand in cis to the V═O group, with the bovine pancreatic ribonuclease (RNase A) were studied. While electrospray ionization-mass spectrometry, circular dichroism, and ultraviolet–visible absorption spectroscopy substantiate the interaction between the metal moiety and RNase A, electron paramagnetic resonance (EPR) allows us to determine that a carboxylate group, stemming from Asp or Glu residues, and imidazole nitrogen from His residues are involved in the V binding at acidic and physiological pH, respectively. Crystallographic data demonstrate that the VIVO(pic)2 moiety coordinates the side chain of Glu111 of RNase A, by substituting the equatorial water molecule at acidic pH. Computational methods confirm that Glu111 is the most affine residue and interacts favorably with the OC-6-23-Δ enantiomer establishing an extended network of hydrogen bonds and van der Waals stabilizations. By increasing the pH around neutrality, with the deprotonation of histidine side chains, the binding of the V complex to His105 and His119 could occur, with that to His105 which should be preferred when compared to that to the catalytically important His119. The binding of the V compound affects the enzymatic activity of RNase A, but it does not alter its overall structure and stability.
... ANG is a secreted growth factor present in normal human tissues and plasma, or amniotic fluid [3,4], so called because it promotes, together with other proteins, chemokines, factors, and cells [5], the neo-formation of vessels [1,6]. ANG is also known as ribonuclease 5 (RNase 5) [7] because it displays the typical size and fold of the secretory pancreatic-type (pt)-RNases [8], and its catalytic triad is composed of His13, Lys40, and His114 [9,10], corresponding to the H12, K41, and H119 residues in bovine pancreatic RNase A, the super-family archetype [11]. ANG and RNase A share only 33% sequence identity and 65% sequence similarity [1], and ANG lacks the C65-C72 short disulfide but possesses an extra 3 10 -helix at its C-terminus [8]. ...
Human Angiogenin (hANG, or ANG, 14.1 kDa) promotes vessel formation and is also called RNase 5 because it is included in the pancreatic-type ribonuclease (pt-RNase) super-family. Although low, its ribonucleolytic activity is crucial for angiogenesis in tumor tissues but also in the physiological development of the Central Nervous System (CNS) neuronal progenitors. Nevertheless, some ANG variants are involved in both neurodegenerative Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Notably, some pt-RNases acquire new biological functions upon oligomerization. Considering neurodegenerative diseases correlation with massive protein aggregation, we analyzed the aggregation propensity of ANG and of three of its pathogenic variants, namely H13A, S28N, and R121C. We found no massive aggregation, but wt-ANG, as well as S28N and R121C variants, can form an enzymatically active dimer, which is called ANG-D. By contrast, the enzymatically inactive H13A-ANG does not dimerize. Corroborated by a specific cross-linking analysis and by the behavior of H13A-ANG that in turn lacks one of the two His active site residues necessary for pt-RNases to self-associate through the three-dimensional domain swapping (3D-DS), we demonstrate that ANG actually dimerizes through 3D-DS. Then, we deduce by size exclusion chromatography (SEC) and modeling that ANG-D forms through the swapping of ANG N-termini. In light of these novelties, we can expect future investigations to unveil other ANG determinants possibly related with the onset and/or development of neurodegenerative pathologies.
... (i) Structural and protective: The aforementioned lipid compounds have a remarkable ability to self-assemble around mRNA [24] forming stable, spherical nanoparticles below 100 nm in diameter, approximately the average size of a SARS-CoV-2 virion. The trafficking of mRNA into such lipid spheres (LNP) protects it against enzymes that would otherwise digest it very quickly, such as RNases [25]. ...
In the context of the current COVID-19 pandemic, traditional, complex and lengthy methods of vaccine development and production would not have been able to ensure proper management of this global public health crisis. Hence, a number of technologies have been developed for obtaining a vaccine quickly and ensuring a large scale production, such as mRNA-based vaccine platforms. The use of mRNA is not a new concept in vaccine development but has leveraged on previous knowledge and technology. The great number of human resources and capital investements for mRNA vaccine development, along with the experience gained from previous studies on infectious diseases, allowed COVID-19 mRNA vaccines to be developed, conditionally approved and commercialy available in less than one year, thanks to decades of basic research. This review critically presents and discusses the COVID-19 mRNA vaccine-induced immunity, and it summarizes the most common anaphylactic and autoimmune adverse effects that have been identified until now after massive vaccination campaigns.
... While identification of SARS-CoV-2 RNA could technically represent "inert" RNA, the possibility is unlikely because inert RNA in the human body is rapidly degraded (Houseley and Tollervey, 2009;Fabre et al., 2014). Nearly every human cell type, and human tears, saliva, mucus, perspiration, and extracellular spaces express RNAase enzymes that rapidly degrade inert RNA (Sorrentino, 2010;Gupta et al., 2013). Indeed, overcoming RNAse activity was a central challenge in mRNA vaccine development (Pardi et al., 2018). ...
The novel virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic of coronavirus disease 2019 (COVID-19). Across the globe, a subset of patients who sustain an acute SARS-CoV-2 infection are developing a wide range of persistent symptoms that do not resolve over the course of many months. These patients are being given the diagnosis Long COVID or Post-acute sequelae of COVID-19 (PASC). It is likely that individual patients with a PASC diagnosis have different underlying biological factors driving their symptoms, none of which are mutually exclusive. This paper details mechanisms by which RNA viruses beyond just SARS-CoV-2 have be connected to long-term health consequences. It also reviews literature on acute COVID-19 and other virus-initiated chronic syndromes such as post-Ebola syndrome or myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) to discuss different scenarios for PASC symptom development. Potential contributors to PASC symptoms include consequences from acute SARS-CoV-2 injury to one or multiple organs, persistent reservoirs of SARS-CoV-2 in certain tissues, re-activation of neurotrophic pathogens such as herpesviruses under conditions of COVID-19 immune dysregulation, SARS-CoV-2 interactions with host microbiome/virome communities, clotting/coagulation issues, dysfunctional brainstem/vagus nerve signaling, ongoing activity of primed immune cells, and autoimmunity due to molecular mimicry between pathogen and host proteins. The individualized nature of PASC symptoms suggests that different therapeutic approaches may be required to best manage care for specific patients with the diagnosis.
... The vertebrate-specific RNase A superfamily includes in human 13 members, named RNases 1-13, which are secreted proteins and have diverse roles, such as antimicrobial and immunomodulatory [15,18,[198][199][200][201]. Expressed by innate immune cells and targeted to either the extracellular or endolysosomal space, RNase A family members are well fit to provide a safeguard action against intruding pathogens [14,202]. ...
Owing to the recent outbreak of Coronavirus Disease of 2019 (COVID-19), it is urgent to develop effective and safe drugs to treat the present pandemic and prevent other viral infections that might come in the future. Proteins from our own innate immune system can serve as ideal sources of novel drug candidates thanks to their safety and immune regulation versatility. Some host defense RNases equipped with antiviral activity have been reported over time. Here, we try to summarize the currently available information on human RNases that can target viral pathogens, with special focus on enveloped single-stranded RNA (ssRNA) viruses. Overall, host RNases can fight viruses by a combined multifaceted strategy, including the enzymatic target of the viral genome, recognition of virus unique patterns, immune modulation, control of stress granule formation, and induction of autophagy/apoptosis pathways. The review also includes a detailed description of representative enveloped ssRNA viruses and their strategies to interact with the host and evade immune recognition. For comparative purposes, we also provide an exhaustive revision of the currently approved or experimental antiviral drugs. Finally, we sum up the current perspectives of drug development to achieve successful eradication of viral infections.
... The family has eight canonical members (RNASE 1-8) that have a conserved RNA-degrading catalytic domain. Additionally, there are 5 reported non-canonical members (RNASE 9-13), that are involved in male-reproductive functions but do not possess ribonuclease activity (188). Although the major function of several RNase A family members is digestion of dietary RNA, several of them have evolved to perform antibacterial, antiviral and immune modulatory functions (189). ...
Detection of microbial nucleic acids by the innate immune system is mediated by numerous intracellular nucleic acids sensors. Upon the detection of nucleic acids these sensors induce the production of inflammatory cytokines, and thus play a crucial role in the activation of anti-microbial immunity. In addition to microbial genetic material, nucleic acid sensors can also recognize self-nucleic acids exposed extracellularly during turn-over of cells, inefficient efferocytosis, or intracellularly upon mislocalization. Safeguard mechanisms have evolved to dispose of such self-nucleic acids to impede the development of autoinflammatory and autoimmune responses. These safeguard mechanisms involve nucleases that are either specific to DNA (DNases) or RNA (RNases) as well as nucleic acid editing enzymes, whose biochemical properties, expression profiles, functions and mechanisms of action will be detailed in this review. Fully elucidating the role of these enzymes in degrading and/or processing of self-nucleic acids to thwart their immunostimulatory potential is of utmost importance to develop novel therapeutic strategies for patients affected by inflammatory and autoimmune diseases.
... The RNase A superfamily is classified in two subgroups, canonical (RNases 1-8) and non-canonical (RNases 9-13) [86]. The latter subgroup is deficient in RNase activity and, although to date little is known about their physiological functions, it is believed that they may play a role in host defense similar to other canonical members, such as EDN or ECP (for a review see [87,88]). Taking into account the omics results known to date on the cytotoxic action of RNases, it is likely that some members could behave as transcription factors-like or as cellspecific ligands that transduce signals to the target cells ultimately affecting the expression of different coding and/or non-coding RNAs (Figure 1). These roles are well defined for ANG, which presents a ribonucleolytic activity toward model substrates that is 10 −5 -10 −6fold lower than that of RNase A [89]. ...
