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A New Heat Shock Protein That Binds Nucleic Acids
Abstract
We describe the isolation of Hsp15, a new, very abundant heat shock protein that binds to DNA and RNA. Hsp15 is well conserved
and related to a number of RNA-binding proteins, including ribosomal protein S4, RNA pseudouridine synthase, and tyrosyl-tRNA
synthetase. The region shared between these proteins appears to represent a common, but previously unrecognized, RNA binding
motif. Filter binding studies showed that Hsp15 binds to a 17-mer single-stranded RNA with a dissociation constant of 9 μm in 22.5 mm Hepes, pH 7.0, 5 mm MgCl2. A role of Hsp15 in binding nucleic acids puts this protein into a different functional category from that of many other
heat shock proteins that act as molecular chaperones or proteases on protein substrates.
... The heat-shock locus R (hslR, formerly yrfH) gene encodes the heat shock protein 15 (Hsp15), a 15 kDa protein that is upregulated by 43-fold upon temperature upshift (2). Unlike most heat shock proteins that generally function as molecular chaperones or proteases to maintain the native state of proteins, Hsp15 contains an RNAbinding domain, leading to the suggestion that it interacts with an RNA substrate (3,4). Consistently, the crystal structure of E. coli Hsp15 revealed a single compact globular RNA binding domain, which is found in other RNAbinding proteins such as ribosomal protein S4 and threonyl-tRNA synthetase. ...
... Consistently, the crystal structure of E. coli Hsp15 revealed a single compact globular RNA binding domain, which is found in other RNAbinding proteins such as ribosomal protein S4 and threonyl-tRNA synthetase. In addition, E. coli Hsp15 contains a long C-terminal ␣-helix (5), which is absent in Hsp15 homologs from some species, such as Bacillus subtilis ( Figure 1) (3,4). E. coli Hsp15 was shown in vivo to associate with ribosomal 50S subunits, but not 70S ribosomes, and that this association was dependent on the presence of the nascent polypeptide chain (6). ...
In Escherichia coli, the heat shock protein 15 (Hsp15) is part of the cellular response to elevated temperature. Hsp15 interacts with peptidyl-tRNA-50S complexes that arise upon dissociation of translating 70S ribosomes, and is proposed to facilitate their rescue and recycling. A previous structure of E. coli Hsp15 in complex with peptidyl-tRNA-50S complex reported a binding site located at the central protuberance of the 50S subunit. By contrast, recent structures of RqcP, the Hsp15 homolog in Bacillus subtilis, in complex with peptidyl-tRNA-50S complexes have revealed a distinct site positioned between the anticodon-stem-loop (ASL) of the P-site tRNA and H69 of the 23S rRNA. Here we demonstrate that exposure of E. coli cells to heat shock leads to a decrease in 70S ribo-somes and accumulation of 50S subunits, thus identifying a natural substrate for Hsp15 binding. Additionally , we have determined a cryo-EM reconstruction of the Hsp15-50S-peptidyl-tRNA complex isolated from heat shocked E. coli cells, revealing that Hsp15 binds to the 50S-peptidyl-tRNA complex analogously to its B. subtilis homolog RqcP. Collectively, our findings support a model where Hsp15 stabilizes the peptidyl-tRNA in the P-site and thereby promotes access to the A-site for putative rescue factors to release the aberrant nascent polypeptide chain.
... As reported in earlier studies EcHsp15 has a high affinity for free 50Snc-tRNA with K D value less than 5 nM [3]. The measured value of dissociation constant of EcHsp15 with 17 mer RNA was 8.9 ± 3.0 mM [20]. Structure of VcHsp15 indicated a conserved tryptophan residue (Trp17) is encompassed between several Lys, Arg residues near the putative DNA/RNA binding. ...
... The Kd values calculated from the plot indicate positive binding for dTTP (Kd~7.7 ± 0.3 mM) and dGTP (Kd 2.4 ± 0.2 mM) but no or nominal binding for dATP and dCTP. Kd values calculated for 16 mer DNA C3T3C4T4 (Kd~8.0 ± 0.34 mM) in our study closely corresponds to the binding of a 17 mer RNA with EcHsp15 [20]. ...
