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Ubiquitin and the Enigma of Intracellular Protein Degradation
Abstract
Contrary to widespread belief, the regulation and mechanism of degradation for the mass of intracellular proteins (i.e. differential, selective protein turnover) in vertebrate tissues is still a major biological enigma. There is no evidence for the conclusion that ubiquitin plays any role in these processes. The primary function of the ubiquitin-dependent protein degradation pathway appears to lie in the removal of abnormal, misfolded, denatured or foreign proteins in some eukaryotic cells. ATP/ubiquitin-dependent proteolysis probably also plays a role in the degradation of some so-called 'short-lived' proteins. Evidence obtained from the covalent modification of such natural substrates as calmodulin, histones (H2A, H2B) and some cell membrane receptors with ubiquitin indicates that the reversible interconversion of proteins with ubiquitin followed by concomitant functional changes may be of prime importance.
... Ubiquitination is a post-translational modification process by which ubiquitin becomes covalently bound to a substrate protein by enzymatic action (4)(5)(6)(7)(8). The function of ubiquitination is to mark target proteins so that they are recognized and degraded by proteasomes (4)(5)(6)(7)(8). ...
... Ubiquitination is a post-translational modification process by which ubiquitin becomes covalently bound to a substrate protein by enzymatic action (4)(5)(6)(7)(8). The function of ubiquitination is to mark target proteins so that they are recognized and degraded by proteasomes (4)(5)(6)(7)(8). Comprising 76 amino acids, ubiquitin is a small polypeptide with a conserved sequence, which is present universally and ubiquitously in eukaryotic cells (4)(5)(6)(7)(8). ...
... The function of ubiquitination is to mark target proteins so that they are recognized and degraded by proteasomes (4)(5)(6)(7)(8). Comprising 76 amino acids, ubiquitin is a small polypeptide with a conserved sequence, which is present universally and ubiquitously in eukaryotic cells (4)(5)(6)(7)(8). The ubiquitination process begins with an E1 ubiquitin-activating enzyme, which, in an ATP-dependent reaction, activates a single free ubiquitin molecule by forming a thioester bond between the Cys residue of the activity center of E1 and the C-terminus of ubiquitin. ...
Human amniotic epithelial stem cells (HuAECs) exhibit pluripotent characteristics, which are similar to those of embryonic stem cells, and can differentiate into various adult tissues and cells through directed induction. However, in culture, HuAECs tend to lose their pluripotency, and their directed differentiation capability declines with increasing passage number. The stem cell pluripotency factor octamer‑binding protein 4 (Oct4) is an important transcription factor that promotes stem cell self‑proliferation and maintains their pluripotency. Previous studies have demonstrated that WW domain containing E3 ubiquitin protein ligase 2 (WWP2) negatively regulates Oct4 expression and stem cell pluripotency. Therefore, the present study aimed to investigate the regulation of WWP2 by microRNAs (miRs), and to evaluate the expression of the downstream factor Oct4 and the maintenance of HuAEC pluripotency. Bioinformatics analysis identified a complementary binding site for miR‑32 in the 3'untranslated region of the WWP2 gene, thus suggesting that it may be a target gene of miR‑32. Post‑infection of HuAECs with a vector overexpressing miR‑32, the endogenous expression of WWP2 was significantly decreased, whereas Oct4 expression was significantly increased. Furthermore, miR‑32‑infected cells differentiated into β islet‑like cells by directed induction. The results indicated that after induction, HuAECs overexpressing miR‑32 also overexpressed the biomarkers of β islet‑like cells. In addition, the ability to secrete insulin was markedly enhanced in response to glucose stimulation, in cells overexpressing miR‑32. In conclusion, the present study suggested that miR‑32 may effectively inhibit WWP2 expression in HuAECs and promote Oct4 overexpression to maintain their pluripotency.
... Ubiquitin and bone morphogenetic protein 2 (BMP-2) were employed as model proteins in this work. Ubiquitin is a small (8.5 kDa) intracellular signaling protein [2], ideal as a model for coupling to surfaces. Ubiquitin was purchased from Sigma (Munich). ...
... Ubiquitin was purchased from Sigma (Munich). 125 I-ubiquitin was prepared by the chloramine-T method as described previously [2]. Human BMP-2 is a small, basic (IEP: 8.21) protein consisting of two identical polypeptide chains of 114 amino acids each (sequence molecular mass 12905 Da, see Fig. 1) covalently linked together to form the biologically active protein of 25.8 kDa [3]. ...
... The half-activation constants (K 0.5 ) of commercial and self-prepared rhBMP-2 were in the range of 20 ± 75 nM. Radioactive rhBMP-2 was prepared from commercial BMP-2 by labeling with 125 I according to the Bolton-Hunter Method (see ref. [2,8]) and employed for covalent coupling to titanium plates. This labeling proce- In each lane 20 lg of the indicated protein was applied in sample buffer [17] containing mercaptoethanol. ...
Proteins such as the model protein 125I-ubiquitin and recombinant human bone morphogenetic protein 2 (125I-rhBMP-2) were covalently immobilized on titanium surfaces after reaction of the latter with aminopropyltriethoxysilane (APS) and activation with carbonyldiimidazole (CDI). On titanium powder (grain size ∼ 60 μm, 2400 cm2/g) net amounts of 15-60 ng/cm2 of 125I-ubiquitin could be coupled after correction for ca. 25% non-specifically adsorbed protein. Similarly, normal polished and special surface-enhanced titanium and stainless steel plates (size 1 × 10 × 15 mm) were modified as above and coupled with 125I-ubiquitin and 125I-BMP-2. The surface-enhanced metal plates were prepared by a special chromosulfuric acid treatment, probably leading to an etching of the metal surface and to an increase in the thickness of the metal oxide layer, which produced a strong enhancement of the binding capacity for chemical modification reactions. Dynamic contact angle measurements (Wilhelmy) of the clean surface-enhanced plates yielded an advancing contact angle of 18.0 and zero contact angle hysteresis. On such surface-enhanced titanium plates net amounts of 300-500 ng/cm2 of 125I-ubiquitin could be immobilized, values 2-5 fold higher than obtained on normal polished plates. In comparison 60-100ng/ cm2125I-rhBMP-2 could be immobilized on the oxidized plates. The above technique is being applied as a tool for biocoating bone implants with the aim of constructing bone-cell-reactive interfaces with specific biorecognition properties.
... This idea is supported by two lines of evidence. First, ubiquitination occurs on lysines (24), and, by contrast with aptamer 5-, 8-, 10-, and 10M-hect fusions, aptamer 2-and 11-hect fusions, which contain lysines in their V regions, did not ubiquitinate LexA-Cdk2 (not shown), even though aptamers 2 and 11 bind Cdk2 as tightly as the others (Fig. 2). This fact is consistent with the idea that ubiquitination of their V regions blocks their binding. ...
... In fact, no Cdk is known to undergo ubiquitin-dependant proteolysis, although some of its binding partners are degraded by this means (32). A number of other proteins are also ubiquitinated without being degraded, including H2A (24), cyclins in certain cell-cycle phases (33), and some membrane receptors whose ubiquitination signals endocytosis without involving the proteasome (34). ...
... The conjugation of multiple ubiquitin molecules to a target protein is followed by its degradation in a large protease complex, the 26S proteasome (Hershko & Ciechanover 1992;Jentsch 1992;Coux et al. 1996;Hochstrasser 1996a,b). The broad biological roles of the ubiquitin-proteasome proteolytic system have been identified: gene expression, cellular stress response, antigen presentation, DNA repairs, programmed cell death, cell cycle and tumorigenesis (Ciechanover 1994;Jennissen 1995). In addition, ubiquitination is also related to the modulation of receptor-mediated endocytosis and subsequent lysosomal degradation (Hochstrasser 1996a,b). ...
... The covalent attachment of ubiquitin, a highly conserved 76-amino acid polypeptide, to lysine residues of a substrate protein, is required for proteasomal degradation (Hershko & Ciechanover 1992;Jentsch 1992;Ciechanover 1994;Jennissen 1995;Hochstrasser 1996a,b). Protein ubiquitination involves the cascade of three classes of enzymes: the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme Ubc or E2, and the ubiquitin-protein ligase E3. ...
Background
Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport.ResultsThree mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, ≈120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed that both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme.Conclusion
Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.
... In contrast to intracellular proteins, which are constantly being substituted (Jennissen 1995), extracellular matrix proteins are exceptionally long-lived proteins (Sivan et al 2006, Shapiro et al 1991. Thus, it is most probable that the cell mechanical characteristics will be transformed in order to allow the migration and invasion of the cells. ...
... Dissimilar to intracellular proteins, which are persistently replaced (Jennissen, 1995), extracellular matrix proteins are extraordinary long-lived proteins (Shapiro et al., 1991;Sivan et al., 2006). Therefore, it is very likely that the cell mechanical properties will be altered to enable the migration and invasion of the cells. ...
Cell migration and invasion is a key driving factor for providing essential cellular functions under physiological conditions or the malignant progression of tumors following downward the metastatic cascade. Although there has been plentiful of molecules identified to support the migration and invasion of cells, the mechanical aspects have not yet been explored in a combined and systematic manner. In addition, the cellular environment has been classically and frequently assumed to be homogeneous for reasons of simplicity. However, motility assays have led to various models for migration covering only some aspects and supporting factors that in some cases also include mechanical factors. Instead of specific models, in this review, a more or less holistic model for cell motility in 3D is envisioned covering all these different aspects with a special emphasis on the mechanical cues from a biophysical perspective. After introducing the mechanical aspects of cell migration and invasion and presenting the heterogeneity of extracellular matrices, the three distinct directions of cell motility focusing on the mechanical aspects are presented. These three different directions are as follows: firstly, the commonly used invasion tests using structural and structure-based mechanical environmental signals; secondly, the mechano-invasion assay, in which cells are studied by mechanical forces to migrate and invade; and thirdly, cell mechanics, including cytoskeletal and nuclear mechanics, to influence cell migration and invasion. Since the interaction between the cell and the microenvironment is bi-directional in these assays, these should be accounted in migration and invasion approaches focusing on the mechanical aspects. Beyond this, there is also the interaction between the cytoskeleton of the cell and its other compartments, such as the cell nucleus. In specific, a three-element approach is presented for addressing the effect of mechanics on cell migration and invasion by including the effect of the mechano-phenotype of the cytoskeleton, nucleus and the cell's microenvironment into the analysis. In precise terms, the combination of these three research approaches including experimental techniques seems to be promising for revealing bi-directional impacts of mechanical alterations of the cellular microenvironment on cells and internal mechanical fluctuations or changes of cells on the surroundings. Finally, different approaches are discussed and thereby a model for the broad impact of mechanics on cell migration and invasion is evolved.
... The primary role of the ubiquitin-proteasome system (UPS) is to degrade unwanted, misfolded, abnormal, denatured and foreign proteins (Jennissen, 1995). For example, ubiquitin levels are increased in cells by as much as five-fold in order to cope with the increased levels of unfolded proteins following heat shock (Bond and Schlesinger, 1985). ...
Tau is a microtubule-associated protein, normally binding to tubulin, in an interaction mediated by chaperones. Unwanted tau is degraded by proteasomes. The tauopathies are neurodegenerative disorders characterized by a tau-predominant neuropathology. Tauopathies include Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP-17, which can be caused by mutations in the tau gene). Pathological tau is hyperphosphorylated, and has reduced microtubule binding capacity. Tau mutations in the microtubule-binding region have similar effects. In some tauopathies, tau deposition is isoform-specific, and pathogenic mutations can cause isoform imbalances. Oxidative stress may also be a factor in tauopathies. In addition, tau pathology has been associated with an ubiquitin variant, which causes proteasome inhibition. There is evidence of aberrations in the chaperone system in diseased cells, reducing tau-microtubule binding, and degradation. To investigate the role of UBB+1 in tauopathies, a model system was developed with SH-SY5Y neuroblastoma cells, which showed that UBB+1 inhibits the proteasome. UBB+1 expression did not affect tau, but was coupled with a chaperone expression response. Subsequently, contrary to expectations, cells with induced UBB+1 expression coped better with oxidative insults. These proteins were studied in sections from PSP brains; UBB+1 and abnormal tau were only present in the diseased tissue. Chaperones and UBB+1 did not co-localise, indicating a failure in the chaperone response. In cells, mutant-tau localized with microtubules as effectively as wild-type. Through neuronal differentiation, tau concentrations were greatly increased, and phosphorylation was decreased. Proteasome inhibition did not increase tau concentration or phosphorylation, or affect isoform balance. A failure in the chaperone response in the brains of tauopathies could explain an increase in unbound cytosolic-tau, tau aggregation, decreased proteasome activity, and susceptibility to oxidative stress.
... Ubiquitin (Ub) is a highly conserved small protein consisting of 76 amino acids with a molecular mass of ~8.5 kDa that is widely expressed in eukaryotes (92). The process by which one or more molecules of ubiquitin bind target proteins via enzyme catalysis is called ubiquitination, with its fundamental function being protein degradation (93). ...