Approaches to develop effective drugs to kill cancer cells are mainly focused either on the improvement of the currently used chemotherapeutics or on the development of targeted therapies aimed at the selective destruction of cancer cells by steering specific molecules and/or enhancing the immune response. The former strategy is limited by its genotoxicity and severe side effects, while the second one is not always effective due to tumor cell heterogeneity and variability of targets in cancer cells. Between these two strategies, several approaches target different types of RNA in tumor cells. RNA degradation alters gene expression at different levels inducing cell death. However, unlike DNA targeting, it is a pleotropic but a non-genotoxic process. Among the ways to destroy RNA, we find the use of ribonucleases with antitumor properties. In the last few years, there has been a significant progress in the understanding of the mechanism by which these enzymes kill cancer cells and in the development of more effective variants. All the approaches seek to maintain the requirements of the ribonucleases to be specifically cytotoxic for tumor cells. These requirements start with the competence of the enzymes to interact with the cell membrane, a process that is critical for their internalization and selectivity for tumor cells and continue with the downstream effects mainly relying on changes in the RNA molecular profile, which are not only due to the ribonucleolytic activity of these enzymes. Although the great improvements achieved in the antitumor activity by designing new ribonuclease variants, some drawbacks still need to be addressed. In the present review, we will focus on the known mechanisms used by ribonucleases to kill cancer cells and on recent strategies to solve the shortcomings that they show as antitumor agents, mainly their pharmacokinetics.
... 40 Members of the RNase A superfamily share a similar primary sequence and structural similarities such as 6e8 conserved cysteine residues forming characteristics distinct disulfide bonds and conserved histidines, as well as a lysine in the active center to catalyze the ribonuclease activity. 41,42 The ribonucleases are reported in mucosal secretions, in several types of immune cells as well as in major organs. 42 EDN exhibits ribonuclease activity against single stranded RNA viruses including respiratory syncytial virus, 43 hepatitis B virus 44 and HIV. ...
Neutrophils and eosinophils are granulocytes which are characterized by the presence of granules in the cytoplasm. Granules provide a safe storage site for granule proteins that play important roles in the immune function of granulocytes. Upon granulocytes activation, diverse proteins are released from the granules into the extracellular space and contribute to the fight against infections. In this article, we describe granule proteins of both neutrophils and eosinophils able to kill pathogens and review their anticipated mechanism of antimicrobial toxicity. It should be noted that an excess of granules protein release can lead to tissue damage of the host resulting in chronic inflammation and organ dysfunction.
... RNase1 belongs to the RNaseA family, which consists of eight members that have endonuclease activities and which are secreted by a large variety of different tissues and cells (Koczera et al., 2016). Eosinophil (RNase 2, RNase3) or epithelial cell-derived RNase7 serves as an anti-microbial protein, whereas RNase5 (also designated angiogenin) has potent angiogenic functions without having an appreciable ribonucleolytic activity (Cho et al., 2005;Sorrentino, 2010). Extracellular RNases can also be internalized by cells via endosomal pathways (Haigis and Raines, 2003), but due to the action of RNase inhibitor, which binds mammalian RNaseA family members with an extremely high affinity, these endonucleases are immediately inactivated and do not express any intracellular cytotoxic activity (Dickson et al., 2005;Rutkoski and Raines, 2008). ...
Upon vascular injury, tissue damage, ischemia, or microbial infection, intracellular material such as nucleic acids and histones is liberated and comes into contact with the vessel wall and circulating blood cells. Such “Danger-associated molecular patterns” (DAMPs) may thus have an enduring influence on the inflammatory defense process that involves leukocyte recruitment and wound healing reactions. While different species of extracellular RNA (exRNA), including microRNAs and long non-coding RNAs, have been implicated to influence inflammatory processes at different levels, recent in vitro and in vivo work has demonstrated a major impact of ribosomal exRNA as a prominent DAMP on various steps of leukocyte recruitment within the innate immune response. This includes the induction of vascular hyper-permeability and vasogenic edema by exRNA via the activation of the “vascular endothelial growth factor” (VEGF) receptor-2 system, as well as the recruitment of leukocytes to the inflamed endothelium, the M1-type polarization of inflammatory macrophages, or the role of exRNA as a pro-thrombotic cofactor to promote thrombosis. Beyond sterile inflammation, exRNA also augments the docking of bacteria to host cells and the subsequent microbial invasion. Moreover, upon vessel occlusion and ischemia, the shear stress-induced release of exRNA initiates arteriogenesis (i.e., formation of natural vessel bypasses) in a multistep process that resembles leukocyte recruitment. Although exRNA can be counteracted for by natural circulating RNase1, under the conditions mentioned, only the administration of exogenous, thermostable, non-toxic RNase1 provides an effective and safe therapeutic regimen for treating the damaging activities of exRNA. It remains to be investigated whether exRNA may also influence viral infections (including COVID-19), e.g., by supporting the interaction of host cells with viral particles and their subsequent invasion. In fact, as a consequence of the viral infection cycle, massive amounts of exRNA are liberated, which can provoke further tissue damage and enhance virus dissemination. Whether the application of RNase1 in this scenario may help to limit the extent of viral infections like COVID-19 and impact on leukocyte recruitment and emigration steps in immune defense in order to limit the extent of associated cardiovascular diseases remains to be studied.
Nucleic acid (NA)-sensing Toll-like receptors (TLRs) reside in the endosomal compartment of innate immune cells, such as macrophages and dendritic cells. NAs transported to the endosomal compartment are degraded by DNases and RNases. Degradation products, including single-stranded DNA, oligoRNA, and nucleosides, are recognized by TLR7, TLR8, and TLR9 to drive the defense responses against pathogens. NA degradation influences endosomal TLR responses by generating and degrading TLR ligands. TLR ligand accumulation because of impaired NA degradation causes constitutive TLR activation, leading to autoinflammatory and autoimmune diseases. Furthermore, some genes associated with these diseases promote endosomal TLR responses. Therefore, endosomal TLRs are promising therapeutic targets for TLR-mediated inflammatory diseases, and novel drugs targeting TLRs are being developed.
Considerable research has been done in investigating SARS‐CoV‐2 infection, its characteristics, and host immune response. However, debate is still ongoing over the emergence of post‐acute sequelae of SARS‐CoV‐2 infection (PASC). A multitude of long‐lasting symptoms have been reported several weeks after the primary acute SARS‐CoV‐2 infection that resemble several other viral infections. Thousands of research articles have described various post‐COVID‐19 conditions. Yet, the evidence around these ongoing health problems, the reasons behind them, and their molecular underpinnings are scarce. These persistent symptoms are also known as long COVID‐19. The persistence of SARS‐CoV‐2 and/or its components in host tissues can lead to long COVID. For example, the presence of viral nucleocapsid protein and RNA was detected in the skin, appendix, and breast tissues of some long COVID patients. The persistence of viral RNA was reported in multiple anatomic sites, including non‐respiratory tissues such as the adrenal gland, ocular tissue, small intestine, lymph nodes, myocardium, and sciatic nerve. Distinctive viral spike sequence variants were also found in non‐respiratory tissues. Interestingly, prolonged detection of viral subgenomic RNA was observed across all tissues, sometimes in multiple tissues of the same patient, which likely reflects recent but defective viral replication. Moreover, the persistence of SARS‐CoV‐2 RNA was noticed throughout the brain at autopsy, as late as 230 days following symptom onset among unvaccinated patients who died of severe infection. Here, we review the persistence of SARS‐CoV‐2 and its components as an intrinsic factor behind long COVID. We also highlight the immunological consequences of this viral persistence.
Gene therapy is on its way to revolutionize the treatment of both inherited and acquired diseases, by transferring nucleic acids to correct a disease-causing gene in the target cells of patients. In the fight against infectious diseases, mRNA-based therapeutics have proven to be a viable strategy in the recent Covid-19 pandemic. Although a growing number of gene therapies have been approved, the success rate is limited when compared to the large number of preclinical and clinical trials that have been/are being performed. In this review, we highlight some of the hurdles which gene therapies encounter after administration into the human body, with a focus on nucleic acid degradation by nucleases that are extremely abundant in mammalian organs, biological fluids as well as in subcellular compartments. We overview the available strategies to reduce the biodegradation of gene therapeutics after administration, including chemical modifications of the nucleic acids, encapsulation into vectors and co-administration with nuclease inhibitors and discuss which strategies are applied for clinically approved nucleic acid therapeutics. In the final part, we discuss the currently available methods and techniques to qualify and quantify the integrity of nucleic acids, with their own strengths and limitations.
Here, we studied the interaction of [V IV O(8-HQ) 2 ] – a promising antitrypanosomal, antituberculosis and antitumor vanadium complex (VC) formed by 8-hydroxyquinolinato (8-HQ) – with bovine pancreatic ribonuclease (RNase A) by a...
Background: HNP1, LL-37, and HBD1 are antimicrobial against Escherichia coli ATCC 25922 at the standard inoculum but less active at higher inocula.
Methods: The virtual colony count (VCC) microbiological assay was adapted for high inocula and the addition of yeast tRNA and bovine pancreatic ribonuclease A (RNase). 96-well plates were read for 12 hours in a Tecan Infinite M1000 plate reader and photographed under 10x magnification.
Results: Adding tRNA 1:1 wt/wt to HNP1 at the standard inoculum almost completely abrogated activity. Adding RNase 1:1 to HNP1 at the standard inoculum of 5x10 ⁵ CFU/mL did not enhance activity. Increasing the inoculum to 6.25x10 ⁷ CFU/mL almost abrogated HNP1 activity. However, adding RNase 25:1 to HNP1 enhanced activity at the highest tested concentration of HNP1. Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present. HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA. At the high inoculum, LL-37 activity was enhanced by RNase. HBD1 activity was not enhanced by RNase. RNase was not antimicrobial in the absence of antimicrobial peptides. Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides and at the standard inoculum in the presence of HNP1+tRNA and HBD1+tRNA.