... Diverse functions of sHsp are also reported in bacteria and eukaryotes (Table 1; Figure 3). For example, E. coli Hsp15 protects DNA by non-specifically getting associated with it (Korber et al., 1999;Macario et al., 1999). In yeast, Hsp42 initiates aggregation of the unfolded proteins and forces them to precipitate, thus preventing them from acting as nucleation points for the aggregation of native proteins (Ungelenk et al., 2016). ...
Small heat shock proteins (sHsp) are a ubiquitous group of ATP-independent chaperones found in all three domains of life. Although sHsps in bacteria and eukaryotes have been studied extensively, little information was available on their archaeal homologs until recently. Interestingly, archaeal heat shock machinery is strikingly simplified, offering a minimal repertoire of heat shock proteins to mitigate heat stress. sHsps play a crucial role in preventing protein aggregation and holding unfolded protein substrates in a folding-competent form. Besides protein aggregation protection, archaeal sHsps have been shown recently to stabilize membranes and contribute to transferring captured substrate proteins to chaperonin for refolding. Furthermore, recent studies on archaeal sHsps have shown that environment-induced oligomeric plasticity plays a crucial role in maintaining their functional form. Despite being prokaryotes, the archaeal heat shock protein repository shares several features with its highly sophisticated eukaryotic counterpart. The minimal nature of the archaeal heat shock protein repository offers ample scope to explore the function and regulation of heat shock protein(s) to shed light on their evolution. Moreover, similar structural dynamics of archaeal and human sHsps have made the former an excellent system to study different chaperonopathies since archaeal sHsps are more stable under in vitro experiments.
... Cytoplasmic localization of P-Hsp27 regulates, majorly, chaperoning functions which contribute to membrane stability, actin organization, cell migration and cell viability. In the nucleus, P-Hsp27 protects the DNA from damage by responding to genetic stresses and perform its survival functions (Geum et al., 2002;Korber et al., 1999;Rousseau et al., 1997). In the present study, the western blotting data indicated inhibition of phosphorylation of p38MAPK and Hsp27 in response to CID-6033590 treatment. ...
... The spatial variation of HSP27 is related to the adaptation of cells to extracellular stimuli. It has been reported that HSP27 is mainly distributed in cytoplasm under normal conditions but it transfers into cell nuclei [46] to protect nucleic acid and improve cell viability under high heat stress stimuli [47]. In this study, the results show that HSP27 is uniformly distributed in cells under low shear stress stimuli while it accumulates downstream under higher shear stress stimuli. ...
Heat shock protein 27 (HSP27) is a multifunctional protein that undergoes significant changes in its expression and phosphorylation in response to shear stress stimuli, suggesting that it may be involved in mechanotransduction. However, the mechanism of HSP27 affecting tumor cell migration under shear stress is still not clear. In this study, HSP27-enhanced cyan fluorescent protein (ECFP) and HSP27-Ypet plasmids are constructed to visualize the self-polymerization of HSP27 in living cells based on fluorescence resonance energy transfer technology. The results show that shear stress induces polar distribution of HSP27 to regulate the dynamic structure at the cell leading edge. Shear stress also promotes HSP27 depolymerization to small molecules and then regulates polar actin accumulation and focal adhesion kinase (FAK) polar activation, which further promotes tumor cell migration. This study suggests that HSP27 plays an important role in the regulation of shear stress-induced HeLa cell migration, and it also provides a theoretical basis for HSP27 as a potential drug target for metastasis.
... Further, some of the smaller heat shock proteins have flexible C-terminal regions that are rich in acidic amino acids, which when mutated, lead to reduced activity (Edwards, Kueltzo, et al, 2001;. Members of both the large and sHsp protein families interact with polyanions such as nucleic acids and tubulin (Korber, Zander, et al, 1999;Koyasu, Nishida, et al, 1986). It is conceivable that Daxx is part of a family of acidrich domain containing proteins that share a similar function in terms of chaperone activity. ...