The Hippo signaling pathway is considered to be a tissue growth regulator and tumor suppressor pathway that controls cell proliferation, differentiation, survival, regeneration and tissue homeostasis. Defects in Hippo kinases and hyperactivation of transcriptional co-activator with PDZ-binding motif and Yes-associated protein (YAP) may contribute to the development of different types of cancer. The Hippo pathway is regulated in a variety of way, of which ubiquitination is of considerable importance. Ubiquitination is a crucial post‑translational protein modification in cancer cells and is an applicable target for pharmacological intervention. Ubiquitin modifications are involved in regulating various physiological processes and are counteracted by deubiquitination. Imbalanced ubiquitination-deubiquitination is closely associated with tumor initiation and progression. Therefore, the examination of the specific association between the Hippo pathway and ubiquitination is of interest. The present study reviews the modulatory mechanism of ubiquitination-deubiquitination in the Hippo signaling pathway, the recent progress in identifying therapeutic targets and strategies, and the future directions in the field that may contribute to better tumor diagnosis and treatment.
... This lack of consensus between each proteome may be due to difference in protein identification techniques and tissue sources. The categorisation of HPA proteins in organs or tissues for example is reliant on RNA expression, which can indicate protein expression [7][8][9] but is much more reflective of protein turnover than absolute abundance or historical protein synthesis 10,11 . This issue is particularly important in ECM-rich tissues (such as the dermis) where, unlike cellular proteins whose half-lives are a maximum of days 12 , protein half-life can be as long as 80 years e.g. ...
Defining protein composition is a key step in understanding the function of both healthy and diseased biological systems. There is currently little consensus between existing published proteomes in tissues such as the aorta, cartilage and organs such as skin. Lack of agreement as to both the number and identity of proteins may be due to issues in protein extraction, sensitivity/specificity of detection and the use of disparate tissue/cell sources. Here, we developed a method combining bioinformatics and systematic review to screen >32M articles from the Web of Science for evidence of proteins in healthy human skin. The resulting Manchester Proteome ( www.manchesterproteome.manchester.ac.uk ) collates existing evidence which characterises 2,948 skin proteins, 437 unique to our database and 2011 evidenced by both mass spectrometry and immune-based techniques. This approach circumvents the limitations of individual proteomics studies and can be applied to other species, organs, cells or disease-states. Accurate tissue proteomes will aid development of engineered constructs and offer insight into disease treatments by highlighting differences in proteomic composition.
... Even though the exact biologically functions of histone ubiquitination are remained largely obscure, identified information of this mechanism has implicated them many possible features which are strongly related to numerous cellular processes, such as DNA repairing, and cellular differentiation, and meiosis in higher eukaryotes [33][34][35]. Despite such an array of covalent modifications, ubiquitination has also been proposed as a highly dynamic modification which are determined by the activity of isopeptidases called de-ubiquitin enzyme that release the ubiquitin moiety and by the availability of free ubiquitin [36]. ...
The complexity of immune system is tempered by precise regulation to maintain stabilization when exposed to various conditions. A subtle change in gene expression may be magnified when drastic changes are brought about in cellular development and function. Posttranslational modifications (PTMs) timely alter the functional activity of immune system, and work proceeded in these years has begun to throw light upon it. Posttranslational modifications of histone tails have been mentioned in a large scale of biological developments and disease progression, thereby making them a central field to investigate. Conventional assessments of these changes are centered on the transcription factors and cytokines in T cells regulated by variable histone codes to achieve chromatin remodeling, as well as involved in many human diseases, especially autoimmune diseases. We here put forward an essential review of core posttranslational modulations that regulate T cell function and differentiation in the immune system, with a special emphasis on histone modifications in different T helper cell subsets as well as in autoimmune diseases.
... Ubiquitin conjugates to intracellular target proteins for selective degradation (for reviews, Finley et al., 1989;Hershoko and Ciechanover, 1992;Jennissen, 1995). Ubiquitin also links to the ribosomal protein precursors (Redman and Rechsteiner, 1989;Finley et al., 1989), histones (Hunt and Dayhoff, 1977;West and Bonner, 1980), and receptors including growth hor-*Corresponding author. ...
Changes in Ubiquitin-immunoreactivity after nerve growth factor (NGF) treatment were investigated in PCI2h cells. Ubiquitin-immunoreacitivity was increased in the nucleus of NGF-treated cells. The quantitative analysis revealed that, after 7 days of NGF treatment, almost 20% of cells had ubiquitin-immunoreactive nuclei and the frequency was increased thereafter. Levels of free ubiquitin and multi-ubiquitin chains were measured by radioimmunoassay (RIA) and enzyme-linked immunosor-bent assay (ELISA), respectively. Measurements were carried out for four subcellular fractions: urea-and water-soluble extracts of nuclei and cytoplasm. Decrease in free ubiquitin was observed in water-soluble cytoplasmic extracts of NGF-treated cells, though increase in multi-ubiquitin chains in the same fraction was not observed. As for nuclei, increase in multi-ubiquitin chains and concomitant decrease in free ubiquitin were found in the water-soluble extracts after NGF treatment. Levels of multi-ubiqui-tin chains did not change in urea-soluble cytoplasmic extracts as well as nuclear urea-soluble ones after NGF treatment. These results indicated that multi-ubiquitination of nuclear proteins is increased during NGF-induced neuronal differentiation of PC12h cells.
... The hallmark of the ubiquitin-dependent pathway is the covalent attachment of ubiquitin to proteins to form ubiquitin–protein conjugates in a process termed ubiquitinylation . The best-known role of ubiquitinylation is selectively to target proteins for degradation, but ubiquitinylation of some proteins, such as calmodulin, histones H2A and H2B, actin and some membrane receptors, serves a regulatory function without targeting them for cytosolic degradation (Jennissen, 1995). Nematodes rely on proteomic plasticity to remodel themselves during periods of developmental change, to endure varying environmental conditions and to respond to biotic and abiotic stresses. ...
... Ubiquitin (Ub) has 76 amino acids and mainly functions to label proteins for hydrolysis [10]. Small ubiquitin-related modifier (SUMO) consists of about 100 amino acid residues, and modulates protein structure and function by covalent modification of target proteins in eukaryotes. ...
Antimicrobial peptides have become important candidates as new antibiotics against resistant bacterial strains. However, the major industrial manufacture of antimicrobial peptides is chemical synthesis with high costs and in relatively small scale. The Ub-tag and SUMO-tag are useful for increasing the yield of enzymes and other proteins in expression system. In this study, antimicrobial peptide A20L (KWKSFLKTFKSAKKTVLHTLLKAISS), a derivative of V13K in the previous study is used as a template to be expressed in different Ub-tag and human SUMO tag systems to compare the prokaryotic expression approaches of antimicrobial peptide. The antibacterial mechanism of action and membrane specificity of A20L was further studied.
We fused the Ub and SUMO1/2/3/4 with A20L to construct expression plasmids. Ub-A20L and SUMO1/2/34 gene sequences were inserted into the pHUE plasmids and pET-28b+ plasmids, respectively, to construct pHUE-A20L plasmids and pET-28b+-SUMO1/2/3/4-A20L plasmids. These plasmids were transformed into E. coli Rosetta (DE3) and induced with IPTG to express Ub-A20L and SUMO1/2/3/4 fusion proteins. The recombinant proteins were found in the soluble fraction after being over expressed in E. coli Rosetta (DE3). Antibacterial and hemolytic activities and membrane permeabilization ability of A20L were determined. Peptide structure was also studied by circular dichroism experiments.
A20L (KWKSFLKTFKSAKKTVLHTLLKAISS) was successfully expressed by fusion with an ubiquitin tag (Ub-tag) and human SUMO tags (SUMO1/2/3/4-tags). A20L exhibited antimicrobial activity against various Gram-negative and Gram-positive bacteria. Based on the hemolytic activity against human red blood cells, A20L showed good specificity against bacteria. The circular dichroism experiments illustrated that A20L was transferred into an α-helical structure in the presence of hydrophobic environment. The antibacterial mechanism of action and membrane specificity of A20L was further studied using membrane permeabilization experiments and tryptophan fluorescence and quenching experiments in liposomes.
The Ub-tag and human SUMO-tags represent good alternatives to chemical synthesis for the industrial production of antimicrobial peptides with low costs and high yields. The antibacterial mechanism of action of A20L was proved as membrane disruption. A20L showed stronger specificity on liposomes mimicking bacterial membrane than those mimicking eukaryotic cell membrane, which is consistent with the biological activity studies.
... En camarones adultos, el incremento en RPC a las 48 h posteriores al reto con inmunoestimulantes (anexo II), pudo deberse al incremento en las proteínas involucradas en estrés (Jennissen, 1995). Rodríguez et al (2000) reportaron P. vannamei, variaciones hasta de un 40% en el contenido total de proteína en plasma respecto a los valores control, al someter a los organismos a diferentes niveles de proteína. ...
... Ubiquitination can be mono or poly-Ub on lysine residues (Bhaumik et al., 2007, Weake andWorkman, 2008). It has been found to be associated with numerous processes including protein degradation, DNA repair, cellcycle control, stress response, cell differentiation, ribosome biogenesis, peroxisome biogenesis, viral infection, neural/muscular degeneration and transcription (Jason et al., 2002, Finley and Chau, 1991, Jennissen, 1995 Histone ubiquitination is reversible; the levels are determined by the availability of free ubiquitin and enzymatic activities involved in adding or removing the ubiquitin moiety from histones. Addition of a ubiquitin moiety to a protein involves the sequential action of E1, E2, and E3 enzymes whereas deubiquintination is performed by a class of thiol proteases known as ubiquitin-specific proteases (USPs in mammals) (Weake andWorkman, 2008, Nijman et al., 2005). ...
It is well established that epigenetics and chromatin modifications are important factors
that can govern gene activity and nuclear architecture. They are also proven to be
essential for normal embryonic development and cell differentiation. One important
event during mouse development is the establishment of epigenetic reprogramming
which is believed to be essential for normal growth and development, however; the
mechanism is still poorly understood. The general objective of this PhD study was to
investigate the profiles and mechanisms of epigenetic and chromatin modifications
during normal mouse development and in embryonic stem cells. Mouse pre- and postimplantation
embryos and ES cells were used in experiments employing a range of
different methodologies. The dynamics of epigenetic DNA and histone methylation
were captured using laser confocal immunofluorescent microscopy and western
blotting. The activity of epigenetic modifiers was monitored by real-time PCR and
candidate genes were validated using siRNA technology. The present studies
demonstrate that heterochromatin markers H3K9me3, H3K9me2, H4K20me2,
H4K20me3, HP1α and HP1β are reprogrammed during early development.
Demethylation of H3K9me2, H3K9me3 and H4K20me3 took place at two-cell stage
and remethylation occurred at four-cell stage except for H4K20me3. The
reestablishment of H4K20me3 was initially observed in early postimplantation embryos
in extraembryonic tissue, specifically in the mural trophectoderm. In embryonic tissue,
H4K20me3 was not clearly detected until in mid to late postimplantation development.
The mechanism of H3K9me2 and H3K9me3 demethylation might be due to either an
imbalance of epigenetic modifiers or the presence of Jmjd2a and Jmjd1a histone
demethylase postfertilisation. We have also report evidence that HP1α and Suv4-20h
are required in heterochromatin before the recruitment of H4K20me3 during mouse
development and in ES cells. Therefore H4K20me3 removal was believed to involve
the lack of prerequisite heterochromatin complexes such as HP1α and Suv4-20h
enzymes. Furthermore, the presence and levels of H4K20me3 and HP1α might be
strongly associated with cell differentiation and tissue maturation in mouse in vivo
development but not in vitro early differentiated ES cells. Surprisingly, the results
showed that chromatin modifications and their modifiers in ES cells are different from ICM and epiblast. Chromatin modifications H4K20me3 and HP1α were absent from
ICM and epiblast, but were detected in ES cells. Notably, H4K20me3 and HP1α were
established after early incubation of ICM into ES cell medium, but this change was not
dependent on the presence of serum and leukaemia inhibiting factor. Epigenetic
modifier Jmjd2a but not Jmjd1a was found in ICM. Conversely, Jmjd1a is highly
expressed in ES cells while Jmjd2a was inactivated. In addition, the present studies
revealed the substantial role of histone demethylases in development, as it may be
important for epigenetic reprogramming. The results demonstrated that inhibition of
demethylase Jmjd2a and Jmjd1a caused preimplantation embryos to arrest at the twocell
stage while Jmjd2c deficient embryos failed to reach blastocyst. Thus it is possible
that Jmjd2a and Jmjd1a were essential for epigenetic reprogramming while Jmjd2c is
critical for cell fate establishment during blastocyst formation. In conclusion, the global
chromatin signature in ES cells differs from ICM and epiblast; heterochromatin
reprogramming occurs at two-cell stage; maturation of heterochromatin occurs at
postimplantation; and histone demethylases Jmjd1a, Jmjd2a and Jmjd2c are important
in preimplantation development. Results from the present studies could provide crucial
information for developmental biology and stem cell research, and provide as a model
for improvement of reproductive biotechnologies such as somatic cell reprogramming,
and diagnosis of epigenetic abnormalities in early development.