Conclusions: Antimicrobial peptide-ribonuclease combinations have the potential to be active against high cell concentrations, conditions where the antimicrobial agent alone is relatively ineffective.
The evolutionary role of conformational exchange in the emergence and preservation of function within structural homologs remains elusive. While protein engineering has revealed the importance of flexibility in function, productive modulation of atomic-scale dynamics has only been achieved on a finite number of distinct folds. Allosteric control of unique members within dynamically diverse structural families requires a better appreciation of exchange phenomena. Here, we examined the functional and structural role of conformational exchange within eosinophil-associated ribonucleases. Biological and catalytic activity of various EARs was performed in parallel to mapping their conformational behavior on multiple timescales using NMR and computational analyses. Despite functional conservation and conformational seclusion to a specific domain, we show that EARs can display similar or distinct motional profiles, implying divergence rather than conservation of flexibility. Comparing progressively more distant enzymes should unravel how this subfamily has evolved new functions and/or altered their behavior at the molecular level.
Upon microbial infections with the subsequent host response of innate immunity, a variety of fragmented RNA- and DNA-based "Pathogen-associated molecular patterns" (PAMPs) are recognized mainly by endosomal or cytoplasmic host cell "Pattern recognition receptors" (PRRs), particularly "Toll-like receptors" (TLRs). Concomitantly, various self-extracellular RNA species (exRNAs) are present in extracellular body fluids where they contribute to diverse physiological and homeostatic processes. In principle, such exRNAs, including the most abundant one, ribosomal exRNA (rexRNA), are designated as "Danger-associated molecular patterns" (DAMPs) and are prevented by e.g. natural modifications from uncontrolled signaling via TLRs to avoid hyper-inflammatory responses or autoimmunity. Upon cellular stress or tissue damage/necrosis, the levels and composition of released self-exRNA species, either in free form, in complex with proteins or in association with extracellular vesicles (EVs), can change considerably. Among the self-exRNAs, rexRNA is considered as a non-typical DAMP, since it may induce inflammatory responses by cell membrane receptors, both in the absence or presence of PAMPs. Yet, its mode of receptor activation to mount inflammatory responses remains obscure. RexRNA also serves as a universal damaging factor in cardiovascular and other diseases independent of PRRs. In general, RNase1 provides a profound antagonist in these pathologies and in rexRNA-mediated inflammatory cell responses. Based on the extrapolation of the here described aspects of rexRNA-biology, further activities of this molecular entity are hypothesized that may stimulate additional research in this area.
Background: HNP1, LL-37, and HBD1 are antimicrobial against Escherichia coli ATCC 25922 at the standard inoculum but less active at higher inocula.
Methods: The virtual colony count (VCC) microbiological assay was adapted for high inocula and the addition of yeast tRNA and bovine pancreatic ribonuclease A (RNase). 96-well plates were read for 12 hours in a Tecan Infinite M1000 plate reader and photographed under 10x magnification.
Results: Adding tRNA 1:1 wt/wt to HNP1 at the standard inoculum almost completely abrogated activity. Adding RNase 1:1 to HNP1 at the standard inoculum of 5x10 ⁵ CFU/mL did not enhance activity. Increasing the inoculum to 6.25x10 ⁷ CFU/mL almost abrogated HNP1 activity. However, adding RNase 25:1 to HNP1 enhanced activity at the highest tested concentration of HNP1. Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present. HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA. At the high inoculum, LL-37 activity was enhanced by RNase. HBD1 activity was not enhanced by RNase. RNase was not antimicrobial in the absence of antimicrobial peptides. Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides and at the standard inoculum in the presence of HNP1+tRNA and HBD1+tRNA.
Conclusions: Antimicrobial peptide-ribonuclease combinations have the potential to be active against high cell concentrations and biofilms, conditions where the antimicrobial agent alone is relatively ineffective.
Non-invasive acquisition of mRNA data from the skin can be extremely useful for understanding skin physiology and diseases. Inspired by the holocrine process, in which the sebaceous glands secrete cell contents into the sebum, we focused on the possible presence of mRNAs in skin surface lipids (SSLs). We found that measurable levels of human mRNAs exist in SSLs, where the sebum protects them from degradation by RNases. The AmpliSeq transcriptome analysis was modified to measure SSL-RNA levels, and our results revealed that the SSL-RNAs predominantly comprised mRNAs derived from sebaceous glands, the epidermis, and hair follicles. Analysis of SSL-RNAs non-invasively collected from patients with atopic dermatitis revealed increased expression of inflammation-related genes and decreased expression of terminal differentiation-related genes, consistent with the results of previous reports. Further, we found that lipid synthesis-related genes were downregulated in the sebaceous glands of patients with atopic dermatitis. These results indicate that the analysis of SSL-RNAs is a promising strategy to understand the pathophysiology of skin diseases.
In order to overcome limitations of conventional cancer therapy methods, immunotoxins with the capability of target-specific action have been designed and evaluated pre-clinically, and some of them are in clinical studies. Targeting cancer cells via antibodies specific for tumour-associated surface proteins is a new biomedical approach that could provide the selectivity that is lacking in conventional cancer therapy methods such as radiotherapy and chemotherapy. A successful example of an approved immunotoxin is represented by immunoRNases. ImmunoRNases are fusion proteins in which the toxin has been replaced by a ribonuclease. Conjugation of RNase molecule to monoclonal antibody or antibody fragment was shown to enhance specific cell-killing by several orders of magnitude, both in vitro and in animal models. There are several RNases obtained from different mammalian cells that are expected to be less immunogenic and systemically toxic. In fact, RNases are pro-toxins which become toxic only upon their internalization in target cells mediated by the antibody moiety. The structure and large size of the antibody molecules assembled with the immunoRNases have always been a challenge in the application of immunoRNases as an antitoxin. To overcome this obstacle, we have offered a new strategy for the application of immunoRNases as a promising approach for upgrading immunoRNAses with maximum affinity and high stability in the cell, which can ultimately act as an effective large-scale cancer treatment. In this review, we introduce the optimized antibody-like molecules with small size, approximately 10 kD, which are presumed to significantly enhance RNase activity and be a suitable agent with the potential for anti-cancer functionality. In addition, we also discuss new molecular entities such as monobody, anticalin, nonobody and affilin as refined versions in the development of immunoRNases. These small molecules express their functionality with the suitable small size as well as with low immunogenicity in the cell, as a part of immunoRNases.
Radiotherapy in cancer treatment involves the use of ionizing radiation for cancer cell killing. Although radiotherapy has shown significant improvements on cancer recurrence and mortality, several radiation-induced adverse effects have been documented. Of these adverse effects, radiation-induced cardiovascular disease (CVD) is particularly prominent among patients receiving mediastinal radiotherapy, such as breast cancer and Hodgkin's lymphoma patients. A number of mechanisms of radiation-induced CVD pathogenesis have been proposed such as endothelial inflammatory activation, premature endothelial senescence, increased ROS and mitochondrial dysfunction. However, current research seems to point to a so-far unexamined and potentially novel involvement of epigenetics in radiation-induced CVD pathogenesis. Firstly, epigenetic mechanisms have been implicated in CVD pathophysiology. In addition, several studies have shown that ionizing radiation can cause epigenetic modifications, especially DNA methylation alterations. As a result, this review aims to provide a summary of the current literature linking DNA methylation to radiation-induced CVD and thereby explore DNA methylation as a possible contributor to radiation-induced CVD pathogenesis.
Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These “glycoRNAs” were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.
Non-invasive acquisition of mRNA data from the skin would be extremely useful for understanding skin physiology and diseases. Inspired by the holocrine process, in which the sebaceous glands secrete cell contents into the sebum, we focused on the possible presence of mRNAs in skin surface lipids (SSLs). We found that measurable human mRNAs exist in SSLs, where sebum protects them from degradation by RNases. The AmpliSeq transcriptome analysis was modified to measure SSL-RNAs, and our results revealed that SSL-RNAs predominantly contained mRNAs derived from sebaceous glands, epidermis, and hair follicles. Analysis of SSL-RNAs non-invasively collected from patients with atopic dermatitis revealed significantly increased expression of inflammation-related genes and decreased expression of terminal differentiation-related genes, consistent with the results of previous reports. Further, we found that lipid synthesis-related genes were downregulated in the sebaceous glands of patients with atopic dermatitis. These results indicate that the analysis of SSL-RNAs is promising to understand the pathophysiology of skin diseases.
Radiotherapy in cancer treatment involves the use of ionizing radiation for cancer cell killing. Although radiotherapy has shown significant improvements on cancer recurrence and mortality, several radiation-induced adverse effects have been documented. Of these adverse effects, radiation-induced cardiovascular disease (CVD) is particularly prominent among patients receiving mediastinal radiotherapy, such as breast cancer and Hodgkin’s lymphoma patients. A number of mechanisms of radiation-induced CVD pathogenesis have been proposed such as endothelial inflammatory activation, premature endothelial senescence, increased ROS and mitochondrial dysfunction. However, current research seems to point to a so-far unexamined and potentially novel involvement of epigenetics in radiation-induced CVD pathogenesis. Firstly, epigenetic mechanisms have been implicated in CVD pathophysiology. In addition, several studies have shown that ionizing radiation can cause epigenetic modifications, especially DNA methylation alterations. As a result, this review aims to provide a summary of the current literature linking DNA methylation to radiation-induced CVD and thereby explore DNA methylation as a possible contributor to radiation-induced CVD pathogenesis.
The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution.
Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human
genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
Human chorionic gonadotropin (hCG) preparations contain activity against HIV type 1 (HIV-1). However, there has been controversy
about whether some biological activities of hCG β-subunit (hCGβ) preparations are caused by the β-subunit itself or other
proteins present in the preparations. We report here the purification, characterization, and identification of three enzymes
with anti-HIV activity present in the β-core fraction of hCGβ prepared from the urine of pregnant women. The N-terminal amino
acid sequence of one protein is identical to human urinary lysozyme C, and those of the other two are identical to human RNase
A and urinary RNase U. We thus refer to these proteins as AVL (antiviral lysozyme) and AVR (antiviral RNases). In addition
to HIV-1 inhibition, AVL is capable of lysing Micrococcus lysodeikticus. AVR digests a variety of RNA substrates, including RNA from HIV-1-infected cells. We also find that lysozyme from chicken
egg white, human milk, and human neutrophils and RNase A from bovine pancreas possess activity against HIV-1. These findings
may offer additional strategies for the treatment of HIV-1 infection.
Stress-induced phosphorylation of eIF2alpha inhibits global protein synthesis to conserve energy for repair of stress-induced damage. Stress-induced translational arrest is observed in cells expressing a nonphosphorylatable eIF2alpha mutant (S51A), which indicates the existence of an alternative pathway of translational control. In this paper, we show that arsenite, heat shock, or ultraviolet irradiation promotes transfer RNA (tRNA) cleavage and accumulation of tRNA-derived, stress-induced small RNAs (tiRNAs). We show that angiogenin, a secreted ribonuclease, is required for stress-induced production of tiRNAs. Knockdown of angiogenin, but not related ribonucleases, inhibits arsenite-induced tiRNA production and translational arrest. In contrast, knockdown of the angiogenin inhibitor RNH1 enhances tiRNA production and promotes arsenite-induced translational arrest. Moreover, recombinant angiogenin, but not RNase 4 or RNase A, induces tiRNA production and inhibits protein synthesis in the absence of exogenous stress. Finally, transfection of angiogenin-induced tiRNAs promotes phospho-eIF2alpha-independent translational arrest. Our results introduce angiogenin and tiRNAs as components of a phospho-eIF2alpha-independent stress response program.
Missense heterozygous mutations in the coding region of angiogenin (ANG) gene, encoding a 14 kDa angiogenic RNase, were recently found in patients of amyotropic lateral sclerosis (ALS). Functional analyses have shown that these are loss-of-function mutations, implying that angiogenin deficiency is associated with ALS pathogenesis and that increasing ANG expression or angiogenin activity could be a novel approach for ALS therapy.
Review the evidence showing the involvement of angiogenin in motor neuron physiology and function, and provide a rationale for targeting angiogenin in ALS therapy.
Review the current understanding of the mechanism of angiogenin action in connection with ALS genetics, pathogenesis and therapy.
ANG is the first gene whose loss-of-function mutations are associated with ALS pathogenesis. Therapeutic modulation of angiogenin level and activity in the spinal cord, either by systemic delivery of angiogenin protein or through retrograde transport of ANG-encoding viral particles, may be beneficial for ALS patients.
Angiogenin is a 14.4-kDa human plasma protein with 65% homology to RNase A that retains the key active site residues and three of the four RNase A disulfide bonds. We demonstrate that recombinant angiogenin functions as a cytotoxic tRNA-specific RNase in cell-free lysates and when injected into Xenopus oocytes. Inhibition of protein synthesis by angiogenin correlates with degradation of endogenous oocyte tRNA. Exogenous, radiolabeled tRNA is also hydrolyzed by angiogenin, whereas oocyte rRNA and mRNA are not detectably degraded by angiogenin. Protein synthesis was restored to angiogenin-injected oocytes by injecting the RNase inhibitor RNasin plus total Xenopus or calf liver tRNAs, thereby demonstrating that the tRNA degradation induced by angiogenin was the sole cause of cytotoxicity. A similar tRNA-reversible inhibition of protein synthesis was seen in rabbit reticulocyte lysates. Angiogenin therefore appears to be a specific cellular tRNase, whereas five homologues in the RNase A superfamily lack angiogenin's specificity for tRNA. One of these homologues purified from human eosinophils, eosinophil-derived neurotoxin, nonspecifically degrades oocyte RNA similar to RNase A and is also cytotoxic at very low concentrations.
Eosinophil-derived neurotoxin (EDN) and human liver RNase were found to be indistinguishable from each other but distinct from the pancreatic ribonucleases in their nucleolytic activity on polynucleotides or small defined substrates. Antibodies to EDN and liver RNase showed identical cross-reactivities in assays of nuclease inhibition and in a radioimmunoassay. In each instance, EDN and liver RNase were easily distinguished from bovine or human pancreatic RNase. When injected intrathecally into rabbits, 5-10 micrograms of EDN or liver RNase each was neurotoxic as judged by induction of the Gordon phenomenon. Human pancreatic RNase was less neurotoxic, and up to 20-fold higher levels of bovine pancreatic RNase showed no effect. Treatment of EDN, liver RNase, and eosinophil cationic protein with iodoacetic acid at pH 5.5 resulted in inactivation of their RNase activity and also destroyed their neurotoxicity. EDN conformation was not greatly affected by iodoacetate treatment since interaction of the modified protein with antibodies was only slightly altered. We conclude that RNase activity is necessary but not sufficient to induce neurotoxic action.
The major ribonuclease of human liver has been isolated in a four-step procedure. The protein appears homogeneous by several criteria. The amino acid composition and the amino-terminal sequence of the enzyme indicate that the protein is related to human pancreatic ribonuclease and to angiogenin, and that it may be identical with an eosinophil-derived neurotoxin and to a ribonuclease that has been isolated from urine. The catalytic activity of the liver ribonuclease and its sensitivity to iodoacetic acid inactivation also relate the enzyme to the pancreatic RNases, but the liver protein is clearly differentiated by immunological measurements. Antibodies to the liver ribonuclease inhibit its activity, but not that of the human pancreatic enzyme; cross-reactivity in a radioimmunological assay is small but measurable. Immunochemical measurements have been used to examine the distribution of the liver-type protein in other tissues. Inhibition of enzyme activity by anti-liver ribonuclease shows that a cross-reactive enzyme is predominant in extracts of spleen and is a significant component in kidney preparations, while the liver-type protein is almost absent in brain or pancreas homogenates. Cross-reactive ribonuclease is present in serum, but levels are not correlated with any of the disease states examined.
Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) were isolated from lysates of human eosinophil granules by gel filtration and ion exchange chromatography on heparin-Sepharose. Radioimmunoassay, using monoclonal antibodies, of fractions from the heparin-Sepharose chromatography showed one peak of EDN activity and two peaks of ECP activity (termed ECP-1 and ECP-2). EDN, ECP-1, and ECP-2 each exhibited heterogeneity in charge and molecular weight when analyzed by two-dimensional nonequilibrium pH gradient electrophoresis and NaDodSO4/PAGE. Digestion of EDN with endoglycosidase F (endo F) decreased its molecular weight and charge heterogeneity. Thus, END likely contains a single complex oligosaccharide. Endo F digestion of ECP-1 and ECP-2 decreased the molecular weight of both polypeptides, indicating that both likely contain at least one complex oligosaccharide. Amino acid sequence analyses showed that ECP-1 and ECP-2 are identical from residue 1 through residue 59 and that the sequences of EDN and ECP are highly homologous (37 of 55 residues identical). Both EDN and ECP NH2-terminal sequences showed significant homology to RNase, especially in regions of the RNase molecule involved in ligand binding. EDN, ECP-1, and ECP-2 had neurotoxic activity, causing the Gordon phenomenon at doses down to 0.15 micrograms when injected into the cisterna magna; the proteins were comparable in their activities. These results indicate that EDN and ECP are related proteins and suggest that they derived from genes associated with the RNase family.
Eosinophil cationic protein (ECP) is a toxin secreted by activated human eosinophils that has anti-parasitic, antibacterial, and neurotoxic activities; ECP also has ribonuclease activity and structural homology to other mammalian ribonucleases. To determine the relationship between the ribonuclease activity and cytotoxicity of ECP, a method for producing recombinant ECP (rECP) in a prokaryotic expression system was devised. Periplasmic isolates from induced bacterial transfectants contained enzymatically active rECP; micromolar concentrations of rECP were shown to be toxic for Staphylococcus aureus (strain 502A). In contrast, recombinant eosinophil-derived neurotoxin, with 67% amino acid sequence identity to ECP, had little to no toxicity for S. aureus; these findings are analogous to those obtained with purified, granule-derived ECP and eosinophil-derived neurotoxin. Two single base pair mutations were introduced into the coding sequence of rECP (Lys38 to Arg and His128 to Asp) to convert ribonuclease active-site residues into non-functional counterparts. These mutations eliminated the ribonuclease activity of rECP but had no discernible effect on the antibacterial activity of this protein, demonstrating that ribonuclease activity and cytotoxicity are, in this case, independent functions of ECP.
The crystal structure of human angiogenin (reported in the preceding paper in this issue) reveals that the site that corresponds to the pyrimidine binding site of RNase A is obstructed by Gln-117. Mutation of this residue to Ala and Gly is here found to increase activity 11- to 18-fold and 21- to 30-fold, respectively, toward dinucleotide, polynucleotide, and cyclic nucleotide substrates, but without changing specificity. The enhanced activity of Q117G toward CpA is due to a 5-fold decrease in Km and a 6-fold increase in kcat. Its Ki value for 2'-CMP is 5-fold lower than that of native angiogenin, whereas its Ki value for 5'-AMP is unchanged. It has been reported previously that mutating Asp-116 to Ala increases activity 15-fold. The double mutant D116A/Q117A is shown to be only slightly more active than each individual mutant. The present results demonstrate that Gln-117 impedes the ribonucleolytic activity of angiogenin, as predicted by x-ray crystallography. Moreover, they suggest that prior to or during catalysis angiogenin must undergo a conformational change to reorient the C-terminal segment that contains this residue, and that a similar reorganization is required for the mutants as well. This view is supported by molecular modeling of an angiogenin-uridine vanadate complex. These in vitro findings have implications for the angiogenic activity of angiogenin in vivo.