Daxx, a multifunctional protein with a diverse set of proposed functions, is ubiquitously expressed and highly conserved through evolution. A primarily nuclear protein, Daxx is able to regulate apoptosis, transcription, and cellular proliferation. Despite many studies into the function of Daxx, its precise role in the cell remains enigmatic. Herein, evidence is presented to expand upon the known anti-apoptotic function of Daxx, to establish Daxx as a novel molecular chaperone, and to further its repertoire of transcriptional targets. As an apoptotic inhibitor, Daxx is known to regulate p53 by stabilizing its main negative regulator, Mdm2, via formation of a ternary complex between Daxx, Mdm2, and Hausp. The present study reveals that DNA damage-induced phosphorylation of Daxx is an important step in the disruption of the Daxx-Mdm2-Hausp complex, allowing for p53 activation. A novel activity for Daxx is presented whereby it is able to modulate protein folding in vitro and in vivo. Daxx can refold and reactivate denatured substrates using its paired amphipathic helical and acid-rich domains. This finding was extended to p53, where Daxx was able to solubilize misfolded p53 both in vitro and in vivo. Further, this finding may provide a biochemical rationale as to the varied functionality of Daxx in the cell. Finally, a novel transcriptional target, Cdk6, is described for Daxx. Microarray analysis indicated that Cdk6 is a strongly downregulated gene upon Daxx silencing. Depletion of Daxx in various cancer and primary cell lines led to a decrease in Cdk6 protein levels. Additionally, Daxx can affect transcription of Cdk6, and chromatin immunoprecipitation reveals that Daxx binds to the Cdk6 promoter. Together, these results indicate that Daxx has potential functional plasticity and is involved in an array of cellular functions; in fact, cellular homeostasis relies on the proper execution of multiple biological processes. Elucidation of the biology of Daxx would not only provide insight into the regulation of these processes, but may also establish Daxx as a relevant therapeutic target.
... Once fixed, further adaptive changes in the protein and intron RNA led to specific binding and more efficient protein-dependent splicing. The CTDs of bacterial and mitochondrial TyrRSs have a fold similar to ribosomal protein S4, a promiscuous RNA-binding domain that has high non-specific RNA-binding activity and has been incorporated into a variety of proteins that bind structurally different RNAs (64)(65)(66)(67). ...
The mitochondrial tyrosyl-tRNA synthetases (mtTyrRSs) of Pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mt tRNATyr and promote the splicing of autocatalytic group I introns. Previous studies showed that one of these proteins, Neurospora crassa CYT-18, binds group I introns by using both its N-terminal catalytic and C-terminal anticodon-binding domains and that the catalytic domain uses a newly evolved group I intron-binding surface, which includes an N-terminal extension and two small insertions (insertions 1 and 2) with distinctive features not found in non-splicing mtTyrRSs. To explore how this RNA-binding surface diverged to accommodate different group I introns in other Pezizomycotina fungi, we determined X-ray crystal structures of C-terminally truncated Aspergillus nidulans and Coccidioides posadasii mtTyrRSs. Comparisons with previous N. crassa CYT-18 structures and a structural model of the A. fumigatus mtTyrRS showed that the overall topology of the group I intron-binding surface is conserved, but with variations in key intron-binding regions, particularly the Pezizomycotina-specific insertions. These insertions, which arose by expansion of flexible termini or internal loops, show greater variation in structure and amino acids potentially involved in group I intron binding than do neighboring protein core regions that also function in intron binding but may be more constrained to preserve mtTyrRS activity. Our results suggest a structural basis for the intron-specificity of different Pezizomycotina mtTyrRSs, highlight flexible terminal and loop regions as major sites for enzyme diversification, and identify targets for therapeutic intervention by disrupting an essential RNA-protein interaction in pathogenic fungi.
Trialeurodes vaporariorum, commonly known as the greenhouse whitefly, severely infests important crops and serves as a vector for apple scar skin viroid (ASSVd). This vector-mediated transmission may cause the spread of infection to other herbaceous crops. For effective management of ASSVd, it is important to explore the whitefly’s proteins, which interact with ASSVd RNA and are thereby involved in its transmission. In this study, it was found that a small heat shock protein (sHsp) from T. vaporariorum, which is expressed under stress, binds to ASSVd RNA. The sHsp gene is 606 bp in length and encodes for 202 amino acids, with a molecular weight of 22.98 kDa and an isoelectric point of 8.95. Intermolecular interaction was confirmed through in silico analysis, using electrophoretic mobility shift assays (EMSAs) and northwestern assays. The sHsp22.98 protein was found to exist in both monomeric and dimeric forms, and both forms showed strong binding to ASSVd RNA. To investigate the role of sHsp22.98 during ASSVd infection, transient silencing of sHsp22.98 was conducted, using a tobacco rattle virus (TRV)-based virus-induced gene silencing system. The sHsp22.98-silenced whiteflies showed an approximate 50% decrease in ASSVd transmission. These results suggest that sHsp22.98 from T. vaporariorum is associated with viroid RNA and plays a significant role in transmission.