... Erst wenn durch mehrfache Beschädigung und durch unzureichende Reparatur die physiologisch notwendige Konfiguration verloren geht, wenn die spezifischen Funktionsstellen der Proteinmoleküle, die Regulationszentren ihrer Aufgabe nicht mehr nachkommen können, dann wird dieses Protein «aus dem Verkehr gezogen» und zerlegt. Die letzte zusammenfassende Darstellung von Jennissen 1995[15] trägt den Titel «Ubiquitin and the enigma of intracellular protein degradation». Die hier so benannte intrazelluläre Proteindegradation bleibt natürlich «rätselhaft», wenn Jennissen ausdrücklich von der dogmatisierten These ausgeht, der Proteinabbau sei zufallsbestimmt. ...
Nature’s Healing Power – Natural Selection in Protein TurnoverFor millennia, medical practitioners, healthy people and the sick have known that there is an inner force that keeps us sound and also cures us. However, neither medicine nor natural sciences have been able to define this curative power. It can not be captured by experiments or assigned with dimensions or numbers. The answer can be found in the quintessential process of all living systems, the interaction of genes and proteins in every single cell. Genes encode proteins; proteins are the ubiquitous instruments of life. Protein molecules undergo rapid turnover: their median lifetime is about 2 days, and in every second so many proteins are synthesized in each of us that their number equals the number of seconds that have passed since the big bang 15 billion years ago. The biochemical dogma that says that after their synthesis proteins are degraded at random – blindly, quasi – is wrong. The experimental results have not been interpreted correctly. In the first instance, proteins are stable and functional, since they have to fulfill numerous tasks. Only aged, severely damaged, non-functioning proteins are discarded. Protein turnover is subject to selection, not to chance. By selecting fresh, functional proteins, the quality of cellular proteins is kept at a high level. The principle of selection in protein turnover is the crucial component in the scientific groundwork of naturopathy therapeutics. With natural influencies and measures (e.g. food processing, intermediate metabolism, exercise, light, warmth, coldness) the steady process of regeneration is accelerated – in contrast to medical therapy, which restricts and blocks protein activities. Natural healing power and self-organization energy are basic phenomena of a therapeutically used physiology. Naturopathy is natural science.
... Both collagen and elastin are stabilised by these enzymatic cross-links; however in elastin the function of these cross-links is to restrain excessive stretching, whilst in collagen the cross-links act by rendering the fibrils virtually inextensible (Bailey 2001). Both elastic fibres and fibrillar collagens are uniquely long-lived (Sivan et al. 2008; Shapiro et al. 1991) compared to intracellular proteins (Jennissen 1995). The low turnover rate of these structural ECM proteins exposes them to degradation by a range of processes; enzymatic, chemical or biophysical. ...
With increasing age, dynamic tissues such as lungs, blood vessels and skin lose their ability to both deform and recoil, culminating in tissue stiffening. This loss of tissue elasticity, which profoundly impacts tissue function and thus morbidity, may be due not only to changes in the relative abundance of key extracellular matrix proteins within tissues but also to their accumulation of post-translational modifications. Whilst to date attention has focussed primarily on the age-related non-enzymatic formation of advanced glycation end products, the accumulation of pathological enzyme-mediated cross-links may also lead to age-related tissue stiffening. The lysyl oxidase (LOX) family of enzymes are constitutively expressed in adult tissues and are known to drive the catalysis of cross-links in both fibrillar collagens and elastin. Although immunochemical approaches are commonly used to localise the inactive pro-enzyme of LOX, and biochemical methods are employed to quantify activity in homogenised tissue, they do not allow for the in situ localisation of the enzyme. Thus, we have developed a novel assay to both detect and localise LOX enzyme activity in situ. LOX family members are amine oxidases and this assay uses the principle that an amine substrate in the presence of this class of enzyme will be oxidised to an aldehyde and hydrogen peroxide (H(2)O(2)). In turn, H(2)O(2), when combined with luminol and horseradish peroxidase, will produce a light-emitting reaction that can be detected by film autoradiography. The development of a technique to localise specific amine oxidase activity in tissue sections may provide crucial additional information on the exact role played by this class of enzymes in mediating age-related tissue stiffening.
... A variety of intracellular proteins are selectively multiubiquitinated for further destruction by 26S proteasome, 1,2 but there is virtually no information regarding the involvement of the ubiquitin system in PGM-B degradation. Several cellular proteins, including histones H2A, H2B and H1, 32,33 G-protein coupled receptors, 34 membrane-bound transporters, 35,36 ubiquitin-conjugating enzymes, 18,37 a yeast kinetochore protein Cbf2p, 38 calmodulin 32 and retrovirus proteins 39 are established as substrates for monoubiquitination in vivo. They are not targeted for destruction by 26S proteasome and the monoubiquitination to these proteins regulates diverse cellular events, such as histone regulation, endocytosis and virus budding, etc. 33 PGM-B does not share common features with them, but we speculate that this modification might affect activity or location 40 of the enzyme. ...
Ubiquitin-conjugated proteins in human colorectal cancer tissues were analyzed by the immunoprecipitation with the antibody FK2 against conjugated ubiquitin followed with SDS-PAGE. In these immunoprecipitable proteins, a 38-kDa protein was abundant in the tumor regions but almost absent in the adjacent normal regions in 17/26 patients, thus we attempted to purify it. Using immunoaffinity chromatography with the antibody FK2 followed by gel filtration and SDS-PAGE, approximately 10 pmol of this protein was separated from 34 g of the pooled cancerous tissue and transferred onto a PVDF membrane. The 38-kDa protein was further digested with Achromobacter protease I, resulting in several peptide fragments. Amino acid sequences of these peptides showed complete sequence identity to those derived from either ubiquitin or phosphoglycerate mutase-B, suggesting that the 38-kDa protein is monoubiquitinated phosphoglycerate mutase-B, whose calculated mass is 37,369 Da. Western blot using an antibody against phosphoglycerate mutase-B revealed the presence of the 38-kDa protein in the anti-ubiquitin immunoprecipitates derived from the tumor regions, but not from normal counterparts. In addition, part of non-ubiquitinated phosphoglycerate mutase-B (29 kDa) was also found in the anti-ubiquitin immunoprecipitates, whose levels were higher in the tumor regions than in the adjacent normal regions. These results suggest that monoubiquitination of phosphoglycerate mutase-B as well as formation of a noncovalent complex containing ubiquitin and phosphoglycerate mutase-B increases in colorectal cancer and novel modification of phosphoglycerate mutase-B might have a pathophysiological role. © 2001 Wiley-Liss, Inc.
... PWV is related to vascular wall stiffness through the Moens-Korteweg equation, which in its simplest form is 1 where E a and h are respectively the mean Young's modulus and thickness of the arterial wall. The lumen radius is given by r and ρ is the blood density. ...
The quality of life of ageing populations is increasingly determined by age-related changes to the mechanical properties of numerous biological tissues. Degradation and mechanical failure of these tissues has a profound effect on human morbidity and mortality. Soft tissues have complex and intricate structures and, similar to engineering materials, their mechanical properties are controlled by their microstructure. Thus age-relate changes in mechanical behavior are determined by changes in the properties and relative quantities of microstructural tissue components. This review focuses on the cardiovascular system; it discusses the techniques used both in vivo and ex vivo to determine the age-related changes in the mechanical properties of arteries.
... Absence of functional stress proteins leads to accumulation of aggregates of misfolded proteins and ultimately to cell death. These points have all been reviewed extensively (e.g., Morimoto et al., 1990Morimoto et al., , 1994bFinley and Chau, 1991;Parsell and Lindquist, 1993;Arrigo and Mehlin, 1994;Ciechanover and Schwartz, 1994;Jennissen, 1995;Hartl, 1996;Peters et al., 1997;Varshavsky, 1997;Morimoto, 1998;Morimoto and Santoro, 1998;Hicke, 1999). ...
Heat shock, or stress, proteins (HSPs) are induced in response to conditions that cause protein denaturation. Activation of cellular stress responses as a protective and survival mechanism is often associated with chemical exposure. One interface between the body and the external environment and chemical or biological agents therein is the olfactory epithelium (OE). To determine whether environmental odorants affect OE HSP expression, rats were exposed to a variety of odorants added to the cage bedding. Odorant exposure led to transient, selective induction of HSP70, HSC70, HSP25, and ubiquitin immunoreactivities (IRs) in supporting cells and subepithelial Bowman's gland acinar cells, two OE non-neuronal cell populations involved with inhalant biotransformation, detoxification, and maintenance of overall OE integrity. Responses exhibited odor specificity and dose dependency. HSP70 and HSC70 IRs occurred throughout the apical region of supporting cells; ubiquitin IR was confined to a supranuclear cone-shaped region. Electron microscopic examination confirmed these observations and, additionally, revealed odor-induced formation of dense vesicular arrays in the cone-like regions. HSP25 IR occurred throughout the entire supporting cell cytoplasm. In contrast to classical stress responses, in which the entire array of stress proteins is induced, no increases in HSP40 and HSP90 IRs were observed. Extended exposure to higher odorant doses caused prolonged activation of the same HSP subset in the non-neuronal cells and severe morphological damage in both supporting cells and olfactory receptor neurons (ORNs), suggesting that non-neuronal cytoprotective stress response mechanisms had been overwhelmed and could no longer adequately maintain OE integrity. Significantly, ORNs showed no stress responses in any of our studies. These findings suggest a novel role for these HSPs in olfaction and, in turn, possible involvement in other normal neurophysiological processes.
... UVR absorbing epidermal molecules such as melanin and UCA appear to play a protective role in absorbing UVR and both DNA and short-lived intracellular proteins are, at least partially, protected from the long term effects of UV-mediated damage by endogenous mechanisms which detect and repair or rapidly replace defective molecules [15,46,60]. In contrast, individual dermal ECM proteins and supra-molecular assemblies must continue to function in potentially harmful environments for many years [61]. ...
Human skin, in common with other organs, ages as a consequence of the passage of time, but in areas exposed to solar ultraviolet radiation, the effects of this intrinsic ageing process are exacerbated. In particular, both the severity and speed of onset of age-related changes, such as wrinkle formation and loss of elasticity, are enhanced in photoaged (also termed extrinsically aged) as compared with aged, photoprotected, skin. The anatomy of skin is characterised by two major layers: an outer, avascular, yet highly cellular and dynamic epidermis and an underlying vascularised, comparatively static and cell-poor, dermis. The structural consequences of photoageing are mainly evident in the extracellular matrix-rich but cell-poor dermis where key extracellular matrix proteins are particularly susceptible to photodamage. Most investigations to date have concentrated on the cell as both a target for and mediator of, ultraviolet radiation-induced photoageing. As the main effectors of dermal remodelling produced by cells (extracellular proteases) generally have low substrate specificity, we recently suggested that the differential susceptibility of key extracellular matrix proteins to the processes of photoageing may be due to direct, as opposed to cell-mediated, photodamage.
In this review, we discuss the experimental evidence for ultraviolet radiation (and related reactive oxygen species)-mediated differential degradation of normally long lived dermal proteins including the fibrillar collagens, elastic fibre components, glycoproteins and proteoglycans. Whilst these components exhibit highly diverse primary and hence macro- and supra-molecular structures, we present evidence that amino acid composition alone may be a useful predictor of age-related protein degradation in both photoexposed and, as a consequence of differential oxidation sensitivity, photoprotected, tissues.
... To compensate for decreased functional protein levels caused by stress, cells will Ž . increase production of these same proteins Jennissen, 1995;Iwai, 1999 . Thus, measuring ubiquitin level indexes the structural integrity of the protein component of the Ž . ...
We developed a Molecular Biomarker System (MBS) to assess the physiological status of mud snails (Ilyanassa obsoleta) challenged by exposure to high temperature, cadmium, atrazine, endosulfan and the water-accommodating fraction of bunker fuel #2. The MBS is used to assay specific cellular parameters of the gastropod cell that are indicative of a non-stressed or stressed condition. The MBS distinguished among responses to each stressor and to non-stressed control conditions. For example, the biomarkers metallothionein and cytochrome P450 2E1 homologue distinguished between metal and non-metal stresses. MBS data from this study corroborate toxicological studies of organismal responses to endosulfan, atrazine, fuel and cadmium stresses. The MBS technology aids in the accurate diagnosis of the snail's health condition because the physiological significance of the changes of each biomarker is well known. This technology is particularly relevant for environmental monitoring because gastropods are used as key indicator species in many estuarine, marine, freshwater and terrestrial ecosystems. Finally, the Molecular Biomarker System technology is relatively inexpensive, easy to implement, precise and can be quickly adapted to an automated, high-throughput system for large sample analysis.