Angiogenin, a potent inducer of neovascularization, is the only angiogenic molecule known to exhibit ribonucleolytic activity. Its overall structure, as determined at 2.4 A, is similar to that of pancreatic ribonuclease A, but it differs markedly in several distinct areas, particularly the ribonucleolytic active center and the putative receptor binding site, both of which are critically involved in biological function. Most strikingly, the site that is spatially analogous to that for pyrimidine binding in ribonuclease A differs significantly in conformation and is "obstructed" by glutamine-117. Movement of this and adjacent residues may be required for substrate binding to angiogenin and, hence, constitute a key part of its mechanism of action.
Mammalian pancreatic ribonucleases form a family of homologous proteins that has been extensively investigated. The primary structures of these enzymes were used to derive phylogenetic trees. These analyses indicate that the presence of three strictly homologous enzymes in the bovine species (the pancreatic, seminal, and cerebral ribonucleases) is due to gene duplication events which occurred during the evolution of ancestral ruminants.
In this paper we present evidence that confirms this finding and that suggests an overall structural conservation of the putative ribonuclease genes in ruminant species.
We could also demonstrate that the sequences related to ox ribonuclease coding regions present in genomic DNA of the giraffe species are the orthologues of the bovine genes encoding the three ribonucleases mentioned above.
A dose-dependent decrease in infectivity was observed on introduction of eosinophils into suspensions of respiratory syncytial
virus group B (RSV-B). This antiviral effect was reversed by ribo-nuclease inhibitor, suggesting a role for the eosinophil
secretory ribonucleases. Recombinant eosino-phil- derived neurotoxin (rhEDN), the major eosinophil ribonuclease, promoted
a dose-dependent decrease in RSV-B infectivity, with a 40-fold reduction observed in response to 50 nM rhEDN. Ribonucleolytically inactivated rhEDN (rhEDNdK38) had no antiviral activity. Semiquantitative reverse transcriptase-polymerase chain reaction demonstrated loss of viral genomic
RNA in response to rhEDN, suggesting that this protein promotes the direct ribonucleolytic destruction of extracellular virions.
Ribonuclease A had no antiviral activity even at ∼ 1000-fold higher concentrations, suggesting that rhEDN has unique features
other than ribonuclease activity that are crucial to its effectiveness. These results suggest that rhEDN may have potential
as a therapeutic agent for prevention or treatment of disease caused by RSV.
We have demonstrated that the human eosinophil-derived neurotoxin (EDN, RNase 2), a rapidly evolving secretory protein derived from eosinophilic leukocytes, mediates the ribonucleolytic destruction of extracellular virions of the single-stranded RNA virus respiratory syncytial virus (RSV). While RNase activity is crucial to antiviral activity, it is clearly not sufficient, as our results suggest that EDN has unique structural features apart from RNase activity that are necessary to promote antiviral activity. We demonstrate here that the interaction between EDN and extracellular virions of RSV is both saturatable and specific. Increasing concentrations of the antivirally inactivated, ribonucleolytically inactivated point mutant form of recombinant human EDN, rhEDNdK38, inhibits rhEDN's antiviral activity, while increasing concentrations of the related RNase, recombinant human RNase k6, have no effect whatsoever. Interestingly, acquisition of antiviral activity parallels the evolutionary development of the primate EDN lineage, having emerged some time after the divergence of the Old World from the New World monkeys. Using this information, we created ribonucleolytically active chimeras of human and New World monkey orthologs of EDN and, by evaluating their antiviral activity, we have identified an N-terminal segment of human EDN that contains one or more of the sequence elements that mediate its specific interaction with RSV.
The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
This chapter focuses on structure and functions of angiogenin. In many ways, angiogenin is the most unusual member of the ribonuclease superfamily. It shares 33% sequence identity with bovine pancreatic RNase A and has structurally equivalent counterparts for the two histidines and one lysine that comprise the catalytic residues for ribonucleolytic activity. It cleaves preferentially on the 3′ side of pyrimidines to generate a cyclic phosphate product that is subsequently hydrolyzed, but it is 4–6 orders of magnitude less active in routine assays. Despite such seemingly minuscule potency, this ribonucleolytic activity is absolutely critical to the biological function of angiogenin. This chapter discusses in depth about angiogenesis and angiogenic molecules, human angiogenin, and bovine angiogenin. Characterization as a member of the ribonuclease family is explained. The chapter also elaborates the relationship of RNase activity and angiogenic activity. The chapter discusses about binding to endothelial cells, induction of second messengers, and effect on endothelial cell growth. The chapter concludes with a discussion on antiangiogenin antibody suppression of tumor growth.
Eosinophil cationic protein (ECP) is located in the matrix of the eosinophil's large specific granule and has marked toxicity for a variety of helminth parasites, hemoflagellates, bacteria, single-stranded RNA virus, and mammalian cells and tissues. It belongs to the bovine pancreatic ribonuclease A (RNase A) family and exhibits ribonucleolytic activity which is about 100-fold lower than that of a related eosinophil ribonuclease, the eosinophil-derived neurotoxin (EDN). The crystal structure of human ECP, determined at 2.4 A, is similar to that of RNase A and EDN. It reveals that residues Gln-14, His-15, Lys-38, Thr-42, and His-128 at the active site are conserved as in all other RNase A homologues. Nevertheless, evidence for considerable divergence of ECP is also implicit in the structure. Amino acid residues Arg-7, Trp-10, Asn-39, His-64, and His-82 appear to play a key part in the substrate specificity and low catalytic activity of ECP. The structure also shows how the cationic residues are distributed on the surface of the ECP molecule that may have implications for an understanding of the cytotoxicity of this enzyme.
Ribonucleases of the T2 family are found in the genomes of protozoans, plants, bacteria, animals and viruses. A broad range of biological roles for these ribonucleases have been suggested, including scavenging of nucleic acids, degradation of self-RNA, serving as extra- or intracellular cytotoxins, and modulating host immune responses. Recently, RNaseT2 family members have been implicated in human pathologies such as cancer and parasitic diseases. Interestingly, certain functions of RNaseT2 family members are independent of their nuclease activity, suggesting that these proteins have additional functions. Moreover, humans lacking RNASET2 manifest a defect in neurological development, perhaps due to aberrant control of the immune system. We review the basic structure and function of RNaseT2 family members and their biological roles.
Human angiogenin (ANG) is a homologue of bovine pancreatic ribonuclease (RNase A) that induces neovascularization. ANG is the only human angiogenic factor that possesses ribonucleolytic activity. To stimulate blood vessel growth, ANG must be transported to the nucleus and must retain its catalytic activity. Like other mammalian homologues of RNase A, ANG forms a femtomolar complex with the cytosolic ribonuclease inhibitor protein (RI). To determine whether RI affects ANG-induced angiogenesis, we created G85R/G86R ANG, which possesses 10(6)-fold lower affinity for RI but retains wild-type ribonucleolytic activity. The neovascularization of rabbit corneas by G85R/G86R ANG was more pronounced and more rapid than by wild-type ANG. These findings provide the first direct evidence that RI serves to regulate the biological activity of ANG in vivo.
tRNAs play a central role in protein translation, acting as the carrier of amino acids. By cloning microRNAs, we unexpectedly obtained some tRNA fragments generated by tRNA cleavage in the anticodon loop. These tRNA fragments are present in many cell lines and different mouse tissues. In addition, various stress conditions can induce this tRNA cleavage event in mammalian cells. More importantly, angiogenin (ANG), a member of RNase A superfamily, appears to be the nuclease which cleaves tRNAs into tRNA halves in vitro and in vivo. These results imply that angiogenin plays an important physiological role in cell stress response, except for the known function of inducing angiogenesis.
The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.
Three ribonucleases (RNases) with different molecular masses were isolated from human kidney. The enzymes were purified to an electrophoretically homogeneous state, and their respective molecular masses were found to be 18,000 (tentatively named RNase HK-1), 20,000 (RNase HK-2A), and 22,000 (RNase HK-2B) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the amino acid compositions, amino-terminal sequences, and enzymological properties of the enzymes indicate that RNase HK-1 is related to "nonsecretory" RNase, and that RNases HK-2A and HK-2B are both related to "secretory" RNase. Furthermore, RNase HK-1 showed cross-reactivity with an antibody specific to nonsecretory RNase from human urine, whereas RNases HK-2A and HK-2B showed cross-reactivity with another antibody specific to human urine secretory RNase. However, the carbohydrate compositions of RNases HK-2A and HK-2B were markedly different from that of the secretory urine RNase. This finding seems to indicate that the kidney is not the origin of the urine enzyme.
A ribonuclease has been isolated from human spleen (RNase HS) by means of acid extraction, ammonium sulphate fractionation, successive column chromatographies on CM-cellulose, heparin-actigel, and poly(G)-agarose, and double gel-filtration on Sephadex G-75. The purified preparation was homogeneous as judged by SDS/PAGE. RNase HS was found to be a glycoprotein, containing three fucose, one mannose and five glucosamine residues/molecule, with a molecular mass of 17 kDa as determined by both SDS/PAGE and gel filtration. The catalytic properties and structural features, including its amino acid composition and the amino acid sequence of the N-terminal 35 residues, indicated that the enzyme was strictly related to nonsecretory RNase isolated from human urine and liver. In particular, the amino acid sequence of the N-terminal was identical with that of urine nonsecretory RNase and eosinophil-derived neurotoxin. Furthermore, analyses using three different antibodies specific to RNase HS, urine nonsecretory RNase and urine secretory RNase, indicated that RNase HS was not immunologically distinguishable from urine nonsecretory RNase, but clearly so from urine secretory RNase. However, the carbohydrate compositions of RNase HS and urine nonsecretory RNase were found to differ. It therefore remains to be resolved whether or not the tissue of origin of nonsecretory RNase in urine is the spleen.