The majority of ixodid ticks display host-specificity to varying extents. Feeding on different hosts affects their development and reproduction. Consequences can be analyzed at the level of the egg, as it is the initial stage of tick development. Tick egg proteins are abundant and diverse, providing nutrients for embryonic development. However, studies on tick egg profiles are scarce. In this study, we aimed to analyze whether feeding Haemaphysalis qinghaiensis ticks on the yaks (Bos grunniens) and domestic sheep (Ovis aries) has an impact on the variety and variability of the egg proteome. Detached engorged females were used to lay eggs, which were then collected, dewaxed, and subjected to protein extraction. The extracted egg proteins were enzymatically digested using Filter-Aided Sample Preparation (FASP), and the unique peptides were separated and detected by Liquid Chromatography-tandem Mass Spectrometry (LC–MS/MS). The MS data were searched against the previously constructed whole tick transcriptome library of H. qinghaiensis, and the UniProt database for the identification of tick-derived egg proteins. The analysis revealed 49 and 53 high-confidence proteins identified in eggs collected from B. grunniens (EggBg) and O. aries (EggOa), respectively. Of these, 46 high-confidence proteins were common to both egg types, while three were unique to EggBg and seven to EggOa. All the identified proteins mainly belonged to enzymes, enzyme inhibitors, transporters, and proteins with unknown functions. The differential abundance analysis showed that nine proteins were significantly more present in EggBg, while six were significantly more present in EggOa. Overall, enzymes were the most diverse group, while vitellogenin (Vg) was the most abundant. Blood meal uptake on different hosts has a certain effect on the egg proteome composition and the abundance of some proteins, but it may also lead to compensation of protein roles.
Aging is a dynamic, time-dependent process that is characterized by a gradual accumulation of cell damage. Continual functional decline in the intrinsic ability of living organisms to accurately regulate homeostasis leads to increased susceptibility and vulnerability to diseases. Many efforts have been put forth to understand and prevent the effects of aging. Thus, the major cellular and molecular hallmarks of aging have been identified, and their relationships to age-related diseases and malfunctions have been explored. Here, we use data from the CAS Content Collection to analyze the publication landscape of recent aging-related research. We review the advances in knowledge and delineate trends in research advancements on aging factors and attributes across time and geography. We also review the current concepts related to the major aging hallmarks on the molecular, cellular, and organismic level, age-associated diseases, with attention to brain aging and brain health, as well as the major biochemical processes associated with aging. Major age-related diseases have been outlined, and their correlations with the major aging features and attributes are explored. We hope this review will be helpful for apprehending the current knowledge in the field of aging mechanisms and progression, in an effort to further solve the remaining challenges and fulfill its potential.
Extracellular matrix (ECM) proteins confer biomechanical properties, maintain cell phenotype and mediate tissue repair (via release of sequestered cytokines and proteases). In contrast to intracellular proteomes, where proteins are monitored and replaced over short time periods, many ECM proteins function for years (decades in humans) without replacement. The longevity of abundant ECM proteins, such as collagen I and elastin, leaves them vulnerable to damage accumulation and their host organs prone to chronic, age-related diseases. However, ECM protein fragmentation can potentially produce peptide cytokines (matrikines) which may exacerbate and/or ameliorate age- and disease-related ECM remodelling. In this review, we discuss ECM composition, function and degradation and highlight examples of endogenous matrikines. We then critically and comprehensively analyse published studies of matrix-derived peptides used as topical skin treatments, before considering the potential for improvements in the discovery and delivery of novel matrix-derived peptides to skin and internal organs. From this, we concluded that while the translational impact of matrix-derived peptide therapeutics is evident, the mechanisms of action of these peptides are poorly defined. Further, well-designed, multimodal studies are required.
Cerebral ischemia-induced hyperglycemia has been reported to accentuate neurological damage following focal or global cerebral ischemia. Hyperglycemia found in rats following focal brain ischemia occurs in the first 24 hours and has been claimed to be caused by increased liver gluconeogenesis and insulin resistance. However, liver gluconeogenesis and the mechanisms leading to hyperglycemia after global cerebral ischemia remain uncertain. This study investigated the glycemic homeostasis and hepatic metabolism in rats after transient four-vessel occlusion (4-VO)-induced global cerebral ischemia, an event that mimics to a certain degree the situation during cardiac arrest. Several metabolic fluxes were measured in perfused livers. Activities and mRNA expressions of hepatic glycolysis and glyconeogenesis rate-limiting enzymes were assessed liver as well as respiratory activity of isolated mitochondria. Global cerebral ischemia was associated to hyperglycemia and hyperinsulinemia 24 hours after ischemia. Insulin resistance developed later and was prominent after the 5th day. Hepatic anabolism and catabolism were both modified in a complex and time-dependent way. Gluconeogenesis, β-oxidation, ketogenesis and glycolysis were diminished at 24 hours after ischemia. At 5 days after ischemia glycolysis had normalized, but gluconeogenesis, ketogenesis and β-oxidation were accelerated. The overall metabolic modifications suggest that a condition of depressed metabolism was established in response to the new conditions generated by the cerebral global ischemia. Whether the modifications in the liver metabolism found in rats after the ischemic insult can be translated to individuals following global brain ischemia remains uncertain, but the results of this study are hoped to encourage further investigations.
Aluminium (Al) toxicity is a primary limitation of crop production in acid soils, which take over 40% of arable soil worldwide. In previous studies, a series of genes have been identified to regulate the plant Al resistance or tolerance. However, none of E3 ubiquitin ligase, the key factor of ubiquitination that plays an important role in plant growth and development, has been characterized for Al response in Arabidopsis. In this study, an E3 ubiquition ligase gene AtATRF1 (Al Tolerance RING Finger 1), a homolog of RAD18 interacting RAD6 to repair the damaged DNA in human and yeast, is isolated from Arabidopsis. It encodes a predicted protein of 296 amino acids with a C3HC4 type RING finger domain. The expression of AtATRF1 is induced by Al, and the transgenic plant overexpressing AtATRF1 enhances the Al tolerance. Similar as RAD18, the AtATRF1 locates in nucleus and regulates the expression of AtATR, which involves in DNA repair and Al response in Arabidopsis. Our results indicate that nuclearlocated AtATRF1 may interact and ubiquitinate the transcriptional regulator of AtATR to mediate the Al tolerance of Arabidopsis.
This chapter reviews the use of in vitro culture systems in neurotoxicology and describes and discusses a range of selected experimental models used in in vitro neurotoxicology. Neurotoxicology aims to predict the effects a substance will have on a neurone in vivo. The chapter discusses the advantages and limitations of several in vitro methodologies available for neurotoxicology testing including primary dissociated neuronal cultures, neuronal cell lines, transfected neuronal cells and neuronal cell lines, hybrid cell line cultures, and co-culture systems. Furthermore, the chapter provides a brief overview of several of the most commonly used techniques for assessing neuronal cell health, such as mitochondrial function measured by MTT, mitochondrial potential, transcellular traffic of solutes, cellular ATP levels, stress response genes, proto-oncogenes and transcription factors, and assessment of proteins that indicate the degree of differentiation of neurones.
The term “sepsis syndrome” describes a systemic inflammatory and hypermetabolic response of the body on the cellular, organ, and organ systems level to a variety of microbial stimuli and to stimuli other than exogenous microbial agents. The latter may include but are not limited to accidental blunt and penetrating injuries, surgical trauma, burns, pancreatitis, inflammatory bowel disease, and others. If bacteria, viruses, or fungi are involved and the sepsis syndrome is caused by these microbes, the term “sepsis” is used. Systemic inflammatory response syndrome (SIRS) is an expression that has been used recently as a common denominator for such inflammatory states independent of their cause. Infection denotes a (locally confined) inflammatory response to the presence of microorganisms or the invasion of normally sterile host tissue by those organisms, and the term “bacteremia” means the presence of bacteria in the bloodstream, indicating an infectious focus with spillover of bacteria into the circulation. Conditions such as viremia and fungemia can be similarly defined.1, 2
The 26 S proteasome is a large multisubunit complex that degrades intracellular proteins in a nucleotide-dependent reaction. The enzyme is composed of about 30 different proteins. Fourteen subunits are provided by the proteasome and an additional 16 or more subunits are present in a particle that I will call the regulatory complex. The 26 S enzyme is the only ATP-dependent protease so far identified in the nuclear and cytosolic compartments of eukaryotic cells—a surprising observation in view of the fact that at least four ATP-dependent proteases are present in E. coli cells (i. e., Lon, Clp, Hf1, and Hs1V). The 26 S proteasome has been shown to be responsible for the degradation of more than 20 specific proteins. Among the known substrates are a variety of important regulatory proteins including transcription factors, cyclins, and cdk inhibitors. The enzyme may well be primarily responsible for the selective degradation of eukaryotic proteins.
IntroductionProtein and amino acid turnoverProtein synthesisRegulation of the translation phase of protein synthesisPost-translational eventsProtein degradationSelectivity of protein turnoverAn integration of these processes of turnover with respect to amino acid metabolismRegulation of amino acid metabolismAmino acid synthesis: the dispensable amino acidsIn vivo aspects of protein and amino acid turnoverIn vivo rates of protein turnoverMechanisms and factors responsible for alterations in protein turnoverInterorgan amino acid metabolismAmino acid and peptide transportDisposal of dietary amino acids and roles of specific organsCatabolic illnessesNon-proteinogenic metabolic functions of amino acidsPerspectives on the futureFurther reading
Live yeast cell derivatives (LYCD) effectively elicited antioxidant activity of superoxide dismutase (SOD) in American white shrimp (Litopenaeus vannamei). Juvenile shrimp were immersed in LYCD solutions for different lengths of time. SOD activity in shrimp immersed in LYCD for 6 h did not significantly (p>0.05) increase in muscle, but did in hemocytes (p<0.05), with SOD activity increasing with LYCD concentration. The highest SOD activity in hemocytes was similar to that in muscle (1.84 and 1.86 times that of control, respectively). Hemocyte count increased significantly 72 h after challenge with 50 μl/ml LYCD. The relative soluble protein in the muscle equaled that of hemocytes. Our results show the immunostimulant effects of LYCD in L. vannamei.
Amyotrophic lateral sclerosis (ALS) is classified into distinct subtypes mainly based on clinicopathological features, in addition to epidemiologic and genetic backgrounds. In addition to sporadic ALS with classical pathology, characteristics in the clinical features, in the histological findings and their topographical distribution, and in the molecular pathology, especially the intracytoplasmic neuronal inclusions, enable us to identify the following subtypes: ALS with dementia (ALS-D), ALS in the Western Pacific, ALS with multi-system degeneration, familial ALS, and superoxide dismutase 1-linked ALS. These subtypes not infrequently exhibit various types of extra-motor system degeneration, and even multisystem pathology. Some of the subtypes (for instance ALS-D or familial ALS) can be deduced, to a certain extent, from characteristic neuronal inclusions such as ALS-type ubiquitinated inclusions, Lewy body-like hyaline inclusions (LBHIs), or ubiquitinated intracytoplasmic neuronal inclusions as typically...
Maritime pine somatic embryos require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome (2D-SDS-PAGE with MS identification) of immature somatic embryos, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous ABA (GC-MS), soluble sugars (HPLC), starch, and confocal laser microscope observations. This multi-scale, integrated analysis was used to unravel early molecular and physiological events involved in somatic embryo development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation which may be antagonistic to somatic embryo development. Under favorable conditions (9G), somatic embryos adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of somatic embryo development whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in development of pine somatic embryos following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.
Significance:
Chronic exposure to environmental ultraviolet radiation (UVR) plays a key role in both photocarcinogenesis and induction of accelerated skin aging. Although the spatiotemporal consequences of UVR exposure for the composition and architecture of the dermal extracellular matrix (ECM) are well characterized, the pathogenesis of photoaging remains poorly defined. Given the compelling evidence for the role of reactive oxygen species (ROS) as mediators of photoaging, UVR-exposed human skin may be an accessible model system in which to characterize the role of oxidative damage in both internal and external tissues.
Recent advances:
Although the cell-mediated degradation of dermal components via UVR-induced expression of ECM proteases has long been identified as an integral part of the photoaging pathway, the relative importance and identity of cellular and extracellular photosensitizers (direct hit and bystanders models, respectively) in initiating this enzymatic activity is unclear. Recently, both age-related protein glycation and relative amino-acid composition have been identified as potential risk factors for photo-ionization and/or photo-sensitization. Here, we propose a selective multi-hit model of photoaging.
Critical issues:
Bioinformatic analyses can be employed to identify candidate UVR targets/photosensitizers, but the action of UVR on protein structure and/or ROS production should be verified experimentally. Crucially, in the case of biochemically active ECM components such as fibronectin and fibrillin, the downstream effects of photo-degradation on tissue homeostasis remain to be confirmed.
Future directions:
Both topical antioxidants and inhibitors of detrimental cell signaling may be effective in abrogating the effects of specific UVR-mediated protein degradation in the dermis.
The activity of the stomatal key enzyme phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) is known to be decreased during ABA-induced stomatal closure, in parallel the level of the second messenger inositol 1,4,5 trisphosphate (ins(1,4,5)P3) is increased. Since ins(1,4,5)P3 itself induces stomatal closure, the effect of the second messenger on protein-selective degradation of PEPCase and the specificity for PEPCase guard cell isoforms was investigated. PEPCase was immunoprecipitated from ins(1,4,5)P3 treated epidermal crude extracts. Analysis of the precipitates gave hints to an ins(1,4,5)P3 — induced selective ubiquitin-dependent proteolysis preferentially of guard cell specific PEPCase isoforms (110,112,116 kDa). The role of the second messenger ins(1,4,5)P3 as a possible mediator between signal-mediated cascades and the ubiquitin-specific PEPCase degradation is discussed on the basis of PEPCase functioning as a target for selected proteolysis during stomatal closure. Moreover, the diurnal rhythmicity of guard cell protein turnover was analysed, especially with respect to ubiquitin-dependent processes.