Several clones of human eosinophil-derived neurotoxin (EDN) cDNA have been isolated from a lambda gt10 cDNA library prepared from mRNA derived from noninduced HL-60 cells. The amino acid (aa) sequence deduced from the coding sequence of the EDN cDNA is identical to the aa sequence of urinary nonsecretory RNase. Comparison of the aa and/or nucleotide (nt) sequences of EDN and other proteins possessing ribonucleolytic activity, namely bovine seminal RNase, human and rat pancreatic RNases, eosinophil cationic protein (ECP), and human angiogenin, shows extensive identity at half-cystine residues and at aa of active sites. Differences in aa sequences at the active sites are often the result of single nt changes in the codons. The data presented here support the concept of a RNase gene superfamily containing secretory and nonsecretory RNases, angiogenin, EDN and ECP.
Human eosinophil granules contain several basic proteins including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and major basic protein (MBP). ECP and MBP are potent helminthotoxins while EDN is less so. Both ECP and EDN possess neurotoxic and ribonuclease activities. A clone representing ECP mRNA was isolated from an eosinophil lambda ZAP cDNA library. The cDNA sequence codes for a preprotein of 160 amino acids and a protein of 133 amino acids, the amino terminus of which is identical to the known partial amino acid sequence of ECP. The ECP nucleotide sequence shows similarity to EDN, rat pancreatic ribonuclease, and human angiogenin; all are members of the ribonuclease gene superfamily. Although the deduced amino acid sequence of ECP shares identical active site and substrate binding site residues with EDN, angiogenin, and human pancreatic ribonuclease, the ribonuclease activity of ECP is 50 to 100 times less than that of EDN possibly because of the lack of a positively charged residue at human pancreatic ribonuclease position 122. The calculated isoelectric point (10.8), electronic charge (14.5), and cationic charge distribution of ECP are different from those of EDN but similar to those of MBP, which may account in part for the greater helminthotoxic activity of ECP when compared to EDN. These data suggest that ECP and EDN are derived from a common ancestral ribonuclease gene and that ECP has evolved into a potent helminthotoxin similar in some respects to MBP, while losing much of its ribonuclease activity.
The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material. Angiogenin, obtained from conditioned media of a human colonic adenocarcinoma cell line, is a single-chain protein consisting of 123 amino acids with the following sequences: less than Glu1-Asp-Asn-Ser-Arg-Tyr-Thr-His- Phe-Leu-Thr-Gln-His-Tyr-Asp15-Ala-Lys-Pro-Gln-Gly-Arg-Asp-Asp- Arg-Tyr-Cys-Glu-Ser-Ile-Met30- Arg-Arg-Arg-Gly-Leu-Thr-Ser-Pro-Cys-Lys-Asp-Ile-Asn-Thr- Phe45-Ile-His-Gly-Asn-Lys-Arg-Ser -Ile-Lys-Ala-Ile-Cys-Glu-Asn-Lys60-Asn-Gly-Asn-Pro-His-Arg-Glu-Asn -Leu-Arg-Ile -Ser-Lys-Ser-Ser75 -Phe-Gln-Val-Thr-Thr-Cys-Lys-Leu-His-Gly-Gly-Ser-Pro-Trp-Pro90-Pro -Cys-Gln-Tyr -Arg-Ala-Thr-Ala -Gly-Phe-Arg-Asn-Val-Val-Val105-Ala-Cys-Glu-Asn-Gly-Leu-Pro-Val- His-Leu-Asp-Gln-Ser-Ile-Phe120-Arg-Arg-Pro123-OH. Three disulfide bonds link the half-cystinyl residues 26-81, 39-92, and 57-107. The sequence is homologous to that of the pancreatic ribonucleases with 35% identity and many of the remaining residues conservatively replaced. Similarities are especially apparent around the major active-site residues His-12, Lys-41, and His-119 of ribonuclease which are conserved as are three of the four disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)
The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)
The structure of phosphate-free bovine ribonuclease A has been refined at 1.26-A resolution by a restrained least-squares procedure to a final R factor of 0.15. X-ray diffraction data were collected with an electronic position-sensitive detector. The final model consists of all atoms in the polypeptide chain including hydrogens, 188 water sites with full or partial occupancy, and a single molecule of 2-methyl-2-propanol. Thirteen side chains were modeled with two alternate conformations. Major changes to the active site include the addition of two waters in the phosphate-binding pocket, disordering of Gln-11, and tilting of the imidazole ring of His-119. The structure of the protein and of the associated solvent was extensively compared with three other high-resolution, refined structures of this enzyme.
A ribonuclease was isolated from serum-free supernatants of the human colon adenocarcinoma cell line HT-29. It was purified by cation-exchange and C18 reversed-phase high-performance liquid chromatography. The protein is basic, has a molecular weight of approximately 16,000, and has an amino acid composition that is significantly different from that of human pancreatic ribonuclease. The amino terminus is blocked, and the carboxyl-terminal residue is glycine. The catalytic properties of this ribonuclease resemble those of the pancreatic ribonucleases in numerous respects. Thus, it exhibits a pH optimum of approximately 6 for dinucleotide cleavage and employs a two-step mechanism in which transphosphorylation to a cyclic 2',3'-phosphate is followed by slower hydrolysis to produce a 3'-phosphate. It does not cleave NpN' substrates in which adenosine or guanosine is at the N position and prefers purines at the N' position. Like bovine ribonuclease A, the HT-29-derived ribonuclease is inactivated by reductive methylation or by treatment with iodoacetate at pH 5.5 and is strongly inhibited by the human placental ribonuclease inhibitor. However, in contrast, the tumor enzyme does not cleave CpN bonds at an appreciable rate and prefers poly(uridylic acid) as substrate 1000-fold over poly(cytidylic acid). It also hydrolyzes cytidine cyclic 2',3'-phosphate at least 100 times more slowly than uridine cyclic 2',3'-phosphate and is inhibited much less strongly by cytidine 2'-monophosphate than by uridine 2'-monophosphate. Other ribonucleases known to prefer poly(uridylic acid) were isolated both from human serum and from liver and were compared with the tumor enzyme. The physical, functional, and chromatographic properties of the serum ribonuclease are essentially identical with those of the tumor enzyme. The liver enzymes, however, differ markedly from the HT-29 ribonuclease. The potential utility of the tumor ribonuclease in the diagnosis of cancer is considered.
• In order to determine the distribution of two human urinary RNase (RNase Us and RNase UL)-like enzymes in human tissues and body fluids, enzyme immunoassay systems were established using rabbit anti-RNase sera. The sensitivity of the assay systems was of similar order to that of radioimmunoassay systems previously reported.
• In the enzyme immunoassay, the cross reactivities of anti-RNase UL serum towards RNase Us, bovine kidney RNase K2 bovine RNase A, and bovine seminal RNase V8 were less than 1%. The cross reactivity of anti-RNase Us-serum towards RNase UL was less than 0.5% and cross reactivities were minimal for RNase A, RNase K2, and RNase V8
• The RNase levels in human organs and body fluids were measured by enzyme immunoassay. In milk, semen and saliva, only RNase UL-like enzyme was found. Both RNase Us- and RNase UL-like enzymes were found in kidney, stomach, and pancreas and the RNase Us/RNase UL ratios were 0.49, 1.35, and 0.34, respectively. In lung, liver, spleen, and leukocytes, most of the RNase activity was accounted for by RNase Us-like enzyme. The activity of RNase Us-like enzyme was especially high in lung, spleen, and leukocytes.
• The crude extracts of several tissues and body fluids were separated by phosphocellulose column chromatography and the contents of the two urinary RNase-like enzymes were determined by enzyme immunoassay. In stomach, kidney, pancreas, and serum, both enzymes were present in multiple forms. In spleen and lung, both the major RNase (RNase Us) and minor RNase (RNase UL) existed in two forms. A single peak of RNase was found in liver and in leukocytes.
• The activity staining of liver, leukocytes, spleen, and lung RNases indicated that RNase Us-like enzyme in these tissues is also present in multiple forms.
Angiogenin, a potent blood vessel inducing protein, was previously isolated from medium conditioned by a human adenocarcinoma cell line [Fett, J. W., Strydom, D.J., Lobb, R.R., Alderman, E.M., Bethune, J.L., Riordan, J.F., & Vallee, B.L. (1985) Biochemistry 24, 5480-5486]. We now report that a protein which is physically and functionally identical with angiogenin is present in normal human plasma and can be purified to homogeneity by CM 52 and Mono S cation-exchange chromatography. The plasma-derived angiogenin exhibits the same angiogenic and ribonucleolytic activities, amino acid composition, molecular weight, immunoreactivity, and chromatographic behavior as the tumor cell derived protein. Peptide mapping and sequencing studies indicate chemical identity of the two proteins. The present yield of angiogenin from either plasma or serum is 60-150 micrograms/L. These findings demonstrate that angiogenin is not a tumor-specific product and provide further opportunities for the investigation of the role and mechanism of action of angiogenin and its potential diagnostic or prognostic utility.
The eosinophil granule contains a series of basic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Both EDN and ECP are neurotoxins and helminthotoxins. Comparison of the partial N-terminal amino acid sequences of EDN and ECP showed 67% identity; surprisingly, they also showed structural homology to pancreatic ribonuclease (RNase). Therefore, we determined whether EDN and ECP possess RNase enzymatic activity. By spectrophotometric assay of acid soluble nucleotides formed from yeast RNA, purified EDN showed RNase activity similar to bovine pancreatic RNase, whereas ECP was 50 to 100 times less active. The RNase activity associated with ECP was not significantly inhibited after exposure of ECP to polyclonal or monoclonal antibody to EDN. These results indicate that EDN and ECP both possess RNase activity, the RNase activity of EDN and ECP is specific, and EDN and ECP have maintained not only structural but also functional homology to pancreatic RNase.