This chapter presents a structural overview of the modifications of a primary structure and discusses the modifications at the posttranslational level that contribute to histone variability. Different contributions of histones and DNA to the stability and dynamics of the chromatin nucleoprotein complex are also discussed in the chapter. The exploration of the informational coding hypothesis has grown exponentially over the past few years, and the results obtained have proven to be very rewarding in terms of connecting histone structure to function. The availability of a powerful DNA template in combination with the use of recombinant histones allows the reconstitution of highly homogeneous chromatin structures that behave like native complexes of equivalent nucleosome loading. Such complexes are amenable to analysis by a wide variety of biophysical techniques providing invaluable tools in ascertaining the role played by histone variability on a chromatin structure.
Peptide aptamers are proteins selected from combinatorial
libraries that display conformationally constrained variable regions.
Peptide aptamers can disrupt specific protein interactions and thus
represent a useful method for manipulating protein function in
vivo. Here, we describe aptamer derivatives that extend the
range of functional manipulations. We isolated an aptamer with
increased affinity for its Cdk2 target by mutagenizing an existing
aptamer and identifying tighter binding mutants with calibrated
two-hybrid reporter genes. We used this and other anti-Cdk2 aptamers as
recognition domains in chimeric proteins that contained other
functional moieties. Aptamers fused to the catalytic domain of a
ubiquitin ligase specifically decorated LexA-Cdk2 with ubiquitin
moieties in vivo. Aptamers against Cdk2 and another
protein, Ste5, that carried a nuclear localization sequence transported
their targets into the nucleus. These experiments indicate that fusion
proteins containing aptameric recognition moieties will be useful for
specific modification of protein function in vivo.
A general problem in metazoan biology is the identification of the specific ligands for transmembrane receptors. One goal of the original proposal was to selectively modify the physiology of the budding yeast Saccharomyces cerevisiae to speed the identification and study of ligand/receptor interactions - in particular that of erB2 given its involvement in breast cancer. Significant progress was made on development of a system that allows a rapid readout for interaction of a receptor with its corresponding ligand. A second approach involves a novel search for small interacting peptide aptamers that could be used as ligand mimics or inhibitors. Peptide aptamer libraries have been characterized and in doing so, a novel peptide effector of the cell cycle regulator Cdk4 was characterized.
Ubiquitination of key cellular proteins involved in signal transduction, gene transcription and cell-cycle regulation usually condemns those proteins to proteasomal or lysosomal degradation. Additionally, cycles of reversible ubiquitination regulate the function of certain proteins in a manner analogous to phosphorylation. In this short review we describe the current methodology for measuring ubiquitin and ubiquitination, provide examples which illustrate how various techniques have been used to study protein ubiquination, alert the readers of pitfalls to avoid, and offer guidelines to investigators newly interested in this novel post-translational protein modification.
In the present paper, we studied the participation of the 20S proteasome, the proteolytic component of the ubiquitin–proteasome pathway, in the remobilization of bulk proteins in senescing wheat leaves. The detached leaves of 15-d-old plants were incubated in darkness for several days, and various proteolytic activities were analysed in soluble extracts prepared at 0, 48 and 96 h after detachment. The endoproteolytic activity, measured at pH 7.5 and 5.4, increased more than 10-fold and the total peptidasic activity increased up to 5-fold after 96 h of incubation in the dark, when expressed as specific activity. In the same period, the leaf-protein content decreased to less than 50% of that present at the initial time. The 20S proteasome chymotrypsin-like activity remained constant when it was expressed as activity per leaf fresh weight and resulted 2-fold higher in terms of specific activity. The western blot analysis showed that the amount of 20S proteasome protein and ubiquitin–protein conjugates also remained constant until 4 d of incubation in darkness. These results indicate that the ubiquitin–proteasome pathway remains functional until the late phases of senescence suggesting that it may participate in the regulatory aspects of the process rather than in the massive protein breakdown.
Hydroxyapatite (HAP) has a great potential as a bone replacement material because of its similarity to the crystal structure of inorganic matrix of bone. Several years ago we showed that porous hydroxyapatite ceramics (Endobon, Merck) can be covalently modified for the immobilization of proteins [Jennissen, H.P. (1999) PCT Patent WO9926674A2] opening the possibility for the immobilization of growth factors and morphogens. Application of this technique to the immobilization of recombinant human bone morphogenetic protein 2 (rhBMP-2) on hydroxyapatite would be of high medical interest, because of the decisive role of bone morphogenetic proteins in bone development and osteogenesis. In this paper it will be shown that the model protein ubiquitin and rhBMP-2 can be immobilized non-covalently and covalently after chemical modification of a hydroxyapatite ceramic surface (Bio-Oss® Cancellous Block, Geistlich, = HAP-wafers). It could be shown that only small amounts of protein are adsorbed to non-functionalized HAP surfaces (control). In contrast ubiquitin and rhBMP-2 can be very efficiently immobilized non-covalently (up to 2.4 mg protein/g HAP) and covalently (up to 9.7 mg protein/g HAP) on porous HAP-wafers. In desorption experiments it is shown that the bound rhBMP-2 is slowly released making such surfaces applicable as long term drug delivery devices for enhancing bone growth and osteointegration of implant materials.
Ubiquitylation is an important regulatory mechanism in the eukaryotic cell. A large volume of experimental data on protein
ubiquitylation has been acquired in recent years. Particular ubiquitylated lysine residues were also identified. This allows
us to analyze co-localization of ubiquitylation sites and functionally important protein domains, following the idea that
ubiquitylation can directly affect protein functional activity. Computational analysis suggests that ubiquitylation can affect
the functional activity of some proteins through direct steric effects. (1) Ubiquitylation can block protein functional domains/active
site or cause accessibility limitations. It also (2) causes steric disturbances for homo-oligomerization and (3) influences
heterologous protein interactions, impeding the binding of target protein with its partners. (4) Interaction with partner
proteins can be disturbed by restricted conformational flexibility. Any of these effects will result in a decrease of target
protein activity. Thus, we suggest a new “loss-of-function” mechanism of protein regulation by ubiquitylation.
Rapid progress in DNA technology has entailed the possibility of readily detecting mutations in disease genes. In contrast to this, techniques to characterize the effects of mutations are still very time consuming. It has turned out that many of the mutations detected in disease genes are missense mutations. Characterization of the effect of these mutations is particularly important in order to establish that they are disease causing and to estimate their severity. We use the experiences with investigation of medium-chain acyl-CoA dehydrogenase deficiency as an example to illustrate that (i) impaired folding is a common effect of missense mutations occurring in genetic diseases, (ii) increasing the level of available chaperones may augment the level of functional mutant protein in vivo, and (iii) one mutation may have multiple effects. The interplay between the chaperones assisting folding and proteases that attack folding intermediates is decisive for how large a proportion of a mutant polypeptide impaired in folding acquires the functional structure. This constitutes a protein quality control system, and the handling of a given mutant protein by this system may vary due to environmental conditions or genetic variability in its components. The possibility that intraindividual differences in the handling of mutant proteins may be a mechanism accounting for phenotypic variability is discussed.
Lens development and response to peroxide stress are associated with dramatic changes in protein ubiquitination, reflecting dynamic changes in activity of the ubiquitin-activating enzyme (E1). Two isoforms of E1 (E1A and E1B) have been identified in lens cells although only one E1 mRNA, containing three potential translational start sites, has been detected. Novel, site-specific antibodies to E1 were generated and the hypothesis that the two isoforms of E1 are translated from alternative initiation codons of a single mRNA was tested. Antibodies raised against E1A-N peptide (Met1to Cys23of E1A) reacted only with E1A by immunoblot and immunoprecipitation. Antibodies raised against E1B-N peptide (Met1to Glu25of E1B or Met41to Glu65of E1A) and E1AB-C peptide (His1030to Arg1058of E1A or His990to Arg1018of E1B) reacted with both E1A and E1B. These results indicate that (1) E1A and E1B contain the same C-terminal residues; (2) E1A contains the N terminal sequence of E1B; and (3) E1B does not contain the N terminal sequence of E1A. The two isoforms of lens E1 are therefore translated from a single mRNA. Specifically, E1A is translated from the first initiation codon, and E1B translated from the second initiation codon. E1A and E1B were affinity-purified, and their ability to ‘charge’ ubiquitin carrier proteins (E2s) with activated ubiquitin was compared in a cell-free system. E1A and E1B were indistinguishable with respect to charging different E2s. However, E1 immunolocalization studies with human lens epithelial cells indicate that E1A and E1B are preferentially localized to the nucleus and cytosol, respectively. This observation suggests that E1A and E1B ubiquitinate different proteins and serve different functions in intact cells.
Calreticulin has mutiple functions, diverse cellular locations, and putative isoforms. It likely maintains integrin avidity by binding α integrin cytoplasmic tails and is a surface lectin which triggers cell spreading. In the present study, we have immunocaptured a cell surface complex from B16 mouse melanoma cells which contains α6β1integrin, two molecular forms of calreticulin, and KDEL docking protein (KDEL-R). One of the calreticulins, “endocalreticulin”, a 52 kDa protein, does not become surface biotinylated, and is probably bound to α integrin cytoplasmic tails; it disappears when B16 cells adhere to laminin, and two ubiquitinated calreticulins appear. One ubiquitinated species, a 125 kDa protein, is restricted to focal contacts whereas a second species, a 75 kDa protein, is in focal contacts and surrounding plasma membrane; it also arises when cells bind non-specific surfaces. The other calreticulin, “ectocalreticulin”, a 62 kDa protein, becomes surface biotinylated, is probably anchored to surface KDEL-R, and cooperates with α6β1integrin, triggering cell spreading. The present results suggest a model in which calreticulin-integrin surface complex functions as a symbiotic unit, transmitting information in both directions across the plasma membrane.
Synaptic terminals are sites of high metabolic activity and thus are particularly vulnerable to oxidative stress. Oxidative damage to proteins can be toxic to neurons and may cause irreversible cell damage and neurodegeneration. A neuroprotective mechanism used by cells to combat oxidative damage is to selectively degrade damaged proteins. Therefore, it is of interest to study the mechanism of degradation of oxidatively damaged proteins in synaptosomes. One way of oxidizing synaptosomal proteins in vitro is by incubating intact synaptosomes in the presence of an oxidizing agent. A problem with this approach is that it may also cause oxidative damage to the machinery required to recognize and degrade oxidized proteins. We have, therefore, introduced a fluorescent macromolecule into synaptosomes to assess the feasibility of using this technique to study how oxidized proteins are degraded and removed from synaptic terminals. Synaptosomes were subjected to electroporation in the presence of FITC labelled-dextran with an average molecular weight of 70 000 (FD-70) and non-specific binding was determined by running parallel experiments in lysed synaptosomes. Following extensive washing, synaptosomes were assayed for the presence of intra-synaptosomal FD-70 by measuring fluorescence in a microplate fluorescence reader. Significant differences in fluorescence were found between intact and lysed synaptosomes with maximal uptake at 100 V/ 1500 μF (approx. 36 pmol/mg protein). To determine if membrane transport was compromised by electroporation, uptake of 3H-arginine was compared in control and electroporated synaptosomes. While untreated electroporated synaptosomes showed a loss of 22% in the ability to transport arginine, preincubation in the presence of 1 mM ATP resulted in a complete restoration of arginine transport. These results show that electroporation is a potentially useful technique for introducing a specific oxidized protein, into synaptic terminals so its metabolic fate can be examined.
In animal chromatin, a fraction of the histone 2A's and 2B's is covalently attached to the protein ubiquitin through an isopeptide linkage. The ubiquitin moieties of the H2A's and H2B's are found to be in rapid equilibrium with the pool of free ubiquitin, both in dividing cells such as L1210 and Chinese hamster ovary cells and in nondividing cells such as unstimulated lymphocytes. The synthesis of ubiquitin and the formation of ubiquitinated histones are not linked to DNA synthesis. All ubiquitinated histones, including the four H2A variants and the H2B's are absent from isolated metaphase chromosomes.
We recently reported (Rogers, S. W., and Rechsteiner, M. (1988) J. Biol. Chem. 263, 19833–19842) the metabolic stabilities of 35 structurally characterized proteins following their injection into HeLa cells. In this study 22 proteins from that set were radioiodinated, and their susceptibilities to proteolysis were measured in reticulocyte lysate. Degradation rates varied from less than 1% per h to almost 25% per h. ATP stimulated the degradation of 21 of the proteins with nucleotide enhancement typically in the range 2- to 3-fold. When structural features of the 22 proteins were compared with their degradation rates in lysate, no correlation was found with respect to charge, size, thermal stability, or N-terminal acylation. Furthermore, relative rates of proteolysis in lysate correlated only marginally with the metabolic stabilities of the 22 proteins as measured 24–48 h after injection into HeLa cells. Degradation rates for the 22 proteins in lysate did, however, show a strong correlation with their rates of turnover immediately after injection into the human cells. Since the enhanced proteolysis observed for many proteins soon after injection is thought to reflect disruption of HeLa cytoskeletal assemblies, this correlation provides further evidence that diffusibility or location can affect intracellular protein stability.
It was found previously that the enzyme ubiquitin-protein ligase (E3) contains specific protein substrate binding sites that are responsible for the selection of proteins for degradation by the ubiquitin system. In the present study, we have tried to gain more insight into the mode of action of E3 by the characterization of other binding sites of this enzyme. Following the ligation of ubiquitin to ¹²⁵I-lysozyme, the conjugates produced are very tightly bound to E3,as indicated by size analysis on glycerol density gradient centrifugation. The strong binding of ubiquitin-protein conjugates to the enzyme may account for the apparently processive addition of multiple molecules of ubiquitin to the protein substrate. Both the protein substrate moiety and the ubiquitin moiety participate in the interaction of ubiquitin-protein conjugates with E3, as indicated by competition with specific agents and by the comparison of the binding of ubiquitin-conjugated protein to that of free protein.
In addition to the binding of its substrates and products, E3 also appears to interact with some of the enzymes with which it acts in concert. When E3 is incubated with the ubiquitin-carrier protein E2,a complex is formed between the two enzymes as analyzed on glycerol gradients. The formation of an E2•E3 complex may facilitate the transfer of activated ubiquitin from E2 to the protein substrate bound to the ligase.
The plant photoreceptor chromoprotein, phytochrome, is rapidly degraded in vivo after photoconversion from a stable red light-absorbing form (Pr) to a far-red light-absorbing form (Pfr). Previously, we demonstrated that during Pfr degradation in etiolated oat seedlings, ubiquitin-phytochrome conjugates, (Ub-P), appear and disappear suggesting that phytochrome is degraded via a ubiquitin-dependent proteolytic pathway (Shanklin, J., Jabben, M., and Vierstra, R. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 359–363). Here, we provide additional kinetic and localization data consistent with this hypothesis by exploiting the unique ability to photoregulate phytochrome degradation in vivo. An assay for the quantitation of Ub-P was developed involving immunoprecipitation of total conjugates with anti-ubiquitin antibodies, followed by the detection of Ub-P with anti-phytochrome antibodies. Using this immunoassay, we found that Ub-P will accumulate to approximately 5% of initial phytochrome during Pfr degradation induced by a saturating red light pulse. Reducing the amount of Pfr produced initially by attenuating the red light pulse, lowered the amount of phytochrome degraded in the following dark period and concomitantly reduced the maximal accumulation of Ub-P. Continuous far-red irradiations that maintained only 4% of phytochrome as Pfr induced rapid phytochrome degradation similar to that induced by a red light pulse converting 86% of Pr to Pfr. The appearance and disappearance of Ub-P were similar for each irradiation indicating that Ub-P accumulation is independent of the level of Pfr provided rapid phytochrome degradation is maintained. Pulse-chase studies employing continuous far-red light followed by darkness showed that Ub-P are continuously synthesized during phytochrome degradation and rapidly disappear once degradation ceases. Ub-P also accumulated during “cycled Pr” degradation induced by the transformation of Pr to Pfr and back to Pr. The commitment to degrade cycled Pr and form Ub-P occurred within seconds after Pfr formation making the cause(s) underlying this phenomenon one of the fastest phytochrome reactions known. Within seconds after Pfr formation, a majority of phytochrome is also known to aggregate in vivo (previously defined as sequestered or pelletable), with aggregated phytochrome preferentially lost during phytochrome degradation. In vitro analysis of aggregated phytochrome indicated that they contain most of the Ub-P. Moreover, the appearance of Ub-P in the aggregated and soluble fractions correlated with the time that phytochrome disappeared from that fraction.(ABSTRACT TRUNCATED AT 400 WORDS)
Previous studies have indicated that at least part of the selection of proteins for degradation takes place at a binding site on ubiquitin-protein ligase, to which the protein substrate is bound prior to ligation to ubiquitin. It was also shown that proteins with free NH2-terminal alpha-NH2 groups bind better to this site than proteins with blocked NH2 termini (Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999). In the present study, we used simple derivatives of amino acids, such as methyl esters, hydroxamates, or dipeptides, to examine the question of whether the protein binding site of the ligase is able to distinguish between different NH2-terminal residues of proteins. Based on specific patterns of inhibition of the binding to ligase by these derivatives, three types of protein substrates could be distinguished. Type I substrates are proteins that have a basic NH2-terminal residue (such as ribonuclease and lysozyme); these are specifically inhibited by derivatives of the 3 basic amino acids (His, Arg, and Lys) with respect to degradation, ligation to ubiquitin, and binding to ligase. Type II substrates (such as beta-lactoglobulin or pepsinogen, that have a Leu residue at the NH2 terminus) are not affected by the above compounds, but are specifically inhibited by derivatives of bulky hydrophobic amino acids (Leu, Trp, Phe, and Tyr). In these cases, the amino acid derivatives apparently act as specific inhibitors of the binding of the NH2-terminal residue of proteins, as indicated by the following observations: (a) derivatives in which the alpha-NH2 group is blocked were inactive and (b) in dipeptides, the inhibitory amino acid residue had to be at the NH2-terminal position. An additional class (Type III) of substrates comprises proteins that have neither basic nor bulky hydrophobic NH2-terminal amino acid residues; the binding of these proteins is not inhibited by homologous amino acid derivatives that have NH2-terminal residues similar to that of the protein. It is concluded that Type I and Type II proteins bind to distinct and separate subsites of the ligase, specific for basic or bulky hydrophobic NH2-terminal residues, respectively. On the other hand, Type III proteins apparently predominantly interact with the ligase at regions of the protein molecule other than the NH2-terminal residue.
The three-dimensional structure of ubiquitin has been determined at 2.8 angstrom resolution. X-ray diffraction data for the native protein and derivatives were collected with an automated diffractometer. Phases were obtained by use of a single isomorphous mercuric acetate derivative. The molecule contains a pronounced hydrophobic core. Prominent secondary structural features include three and one-half turns of alpha -helix, a mixed beta -sheet that contains four strands, and seven reverse turns. The histidine, tyrosine, and two phenylalanine residues are located on the surface of the molecule.
The arginyl transfer ribonucleic acid-protein transferase has been purified nearly 7000-fold from the supernatant fraction
of rabbit liver cytoplasm by a procedure including ammonium sulfate fractionation, precipitation at pH 5.2, chromatography
on carboxymethyl cellulose, and chromatography on diethylaminoethyl cellulose. The purified preparations show one major and
two minor components in polyacrylamide disc gel electrophoresis and 1 mg of protein catalyzes the transfer at 37° of approximately
135 nmoles of arginine per min from tRNA to the amino-terminal aspartic acid residue of bovine serum albumin. Arginine can
be quantitatively transferred from tRNA to protein and is the only amino acid which participates in this reaction. A sulfhydryl
compound is required and maximal rates are obtained at pH 9 with 0.1 m 2-mercaptoethanol or 10 mm dithiothreitol. There is also a requirement for a monovalent cation with an optimal reaction occurring at 0.2 m KCl.
Transfer is completely dependent upon the presence of a suitable acceptor protein. When bovine serum albumin is used in limiting
amounts, 1 molecule of arginine is transferred from tRNA for each molecule of albumin. Bovine thyroglobulin appears to accept
2 molecules of arginine per molecule. Under similar conditions other proteins were far less effective as acceptors.
Radioiodinated histone H3 was incubated with ubiquitin, the ubiquitin-activating enzyme E1, and one of three ubiquitin carrier proteins, reticulocyte E2(20K) or E2(32K) or the yeast RAD6 product. Although the resulting ubiquitin-histone conjugates were synthesized in the absence of the substrate-binding protein E3, they were nevertheless degraded by purified rabbit reticulocyte 26 S protease. In contrast, unmodified histone H3 remained intact upon challenge with the 26 S ubiquitin/ATP-dependent enzyme. Conjugates produced by the RAD6 protein were better proteolytic substrates than those formed by reticulocyte E2 unless ubiquitin molecules with altered lysines were used for conjugate synthesis. Substitution of methylated ubiquitin or ubiquitin molecules in which lysine 48 was converted to arginine by site-directed mutation produced histone conjugates that were degraded at slow but measurable rates. Since methylated ubiquitin molecules are incapable of forming branched polyubiquitin chains, these results demonstrate that neither ubiquitin "trees" nor the substrate binding factor E3 is absolutely required for ubiquitin-dependent degradation of histone H3 in vitro.
Using exogenous 125I-ubiquitin, ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lens epithelial cells and lens cortex tissue. Conjugation was ATP-dependent with the greatest variety and amount of conjugates larger than 150 kDa. In vivo production of ubiquitin-protein conjugates in cultured rabbit and beef lens epithelial cells and rabbit lens tissues of different developmental age was established using immunological detection. There were limited similarities between conjugates found in youngest as opposed to oldest tissue. Cultured rabbit cells contained 27 pmol/mg free ubiquitin and 18 pmol/mg conjugated ubiquitin. Levels of free ubiquitin in lens tissue epithelium, cortex, and core were 36, 5, and 5 pmol/mg, respectively. There were only 2 pmol/mg conjugated ubiquitin in each of these tissues. Hydrolysis of 125I-ubiquitin was catalyzed by supernatants of cultured lens cells, beef and human lens tissues, and reticulocytes. Degradation was greatest in epithelial tissues, and least in core. This corroborates studies which show that proteolytic capabilities are attenuated in older tissue. Decreased initiation of proteolysis by ubiquitination as well as diminished proteolysis in older lens tissue may be related to the accumulation of damaged proteins in aging lens tissue.
Previous studies in a cell-free proteolytic system from reticulocytes indicated that the conjugation of ubiquitin with proteins plays a role in protein breakdown. To examine some of the physiological functions of the ubiquitin conjugation system, and immunochemical method was developed for the isolation of ubiquitin-protein conjugates from intact cells. A specific antiserum was raised against ubiquitin and purified by affinity chromatography on ubiquitin-Sepharose. When cells are labeled with tryptophan (which is missing from ubiquitin), labeled immunoreactive material isolated by the antibody is derived from the protein moiety of ubiquitin-protein conjugates. There is a marked increase in the labeling of ubiquitin-protein conjugates during the formation of abnormal proteins in reticulocytes (induced by the incorporation of amino acid analogs), suggesting that proteins with abnormal structure are more readily conjugated to ubiquitin than most normal proteins. Essentially similar, although less marked, effects of amino acid analogs were observed in Ehrlich ascites cells. When further protein synthesis was blocked with cycloheximide, ubiquitin conjugates decayed more extensively than the corresponding average labeled cellular proteins. This is consistent with the interpretation that a considerable part of ubiquitin conjugates is derived from a pool of rapidly degradable proteins.
The recently characterized ubiquitin activating enzyme catalyzes the first step in ubiquitin-protein isopeptide bond formation and as uch may be central to a
Calmodulin has been radioiodinated by the Bolton-Hunter procedure. This procedure results in incorporation of 1.5 mol of 1b25I/mol of protein and yields a specific radioactivity of 2400 Ci/mmol. The radioiodinated calmodulin retains complete biological activity as determined by its ability to activate calmodulin-deficient cyclic nucleotide phosphodiesterase from rat brain. A sensitive radioimmunoassay for calmodulin has been developed using this 125I-calmodulin as tracer. The assay exhibits a limit of detection of 15 pg and an assay sensitivity of 115 pg with intra- and interassay variabilities of <3% and <5%,. respectively. Purified calmodulins from bovine brain, rat testis, Renilla reniformis (sea pansy) and Arachis hypogaea (peanut) demonstrate identical immunological cross-reactivities. These findings support the structural data revealing the highly conserved amino acid sequence of this protein. A homologous calcium-binding protein, troponin C, required a 665-fold greater protein concentration to cause 50% reduction in binding of 125I-calmodulin. The slope of the troponin C competition curve was different than that for calmodulin suggesting divergence between these two proteins. No immunological cross-reactivity was observed by the muscle calcium-binding protein, parvalbumin, at 50,000-fold excess protein. Adaptation of the radioimmunoassay to cell and tissue extracts reveals that calmodulin levels in various tissues and cells were always greater when assayed by the radioimmunoassay as compared to the phosphodiesterase assay.
Reticulocytes, like other cells, selectively degrade certain abnormal proteins by an energy-dependent process. When isolated rabbit reticulocytes incorporate the valine analog 2-amino-3chlorobutyric acid (ClAbu) in place of valine, they produce an abnormal globin that is degraded with a half-life of 15 min. Normal hemoglobin, in contrast, undergoes little or no breakdown within these cells. Cell-free extracts from reticulocytes have been shown to rapidly hydrolyze these abnormal proteins. The degradative system is located in the 100,000 X g supernatant, has a pH optimum of 7.8, and does not appear to be of lysosomal origin. This breakdown of analog-containing protein was stimulated severalfold by ATP, and slightly by ADP. AMP and adenosine-3':5'-cyclic monophosphate had no significant effect on proteolysis. Experiments with ATP analogs suggest that the terminal high energy phosphate is important in the degradative process. Proteolysis in the cell-free system and in intact reticulocytes was inhibited by the same agents (L-l-tosylamido-2-phenyl-ethylchloromethyl ketone, N-alpha-p-tosyl-L-lysine chloromethyl ketone, N-ethylmaleimide, iodoacetamide, and o-phenanthroline). In addition, the relative rates of degradation of several polypeptides in the cell-free extracts paralleled degradatives rates within cells. Thus, a soluble nonlysosomal proteolytic system appears responsible for the energy-dependent degradation of abnormal proteins in reticulocytes.