The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin. It was purified by cation-exchange and reversed-phase high-performance liquid chromatography; the yield was approximately 0.5 microgram/L of medium. Biological activity of angiogenin was monitored throughout purification by using the chick embryo chorioallantoic membrane assay. Statistical evaluation demonstrates that it displays activity in this system with as little as 35 fmol per egg. Moreover, only 3.5 pmol is required to induce extensive blood vessel growth in the rabbit cornea. The amino acid composition of this basic (isoelectric point greater than 9.5), single-chain protein of molecular weight approximately 14 400 has been determined. The amino terminus is blocked, and the carboxyl-terminal residue is proline.
The physiological function of this much studied pancreatic enzyme has been misunderstood. It is essential only in ruminants and certain other herbivores, where it has a special function.
The primary structure of human (Homo sapiens) pancreatic ribonuclease has been determined by automatic sequencing of the native protein and by analysis of peptides obtained by cleavage with proteolytic enzymes, cyanogen bromide, and hydroxylamine. The following sequence was deduced: (sequence in text). Human pancreatic ribonuclease differs at 37 positions from bovine pancreatic ribonuclease. In addition the human enzyme has three more residues at the C-terminus. About half of the enzyme molecules contain carbohydrate attached to the sequence Asn-Met-Thr (34-36). Two other Asn-X-Ser/Thr sequences are carbohydrate free. Human pancreatic ribonuclease contains many positively charged residues, especially near the N-terminus, while negatively charged residues are more concentrated near the C-terminus.
A ribonuclease, active on single- and double-stranded RNAs, has been isolated from human seminal plasma 3-5 micrograms of enzyme were recovered per ml of seminal plasma, equivalent to 71% of total activity and a 2500-fold purification (measured with poly(A) X poly(U) as substrate) from the initial dialyzed material. Similar amounts of RNAase were found per g (wet weight) of human prostate, where the enzyme appears to be produced. Human seminal RNAase degrades poly(U) 3-times faster than poly(A) X poly(U), and poly(C) or viral single-stranded RNA about 10-times faster than poly(U). Degradation of poly(A) X poly(U), viral double-stranded RNA, and poly(A) by human seminal RNAase is 500-, 380- and 140-times more efficient, respectively, than by bovine RNAase A. The enzyme, a basic protein with maximum absorbance at 276 nm, occurs in two almost equivalent forms, one of which is glycosylated. Mr values of the glycosylated and non-glycosylated form are 21000 and 16000, respectively. The amino-acid composition of the RNAase is very similar to that of human pancreatic RNAase. The same is true for the carbohydrate content of its glycosylated form.
1. Two RNases (RNase UL and RNase US) were purified from the urine of human adults by means of column chromatographies on SP-Sephadex C-50, phospho-cel-lulose and CM-cellulose and gel-filtration on Sephadex G-75 in homogeneous states obtained by SDS-disc electrophoresis.
2. Molecular weights of these RNases determined by gel-filtration were 38,000 and 13,000 for RNase UL and RNase US, respectively.
3. Optimal pH's of urine RNase were 8.0 and 6.75 for RNase UL and RNase US, respectively.
4. Chemical composition of urine RNase was determined. RNase UL contains about 20.7% of neutral sugar and 7.8% of hexosamine. RNase US contains a very small amount of carbohydrate moiety.
5. Base specificity of urine RNase studied with 2′, 3′-cyclic nucleotides and dinu-cleoside phosphates as substrates indicated that both RNase were pyrimidine specific and cytosine preferential enzyme, as is bovine pancreatic RNase A. Although base specificity of RNase UL was qualitatively similar to RNase A, that of RNase US was slightly different. That is, RNase US did not hydrolyze UpU and hydro-lyzed UpC and 2′, 3′-cyclic UMP very slowly.
6. Antigenic properties of human urine RNase were studied by Ouchterlony's double diffusion analysis. RNase UL, RNase US, and RNase A were serologically distinguishable.
The two major ribonuclease (EC 3.1.27.5) present in normal human urine have been highly purified and extensively characterized for their enzymatic, physical, chemical and structural properties. One of the enzymes, RNAase C, is a glycoprotein which exhibits a pH optimum of 8.5 with RNA as the substrate and preferentially degrades the synthetic homoribopolymer poly(C). This enzyme is resolved into multiple components by column electrofocusing. However, prior treatment with neuraminidase results in a single form of RNAase C with an isoelectric point of 10.4, indicating that the charge heterogeneity is the result of variability in sialic acid content. Amino acid composition and NH2- and COOH-terminal sequence analyses of RNAase C show that this enzyme is very similar to mammalian pancreatic RNAases; the data indicate a peptide chain of 126 amino acid residues and a 33% carbohydrate content. The second enzyme isolated from urine, termed RNAase U, is also a glycoprotein which has a pH optimum of 7.0 with RNA as substrate and is virtually inactive against poly(C). RNAase U lacks sialic acid and focuses as a single component with a highly basic isoelectric point of greater than pH 11.0. The NH2- and COOH-terminal sequences of RNAase U show little homology with the pancreatic RNAases. However, the amino acid composition of this enzyme indicates it is very similar to human spleen RNAase.
We have Isolated a unique genomic fragment encoding human ribonuclease 4 (RNase 4) of the mammalian ribonuclease gene family,
whose members Include pancreatic ribonuclease, eosinophil-derived neurotoxin, eosinophll cationic protein and angiogenin.
We have determined that the coding sequence of RNase 4 resides on a single exon found on human chromosome 14. The mRNA encoding
RNase 4 was detected by Northern analysis In a number of human somatic tissues, including pancreas, lung, skeletal muscle,
heart, kidney and placenta, but not brain; liver represents the most abundant source. Interestingly, the mRNA encoding RNase
4 is -2 kb in length, which is approximately twice as large as the mRNAs encoding other members of this gene family. A larger
(-2.4 kb), second transcript was detected in hepatic, pancreatic and renal tissues. The ∼2 kb RNase 4 mRNA was detected in
cells of the human promyelocytlc leukemia line, HL-60, that had been treated with dibutyryl-cAMP to promote neutrophilic differentiation.
In contrast, no mRNA encoding RNase 4 could be detected in cells treated with phorbol myristic acid (PMA), an agent promoting
differentiation toward monocyte/macrophages, suggesting the existence of elements regulating tissue specific expression of
this gene.
Angiogenin is a secreted polypeptide that induces neovascularization in vivo. The expression of angiogenin by human cells in culture was investigated by using a specific radioimmunoassay and by cDNA hybridization. Angiogenin immunoreactivity was widely but differentially produced by anchorage-dependent growing cells including vascular endothelial cells from saphenous and umbilical veins, aortic smooth muscle cells, fibroblasts (from embryos, new-borns and adults), and tumour cells. Endothelial cells from saphenous veins and the endothelium-derived EA.hy926 cell line released immunoreactivity whatever the stage of the culture, including release at the lag phase, during exponential growth and at the confluent phase. However, the rate of accumulation of angiogenin varied as a function of EA.hy926 cell density. As compared to anchored cells, normal peripheral blood cells and tumour cells of myelomonocytic and megakaryocytic origin did not noticeably secrete angiogenin except at low levels. A myeloma cell line supernatant contained as much angiogenin cross-reactivity as did anchored cells, while four tumour T-cell lines expressed the cross-reactivity at different levels, i.e. from undetectable levels to a high level. A 0.9-kb angiogenin messenger RNA was detected by Northern-blot analyses in a variety of representative cells correlating with the presence of immunoreactivity in the cell-culture media. The widespread expression pattern of angiogenin suggests a physiological function that is not restricted to the neovascularization process.
A ribonuclease (RNase) that cleaves specifically on the 3′ side of uridine [Shapiro, R., Fett, J. W., Strydom, D. J. & Vallee, B. L. (1986a) Biochemistry 25, 7255–7264] was purified from human plasma and its amino acid sequence was determined. This protein is a 119-residue single-chain polypeptide cross-linked by four disulfide bonds and has an amino-terminal pyroglutaminyl residue. No post-translational modifications were observed during extensive sequence studies on peptide fragments, except for the amino-terminal pyroglutamic acid and a possible deamidation of Asn66. The protein is homologous to the pancreatic ribonucleases and angiogenin, but differs substantially from both of these proteins; the protein sequence has 43% identity with human pancreatic ribonuclease and 39% identity with human angiogenin, as compared to 35% identity between human angiogenin and pancreatic ribonuclease. It is referred to as RNase 4, based on the nomenclature currently used for the genes of pancreatic RNase (RNase 1) and the eosinophil-derived RNases (RNase 2 and RNase 3).
Virtually all of the RNase active-site components, including the catalytic residues His12, His119 and Lys41, are preserved. However, some invariant residues of RNase 1 are replaced, e.g. Lys7 by arginine, Asp14 by histidine, and Pro42 by arginine. RNase 4 contains a unique two-residue deletion at the position corresponding to amino acids 77 and 78 of pancreatic RNase, and its carboxy-terminal sequence is truncated at position 122. The deletion in angiogenin at position 21 is also found in RNase 4.
RNase 4 is very similar to two RNases isolated from bovine and porcine liver, and together they form a new family in the RNase superfamily. The degree of inter-species similarity (90%) is much greater than within the pancreatic RNase and angiogenin families, which suggests that this ribonuclease could possess a physiologically important function other than general RNA catabolism.