Chromosomal protein A24 has a unique structure inasmuch as it contains histone 2A and a nonhistone polypeptide the sequence of which has been partially determined. Comparative analysis of the ninhydrin-insensitive amino-terminal tryptic peptides of protein A24 and histone 2A and a quantitative analysis of their carboxyl-terminal amino acid indicated that protein A24 has two amino termini and one carboxyl terminus. The amino acid sequence analysis of tryptic peptide 17' of protein A24: (see text) showed it contains tryptic peptide 17 of histone 2A, Lys-Thr-Glu-Ser-His-His-Lys. Lysine 119, the amino terminus of this peptide, which is derived from the histone 2A portion of protein A24, is linked by an isopeptide bond to the carboxyl group of a glycine residue. Accordingly, the branched structure of protein A24 proposed is: (see text).
Immunochemical techniques have been utilized to study the effect of thyroid status on the content and rates of synthesis and degradation of pyruvate carboxylase and pyruvate dehydrogenase in rat liver. Liver from hyperthyroid rats had twice the pyruvate carboxylase activity of normal rats while thyroidectomized rats had about two-thirds of normal activity. Pyruvate dehydrogenase complex activity was unchanged in the hyperthyroid state but was significantly reduced (by a third) in hypothyroid rats. Changes in catalytic activity during altered thyroid status were by immunochemical means to be closely related to the amount of the hepatic enzymes present. Isotopic studies showed that the changes in the content of pyruvate carboxylase and pyruvate dehydrogenase reflected alterations in the rate of the synthesis of the enzymes with the degradation rates little affected by thyroid status. The half-life for pyruvate carboxylase was 4.6 days, and that for pyruvate dehydrogenase, 8.1 days. In both cases, the turnover time was slower than that of the average mitochondrial protein (t1/2 = 3.8 days) for the control animals.
A parametric correlation (p less than 0.01) has been found between the in vitro thermal stability and in vivo turnover rates of nine intracellular proteins. These results are discussed in terms of a "thermodynamic" model for turnover control, in which the rate of intracellular protein degradation is controlled by intramolecular conformation equilibria. A peculiar exception is provided by glyceraldehyde-3-phosphate dehydrogenase which is stable in vivo, but not in vitro.
The E6 protein of human papillomavirus types 16 and 18 (HPV‐16 and HPV‐18) can stably associate with the p53 protein in vitro. In the presence of rabbit reticulocyte lysate, this association leads to the specific degradation of p53 through the ubiquitin‐dependent proteolysis system. We have examined the E6‐p53 complex in more detail and have found that association of E6 with p53 is mediated by an additional cellular factor. This factor is present in rabbit reticulocyte lysate, primary human keratinocytes and in each of five human cell lines examined. The factor is designated E6‐AP, for E6‐associated protein, based on the observation that the E6 proteins of HPV‐16 and 18 can form a stable complex with the factor in the absence of p53, whereas p53 association with the factor can be detected only in the presence of E6. Gel filtration and coprecipitation experiments indicate that E6‐AP is a monomeric protein of approximately 100 kDa.
Multiubiquitination of proteins is a critical step leading to selective degradation for many polypeptides. Therefore, activation‐induced multiubiquitination of cell surface receptors, such as the platelet‐derived growth factor (PDGF) receptor and the T cell antigen (TCR) receptor, may correspond to a degradation pathway for ligand‐receptor complexes. Here we show that the antigen‐induced engagement of high‐affinity immunoglobulin E receptors (Fc epsilon RI) results in the immediate multiubiquitination of Fc epsilon RI beta and gamma chains. This ubiquitination is independent of receptor phosphorylation and is restricted to activated receptors. Surprisingly, receptor multiubiquitination is immediately reversible when receptors are disengaged. Therefore, multiubiquitination and deubiquitination of Fc epsilon RI receptors is controlled at the cell surface by receptor engagement and disengagement. The rapidity, specificity and, most importantly, the reversibility of the activation‐induced receptor multiubiquitination suggest that this process may turn on/off a cell surface receptor signaling function thus far unsuspected.
Ubiquitinated histone H2B (uH2B) has been purified from both calf and pig thymus by exclusion chromatography in 7 M urea. Digestion of uH2B with Staphylococcus aureus V8 protease yielded the peptide 114‐125 containing the ubiquitin moiety. Further digestion of this peptide with trypsin removed the ubiquitin and three H2B residues from the N‐terminus. Edman degradations of both peptides established that ubiquitin is attached to the epsilon‐amino group of lysine 120 in both calf and pig uH2B by an iso‐peptide bond to the C‐terminal glycine 76 of ubiquitin.
The multiple biological functions of the small polypeptide ubiquitin are mirrored by its unparalleled conservation on the amino acid and gene organization level.
During the last years, it has become widely accepted that ubiquitin is an essential component in the ATP‐dependent nonlysosomal protein degradation pathway occurring in all eukaryotic organisms. As turnover, consisting of protein synthesis and disassembly, is a central and vital process for each living cell, ubiquitin‐mediated proteolysis is of enormous physiological value. The components of the ubiquitin ligation system have been characterized skillfully in plant and animal cells, but at the moment many questions remain as to how the high degree of specificity that is necessary for the regulation of intracellular breakdown is ensured. The recent hypotheses and models proposed for the basic mechanisms of protein recognition, conjugation and degradation will be discussed in detail.
The existence of ubiquitin‐protein conjugates which are not rapidly degraded clearly suggested that the role of ubiquitin is not restricted in its implication for protein turnover. Alterations of DNA structure, specific cell recognition mechanisms and cytoskeletal variations were observed as further ubiquitin‐dependent processes which are not directly coupled to protein degradation.
Excerpt
The N-end rule relates the in vivo half-life1 of a protein to the identity of its N-terminal residue (Varshavsky 1992). Similar but distinct versions of the N-end rule have been shown to operate in all organisms examined, from mammals to fungi and bacteria. I summarize the current understanding of the N-end rule pathway and describe some of the recent methods that utilize the N-end rule.
Features of a protein that confer metabolic instability are called degradation signals, or degrons (Varshavsky 1991). The essential component of one degron, the first to be identified, is a destabilizing N-terminal residue of a protein (Bachmair et al. 1986). This signal is called the N-degron. The N-end rule (defined above) results from the existence of N-degrons containing different destabilizing residues (Varshavsky 1992). In eukaryotes, the N-degron comprises two determinants: a destabilizing N-terminal residue and an internal lysine (or lysines) of a substrate. The lysine residue...
Ubiquitin is a highly conserved protein involved in several important regulatory processes through its ATP-dependent, covalent ligation to a variety of eukaryotic target proteins. The authors describe here the characterization of ubiquitin conjugation in wheat germ extracts and the subsequent isolation of enzymes involved in conjugation. With {sup 125}I-ubiquitin as a substrate, wheat germ extracts form conjugates with either endogenous or added proteins. Ubiquitin-activating enzyme (E1) was purified from wheat germ extracts by using a modification of the covalent affinity chromatography procedure of Ciechanover et al. E1 from wheat germ, like that from rabbit reticulocytes, formed thiol ester intermediates with ubiquitin in the presence of ATP. Purified E1 preparations contained three polypeptides of apparent molecular masses of 117, 123, and 126 kDa after NaDodSO{sub 4}-PAGE. Under nondenaturing conditions, these proteins have native molecular masses of {approx}115 kDa, indicating that they exist as monomers. They concluded that all three species were E1 on the basis of their coelution with E1 activity, by immunorecognition by anti-human E1 antibodies, and by the similarity of their peptide maps. Furthermore, antibodies prepared against wheat germ E1's recognized E1 from rabbit reticulocytes. All three wheat germ E1's were detected in crude extracts prepared under conditions that minimized proteolysis, suggesting that the heterogeneity of the purified E1 preparations was not the result of posthomogenization breakdown. The immunological similarity of animal and plant E1's indicates that this conjugation enzyme, like ubiquitin, has been conserved through evolution.
Ubiquitin is a protein found in eukaryotic cells either free or covalently joined to a variety of cytoplasmic and nuclear proteins. Recent biochemical and genetic evidence indicated that conjugation of ubiquitin to short-lived proteins is essential for their selective degradation (in vivo). Also discussed here are recent mechanistic studies of ubiquitin-dependent proteolysis, the unusual organization of the ubiquitin gene, and putative roles of the ubiquitin system in chromosome function and the stress response.
Ubiquitin has been suggested to play a key role in a wide variety of essential cellular functions ranging from differential regulation of gene expression to protein degradation. Recent studies on natural and synthetic ubiquitin gene fusions have led to important discoveries concerning novel functions for the ubiquitin system in cells, mechanisms of proteolytic processing, and the development of a ubiquitin fusion technology for augmenting the expression of heterologous gene products in bacteria and yeast. Furthermore, studies involving site-directed mutagenesis and two-dimensional NMR have proven ubiquitin to be an excellent model for protein engineering and have led to important discoveries concerning its mechanism of action in ATP-dependent proteolysis. Finally, the recent identification and characterization of ubiquitin carboxyl extension proteins as ribosomal proteins has opened up an even newer area of ubiquitin-related research and has helped to explain the mechanisms involved in increasing the expression of heterologous gene products made as ubiquitin fusions.
Phytochrome is rapidly degraded in vivo following photoconversion from the relatively stable red light absorbing form Pr to the far red light absorbing form Pfr. In etiolated seedlings from several species, this photoconversion also induces the accumulation of ubiquitin-phytochrome conjugates (Ub-P), suggesting that Pfr is degraded via a ubiquitin-dependent proteolytic pathway. To understand why Pfr is preferentially conjugated with ubiquitin, Ub-P were partially purified and characterized with the ultimate goal of mapping ubiquitin attachment sites. Ub-P were partially purified by poly(ethylene imine) and ammonium sulfate precipitations followed by hydroxyapatite chromatography and separated from unmodified phytochrome by size-exclusion chromatography. Up-P had an apparent native molecular size of approximately 600 kDa, substantially larger than that of the unmodified Pfr dimer (365 kDa). Ub-P retained the property of spectral photoreversibility. The initial digestion patterns of Ub-P were similar to unmodified phytochrome. In an attempt to identify ubiquitin attachment site(s), Ub-P were probed with a library of anti-oat phytochrome monoclonal antibodies. Of the 16 different anti-phytochrome monoclonal antibodies tested, 3 (O76C, O19F, and O311B) poorly recognized all size classes of Ub-P, indicating that the corresponding epitopes were masked either directly or indirectly as a result of ubiquitin ligation. These epitopes are located between residues 90 and 180 (O76C), 558 and 668 (O19F), and 747 and 830 (O311B) of oat phytochrome. Because the regions recognized by O19F and O311B are near the C-terminal domain containing at least one ubiquitin attachment site, and near amino acid residues that become more accessible when the chromoprotein is in the Pfr form, these regions may be important in the Pfr-dependent ubiquitination of phytochrome.
The structural chromatin protein A24 (uH2A) is a conjugate of histone H2A and a non-histone protein, ubiquitin. Eukaryotic cells contain an enzyme, generically termed isopeptidase, which can cleave A24 stoichiometrically into H2A and ubiquitin in vitro. Isopeptidase, free of proteinase activity, has been partially purified from calf thymus by ion-exchange chromatography, gel filtration and affinity chromatography, and analyzed for its substrate specificity. There are three major types of isopeptide bonds besides the ε-(α-glycyl)lysine bond between H2A and ubiquitin; namely, the disulfide bridge, the aldol and aldimide bonds and the ε-(γ-glutamyl)lysine crosslink. Under conditions where A24 was completely cleaved into H2A and ubiquitin, none of these naturally occurring isopeptide bonds was cleaved by isopeptidase. Furthermore, the bonds formed in vitro by transglutaminase reaction between casein and putrescine, through the γ-NH2 of glutamine residue and the NH2 of putrescine, were not cleaved by the enzyme. The enzyme also failed to cleave the glycyl-lysyl and other orthodox peptide linkages within proteins. Among various proteins examined, the substrates for isopeptidase reaction were confined to conjugates between ubiquitin and other proteins, formed through ε-(α-glycyl)lysine bonds. Since ubiquitin released by isopeptidase is re-usable for an ATP-dependent conjugation with other proteins, its carboxyl terminal -Gly-Gly-COOH most likely is preserved intact, and is not blocked. These results suggest that isopeptidase specifically recognizes and cleaves the ε-(α-glycyl)lysine bond. A possible biological significance of this enzyme is discussed.
Ubiquitin modification of a variety of protein targets within the cell plays important roles in many cellular processes. Among these are regulation of gene expression, regulation of cell cycle and division, involvement in the cellular stress response, modification of cell surface receptors, DNA repair, and biogenesis of mitochondria and ribosomes. The best studied modification occurs in the ubiquitin-dependent proteolytic pathway. Degradation of a protein by the ubiquitin system involves two discrete steps. Initially, multiple ubiquitin molecules are covalently linked in an ATP-dependent mode to the protein substrate. The protein moiety of the conjugate is then degraded by a specific protease into free amino acids with the release of free and reutilizable ubiquitin. This process also requires energy. In addition, stable mono-ubiquitin adducts are also found intracellularly, for example, those involving nucleosomal histories. Despite the considerable progress that has been made in elucidating the mode of action and roles of the ubiquitin system, many problems remain unsolved. For example, very little is known about the cellular substrates of the system and the signals that target them for conjugation and degradation. The scope of this review is to summarize briefly what is currently known on the role of the ubiquitin system in protein turnover, and to discuss in detail the mechanisms involved in selection of substrates for conjugation and in degradation of ubiquitin-conjugated proteins.
Der Eiweißumsatz von Organismen, Zellorganellen und einzelnen Proteinen wird mit der Fähigkeit der Zellfraktionen zur Autoproteolyse verglichen. Dabei wird näher auf das Beispiel der Rattenleber eingegangen und gezeigt, daß in vivo nur ein Zusammenwirken verschiedener Zellräume zur intrazellulären Proteolyse führen kann. Die Rolle der Lysosomen, des intrazellulären pH und einer intrazellulären Denaturierung werden zu bisher nachgewiesenen intrazellulären Endopeptidasen in Beziehung gesetzt. Deren Spezifität wird mit der anderer Peptidasen verglichen. Es wird auf Inhibitoren und die Energieabhängigkeit der Proteolyse hingewiesen. Unterschiede im Anteil oberflächlicher hydrophober Gruppen können eine selektive Autophagie und damit die differenten Umsatzraten verschiedener intrazellulärer Proteine in vivo bedingen. Autophagierte Eiweiße werden durch die Endopeptidasen der Partikelfraktionen zu Peptiden gespalten. Es folgt eine rasche Umwandlung zu freien Aminosäuren durch die hohen Exopeptidaseaktivitäten des Cytosols.
Diese Vorgänge gehören zu den Grundvoraussetzungen für Regulationen des Enzymbestands der Zellen höherer Organismen. Bisher vermitteln die Untersuchungen jedoch erst ein unvollständiges Bild zum Ablauf der intrazellulären Proteolyse, bei dem besonders die Prinzipien ihrer Regulation noch weitgehend ungeklärt sind.
1.1. Two rabbits were injected with an intravenous dose of phenylalanine-3-14C. From these rabbits the following proteins were crystallized or isolated in forms which appeared physically homogeneous: (rabbit-A), aldolase, myosin, L-meromyosin, H-meromyosin, actin, tropomyosin, and serum albumin: (rabbit-B) glyceraldehyde-3-phosphate dehydrogenase, aldolase, actin, tropomyosin, and serum albumin.2.2. Pure phenylalanine and tyrosine, labelled metabolically, were isolated from hydrolysates of these proteins and the specific radioactivities of the amino acids were determined.3.3. From the specific activity ratios, phenylalanine to tyrosine, it seemed likely that H-meromyosin, actin, and the glycolytic enzymes were synthesized from the same acid precursor pools.4.4. The specific activities of amino acids in L- and H-meromyosins were significantly different. It is suggested that subunits of more than one kind in myosin are synthesized independently and turn over metabolically at different rates.5.5. The apparent half-lives of phenylalanine in the individual muscle proteins were estimated by comparison with serum albumin the approximate half-life of which is known. The validity of the assumptions involved in making such comparisons is discussed.
The eye lens is a useful tissue for studying phenomena related to aging since it can be separated into differentially aged or matured zones. This work establishes correlations between ubiquitin-lens protein conjugating capabilities and age, as well as the stage of maturation of bovine lens tissue. When exogenous 125I-ubiquitin was combined with supernatants of epithelial (least mature), cortex, and core (most mature) tissue, ATP-dependent conjugation of 125I-ubiquitin to lens proteins was most effective with the epithelial tissue preparation. Conjugate formation was greatest when lenses were obtained from young animals. Supernatants from cultured bovine lens epithelial (BLE) cells conjugated more 125I-ubiquitin to lens proteins than any tissue preparation. In all cases the predominant conjugates formed in these cell-free assays were of high molecular mass, although conjugates with masses in the 25–70 kDa range were also observed. Lens tissue and cultured BLE cell preparations were also probed with antibodies to ubiquitin to detect in vivo ubiquitin-lens protein conjugates. There was more free ubiquitin and ubiquitin conjugates in tissue from young as compared with older lenses. The greatest levels of conjugates were observed in cultured BLE cells. Specificity in the ubiquitination system is indicated since some of the conjugates formed in vivo appear identical to those formed in the cell-free assays and in reticulocytes using exogenous 125I-ubiquitin. Upon development and maturation of lens tissue (i.e., core as opposed to epithelium), there is accumulation of lower molecular mass conjugates.
Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis. We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein. CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI. Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer. Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability. The deamidated enzyme was more susceptible to proteases and denaturing conditions. Subtilisin cleaved the rabbit enzyme primarily at the Thr139Glu140 bond. The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity. The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation. The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme.
Three iodinated derivatives of ubiquitin have been synthesized and these derivatives have been characterized in the ubiquitin-dependent protein degradation system. With chloramine-T as the oxidant, a derivative containing monoiodotyrosine is formed in the presence of 1 m KI and a derivative containing diiodotyrosine is produced in the presence of 1 mm KI. These derivatives exhibit phenolate ionizations at pH 9.2 and 7.9 with absorbance maxima at 305 and 314 nm, respectively. In addition to modification of the tyrosine residue, these conditions lead to the oxidation of the single methionine residue and iodination of the single histidine residue [M. J. Cox R. Shapira, and K.D. Wilkinson (1986) Anal. Biochem.154, 345–352]. Iodination of ubiquitin under these conditions renders the protein sensitive to hydrolysis by trypsin and results in an enhanced susceptibility to alcohol-induced helix formation. When the derivatives are tested in the ATP: pyrophosphate exchange reaction catalyzed by the ubiquitin adenylating enzyme, they are found to exhibit activity comparable to the native protein. When these derivatives are tested for the ability to act as a cofactor in the ubiquitin-dependent protein degradation system, they are both found to support a rate of protein degradation that is twice that of native ubiquitin. At high concentrations of derivatives, the rate of protein degradation is inhibited, while the steady state level of conjugates increases. Thus, the free derivatives inhibit the protease portion of the reaction, but are fully active in the activation and conjugation portions of the reaction. With iodine as the modification reagent, monoiodination of tyrosine is the predominant reaction. This derivative exhibits activity similar to native ubiquitin. Thus, it appears that modification of the histidine residue is responsible for the increased activity of the more highly iodinated derivatives. The enzymes of the system must recognize different portions of the ubiquitin structure, or different conformations of ubiquitin that are affected by the iodination of the histidine residue. These results suggest a conformational change of the ubiquitin molecule may be important in determining the rate and specificity of proteolysis.
Lens cells must remove obsolete or damaged proteins produced during development, maturation and aging to maintain lens transparency. In reticulocytes removal of abnormal or obsolete proteins is thought to involve a ubiquitin-dependent proteolytic pathway. Two hallmarks of ubiquitin (Ub) dependent proteolysis have previously been demonstrated in lens cell or tissue supernatants: (1) the presence of ubiquitin conjugates, and (2) ATP-dependent proteolysis. Nevertheless, conclusive proof was lacking of a requirement for ubiquitination of substrate proteins for proteolysis. Here we show that in bovine lens epithelial cell (BLEC) supernatant, ATP-dependent proteolysis is also ubiquitin-dependent. Ubiquitin-activating enzyme (E1), the first enzyme in the cascade of ubiquitin ligation, was purified over 3000-fold from a rabbit reticulocyte lysate using Ubiquitin-Sepharose, and showed ATP-PPi exchange activity. Antiserum to E1 was prepared in goats and affinity-purified on Protein G-Sepharose. Western blot analysis revealed that both the goat antiserum and purified antibody (anti-E1(IgG)) recognize specifically E1. Anti-E1(IgG) inhibits 86% of the ATP-dependent degradation of labeled histone H2A in reticulocyte lysate and 75% of the ATP-dependent degradation in BLEC. Upon reconstitution with purified E1, 100% and 80% of proteolysis was restored in reticulocytes and BLEC supernatant, respectively. This confirms that there is a ubiquitin-dependent proteolytic system in lens.
The existence of three different kinds of group specific proteases for pyridoxal enzymes was proved in skeletal muscle, muscle layer and mucous layer of rats, respectively. These enzymes were purified and some properties were compared. These enzymes inactivated all of the apo-pyridoxal enzymes tested, but did not affect non-pyridoxal enzymes.It might be considered that these proteases are one kind of organ specific isozymes. The activities of these specific proteases were found to respond sensitively to dietary conditions. A marked increase of the enzyme activity was observed in the rat fed on non-protein diet or high protein diet. The different roles in intracellular regulation of pyridoxal enzyme levels by these proteases located in different organs were discussed.
Our present understanding of intracellular protein degradation developed from an attempt to understand the metabolic stability of proteins in cells. With the unravelling of its complex biochemical machinery has come a realization that protein degradation plays an important role in the most crucial events in the cell cycle, in signal transduction and in maintaining the integrity of the proper folded state of proteins. In this review, I attempt to trace the curious history of intracellular protein degradation, its novel and elegant biochemistry and to extract the features that might be important for the next era of studies in cell physiology.
After the addition of glucose to acetate- or ethanol-grown yeast cells a small group of selected enzymes is rapidly inactivated. This phenomenon has been called "catabolite inactivation". Among other enzymes participating in gluconeogenesis, fructose-1,6-bisphosphatase is inactivated during this catabolite inactivation process. It was shown by FUNAYAMA et al. (Eur. J. Biochem. 109, 61-66 (1980)) that the mechanism of inactivation is proteolysis. In the present paper evidence is presented that after addition of glucose a covalent conversion of the enzyme protein by phosphorylation of a serine-residue initiates its subsequent proteolysis. It is suggested that the covalent modification triggered by glucose and/or products of its catabolism renders the enzyme susceptible to proteinases and thereby initiates proteolysis of a selected enzyme without the necessity of a specific proteinase present.
Ubiquitin is a singular protein with multiple functions. It is probably the most slowly evolving protein known, is encoded by genes with a unique structure, and provides an intriguing case study for various aspects of molecular evolution. In particular, the multiple ubiquitin-coding repeats which have been characterized in man, yeast and a slime mould graphically illustrate the dynamics of concerted evolution, but cast doubts on the effectiveness of this process for unlinked arrays in this repeat family.
The ATP-dependent proteolytic cell-free system from reticulocytes has been resolved into three components, each of which is absolutely required for acid solubilization of 125I-labeled bovine serum albumin radioactivity. In addition to the previously reported heat-stable polypeptide [Ciechanover, A., Hod, Y. & Hershko, A. (1978) Biochem. Biophys. Res Commun. 81, 1100-1105], we now describe a protein of high molecular weight (approximately 450,000) that is labile at 42 degrees C. The extremely heat-labile factors is remarkably stabilized by ATP. GTP and CTP, which do not stimulate protolysis, do not stabilize this factor. Adenylate nucleotides such as ADP or the nonhydrolyzable beta,gamma imido or methylene analogues of ATP cause stabilization although they do not activate proteolysis. A third protein component of the protease system, stable at 42 degrees C, has been separated from the heat-labile species by salt precipitation. All three components are required with ATP for proteolytic activity, but thus far only the heat-labile factor has been shown to interact directly with ATP.
The principles of chemical kinetics were applied to turnover studies soon after tracers had been introduced into biological experiments, and several reviews on this subject are available. However, rigorous precursor-product analysis of protein turnover was seldom attempted in the past, mostly because it was assumed that the radioactive precursors of proteins are rapidly eliminated from the organisms. The estimate of protein half-life therefore was based on measurements of decay of the protein radioactivity only. Although such experiments clearly confirmed the dynamic state of protein molecules, it was later recognized that this approach leads to a gross overestimate of protein half-lives because the tracer amino acids are not eliminated as fast as had been thought. The omission of precursor specific radioactivity in calculations of turnover rates thus is not justified. Consequently, any meaningful measure of protein turnover requires determination of the specific radioactivity of the amino acid, both in the protein molecule and in the precursor pool.
The degradation of denatured globin in reticulocyte lysates is markedly stimulated by ATP. This system has now been resolved into two components, designated fractions I and II, in the order of their elution from DEAE-cellulose. Fraction II has a neutral protease activity but is stimulated only slightly by ATP, whereas fraction I has no proteolytic activity but restores ATP-dependent proteolysis when combined with fraction II. The active principle of fraction I is remarkably heat-stable, but it is non-dialysable, precipitable with ammonium sulfate and it is destroyed by treatment with proteolytic enzymes. In gel filtration on Sephadex-G-75, it behaves as a single component with a molecular weight of approximately 9,000.