Two RNAases from human cerebrum were purified to an electrophoretically homogeneous state and their molecular masses were 22.0 kDa (tentatively called RNAase HB-1) and 19.0 kDa (RNAase HB-2). Analyses of the amino acid compositions, N-terminal amino acid sequences and catalytic properties of these enzymes provided strong evidence that they were strictly related to the secretory (sec) RNAases, such as the pancreatic enzyme, very similar immunologically to urinary sec RNAase, but clearly distinguishable from urinary non-secretory (nonsec) RNAase. There were several differences between HB-1 and HB-2, namely their immunological reactivities with specific antibodies, heat-stabilities, attached carbohydrate moieties and molecular masses. In particular, HB-2 appeared to be nonglycosylated, in view of its lack of affinity for several conjugated lectins, the absence of hexosamine and no change in electrophoretic mobility before and after peptide:N-glycosidase F digestion, whereas HB-1 and human sec RNAases purified from kidney, pancreas and urine all appeared to be glycosylated, as they moved to the same position as HB-2 when electrophoresed after glycosidase digestion. An antibody against urinary sec RNAase inhibited 75% and 20% of the total activity of the crude cerebral extract against RNA at pH 8.0 and 6.0 respectively, whereas an antibody against urinary nonsec RNAase had no such inhibitory effect. These findings suggest that yet another type(s) of cerebral RNAase, which is unable to cross-react immunologically with sec and nonsec RNAases, may exist. Two RNAases corresponding to HB-1 and HB-2 were identified in fresh cerebrospinal fluid.
The X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin (rEDN) has been determined by molecular replacement methods and refined at 1.83 A resolution to a conventional R-factor ( = sigma magnitute of (magnitute of F(zero)-magnitude of Fc)/ sigma magnitude of F(zero) of 0.152 with excellent stereochemistry. The molecular model of rEDN contains all 1081 non-hydrogen protein atoms, two non-covalently bound sulfate anions and 121 ordered solvent molecules. The polypeptide fold of rEDN is related to those observed in the homologous structures of RNase A, Onconase and angiogenin. rEDN is one of the largest members of the pyrimidine-specific ribonuclease superfamily of vertebrates and has small insertions in four of its seven loop structures and a large insertion from Asp115 to Tyr123. The non-covalently bound SO4(A) and SO4(B) anions occupy phosphate-binding subsites of rEDN. The active site SO4(A) anion makes contacts in rEDN that are similar to those in RNase A and involve the side-chain atoms of Gln14, His15 and His129, and the NH group of Leu130. The SO4(B) anion makes contacts with the side-chain atoms of Arg36 and Asn39 and the main-chain atoms of Asn39 and Gln40. The equivalent residues of RNase A cannot make contacts similar to those observed in rEDN. The SO4(B) binding site of rEDN likely corresponds to the P-1 subsite and may be representative of how other homologous RNases bind the P-1 phosphate.
The discovery of Ribonuclease k6 (RNase k6) was an unexpected result of our ongoing efforts to trace the evolutionary history
of the ribonuclease gene family. The open reading frame of RNase k6, amplified from human genomic DNA, encodes a 150 amino
acid polypeptide with eight cysteines and histidine and lysine residues corresponding to those found in the active site of
the prototype, ribonuclease A. The single-copy gene encoding RNase k6 maps to human chromosome 14 and orthologous sequences
were detected in both primate and non-primate mammalian species. A single mRNA transcript (1.5 kb) was detected in all human
tissues tested, with lung representing the most abundant source. At the cellular level, transcripts encoding RNase k6 were
detected in normal human monocytes and neutrophils (but not in eosinophils) suggesting a role for this ribonuclease in host
defense. Of the five previously identified human ribonucleases of this group, RNase k6 is most closely related to eosinophil-derived
neurotoxin (EDN), with 47% amino acid sequence identity; slight cross-reactivity between RNase k6 and EDN was observed on
Western blots probed with polyclonal anti-EDN anti-serum. The catalytic constants determined, Km = 5.0 µ;M and kcat = 0.13 s−1, indicate that recombinant RNase k6 has 40-fold less ribonuclease activity than recombinant EDN. The identification and characterization
of RNase k6 has extended the ribonuclease gene family and suggests the possibility that there are others awaiting discovery.
Human extracellular ribonucleases (RNase), together with other members of the mammalian RNase superfamily, can be classified into four different enzyme types on the basis of their structural, catalytic and/or biological properties. Their occurrence and main distinctive features have been described, and catalytic differences (action on single- and double-stranded RNAs, dependence of enzyme activity on pH, ionic strength and cations, and hydrolysis of cyclic nucleotides) have been comparatively analyzed and discussed. In addition, some data considered here concerning human nonpancreatic-type RNases may support the suggestion [Chuchillo et al. (1993) FEBS Lett. 333, 207-210] that the enzyme 'ribonuclease', presently classified as 'hydrolase', should be reclassified as 'transferase'.
The presence of four members of the pyrimidine-specific ribonuclease superfamily was demonstrated in rat liver. Three of them (RL1, RL2 and RL3) were purified and showed ribonuclease activity at pH 7.5 with yeast RNA as substrate. RL1 is identical to rat pancreatic ribonuclease (ribonuclease 1). N-terminal sequence analysis showed the presence of the native protein and several N-terminally degraded components. RL2 and RL3 were N-terminally blocked proteins. After acidic cleavage or CNBr digestion, several parts of their sequences were determined. RL2 has high sequence similarity with neurotoxin-type ribonucleases (ribonucleases 2, 3 and 6). The amino acid sequence of rat liver-type ribonuclease (ribonuclease 4) was determined from a liver cDNA library. It differs at about 20% of the amino acid positions from other mammalian liver-type ribonucleases. The sequence of a peptide of RL3 was identical to that derived from the cDNA sequence of the liver-type ribonuclease. A contaminant of the RL3 fraction had a high sequence similarity with mouse and other mammalian angiogenins. Bovine, porcine and rat liver-type ribonucleases showed a strong preference for poly(U) over poly(C). This preference is a unique property of the liver-type enzymes of the ribonuclease superfamily.
Mammalian pancreatic ribonucleases (RNase) form a family of extensively studied homologous proteins. Phylogenetic analyses, based on the primary structures of these enzymes, indicated that the presence of three homologous enzymes (pancreatic, seminal and brain ribonucleases) in the bovine species is due to gene duplication events, which occurred during the evolution of ancestral ruminants. In this paper the sequences are reported of the coding regions of the orthologues of the three bovine secretory ribonucleases in hog deer and roe deer, two deer species belonging to two different subfamilies of the family Cervidae. The sequences of the 3' untranslated regions of the three different secretory RNase genes of these two deer species and giraffe are also presented. Comparison of these and previously determined sequences of ruminant ribonucleases showed that the brain-type enzymes of giraffe and these deer species exhibit variations in their C-terminal extensions. The seminal-type genes of giraffe, hog deer and roe deer show all the features of pseudogenes. Phylogenetic analyses, based on the complete coding regions and parts of the 3' untranslated regions of the three different secretory ribonuclease genes of ox, sheep, giraffe and the two deer species, show that pancreatic, seminal- and brain-type RNases form three separate groups.
The RNase 4 family is unique among RNase enzymes, displaying the highest level of sequence similarity and encompassing the shortest polypeptide chain. It is the only one showing high specificity. The human representative is an intracellular and plasma enzyme, first isolated from colon adenocarcinoma cell line HT-29. The crystal structures of human recombinant RNase 4, unliganded and in complex with d(Up), have been determined, revealing in the unique active site an explanation for the uridine specificity. Arg101, at a position not involved in catalysis in the other RNase enzymes, penetrates the enzyme moiety shaping the recognition pocket, a flip that is mediated by the interaction with the (shorter chain) C-terminal carboxylate group, providing an anchoring point for the O4 atom of the substrate uridine. The bulky Phe42 side-chain forces Asp80 to be in the chi1=-72.49 degrees rotamer, accepting a hydrogen bond from Thr44, further converting the latter into a hydrogen bond acceptor. This favours an interaction with the -NH-donor group of uridine at position 3 over that with the =N-acceptor of cytidine. The two chemical groups that distinguish uracyl from cytosine are used by the enzyme to discriminate between these two bases.
Human angiogenin (Ang), an unusual member of the pancreatic RNase superfamily, is a potent inducer of angiogenesis in vivo. Its ribonucleolytic activity is weak (10(4) to 10(6)-fold lower than that of bovine RNase A), but nonetheless seems to be essential for biological function. Ang has been implicated in the establishment of a wide range of human tumours and has therefore emerged as an important target for the design of new anti-cancer compounds. We report high-resolution crystal structures for native Ang in two different forms (Pyr1 at 1.8 A and Met-1 at 2.0 A resolution) and for two active-site variants, K40Q and H13A, at 2.0 A resolution. The native structures, together with earlier mutational and biochemical data, provide a basis for understanding the unique functional properties of this molecule. The major structural features that underlie the weakness of angiogenin's RNase activity include: (i) the obstruction of the pyrimidine-binding site by Gln117; (ii) the existence of a hydrogen bond between Thr44 and Thr80 that further suppresses the effectiveness of the pyrimidine site; (iii) the absence of a counterpart for the His119-Asp121 hydrogen bond that potentiates catalysis in RNase A (the corresponding aspartate in Ang, Asp116, has been recruited to stabilise the blockage of the pyrimidine site); and (iv) the absence of any precise structural counterparts for two important purine-binding residues of RNase A. Analysis of the native structures has revealed details of the cell-binding region and nuclear localisation signal of Ang that are critical for angiogenicity. The cell-binding site differs dramatically from the corresponding regions of RNase A and two other homologues, eosinophil-derived neurotoxin and onconase, all of which lack angiogenic activity. Determination of the structures of the catalytically inactive variants K40Q and H13A has now allowed a rigorous assessment of the relationship between the ribonucleolytic and biological activities of Ang. No significant change outside the enzymatic active site was observed in K40Q, establishing that the loss of angiogenic activity for this derivative is directly attributable to disruption of the catalytic apparatus. The H13A structure shows some changes beyond the ribonucleolytic site, but sites involved in cell-binding and nuclear translocation are essentially unaffected by the amino acid replacement.