Research on heat shock and other stress proteins (hsps) has revealed a number of intriguing aspects about this remarkable class of molecules. First, hsps are highly abundant proteins in cells even under non-stress conditions. This abundance holds for virtually all organisms or cell types examined. Second, hsps are the most phylogenetically conserved proteins known to biology with an overall primary amino acid sequence homology of some 50 % between Escherichia coli and man. Third, hsps have been implicated in a myriad of cellular processes throughout the years. Although the majority of these biological functions delineate hsps as molecular chaperones, recent evidence suggests that certain heat shock proteins possess a likely ancient, evolutionarily conserved role pointing beyond their classical chaperoning function. This chapter deals with a recently described novel feature of the mammalian 70-kDa super-family of molecular chaperones (hsp70, hsc70, hsp110 and grp170), their inherent RATA binding properties. The monograph highlights the RATA sequence preference as well as the RNA-binding domain of these proteins. The influence of ATP and a peptide substrate on RNA-binding —all key components in chaperoning function- will also be detailed. Although the data were obtained using both hsp/hsc70 and hsp110 as the RATA binding partner, since most of the supporting evidence deals with hsp70/hsc70, discussions of possible in vivo functions will mainly be extended to these widely characterized stress proteins.
The interaction of proteins binding non-specifically to DNA, as well as the properties of many other interacting ligand-lattice systems important in molecular biology, requires a fundamentally different type of theoretical analysis than that provided by the classical Scatchard independent-binding-site treatment. Exact and relatively simple equations describing the binding of both non-interacting and interacting (co-operative) ligands to a homogeneous one-dimensional lattice are derived in terms of ligand site size, intrinsic binding constant and ligand-ligand co-operativity (equations (10) and (15) in the text). The mathematical approach is based on simple conditional probabilities, and reveals some largely unrecognized characteristics of such lattice binding systems. The results indicate that the binding of any non-interacting ligand covering more than one lattice residue results in non-linear (convex downward) Scatchard plots. The introduction of positive ligand-ligand co-operativity antagonizes this non-linearity, and eventually leads to plots of the opposite curvature. The maxima, limiting slopes, and intercepts of such plots can be used to estimate the required binding parameters. The method can be extended to systems involving heterogeneous ligands, and some types of heterogeneous lattices. Procedures for applying the method to a variety of interacting systems are presented, and a preliminary analysis is carried out for some selected sets of data from the literature.
The electrophoretic behavior of closed circular, nicked circular, and linear duplex forms of bacteriophages φx174 and PM2, plasmid ColEl, and rat mitochondrial DNAs has been analyzed by agarose gel electrophoresis as a function of gel concentration, electric-field strength, and ionic conditions. The logarithm of electrophoretic mobility (μ) is a linear function of gel concentration (T) at all agarose concentrations and field strengths tested. The retardation coefficient (dμ/dT = KR) is characteristic of DNA conformation under controlled conditions. Alterations in electric-field strength and ionic conditions lead to predictable changes in the relative migration rates of the various DNA forms, thus defining conditions under which their separation can be optimized. The alkaline titration of a mixture of duplex conformational isomers was carried out on gels at neutral pH. Under conditions where closed circular DNA is stable to denaturation, separation of the linear and circular complementary strands of φX174, Coll, and PM2 was observed. Electrophoresis of closed circular DNA in the presence of the intercalating dye ethidium bromide provided an estimate of superhelix density for PM2 DNA in good agreement with values determined by other methods. In addition, the use of ethidium bromide offers additional control over conditions for optimizing separations of DNA conformational forms. At ethidium bromide concentrations above that necessary to remove all negative superhelical turns, the relative change in the electrophoretic mobility with increasing dye concentration appears to be distinguishably different for the closed circular, nicked circular, and linear duplex forms of PM2 DNA. The combined results of this investigation define several experimental conditions and strategies for the application of agarose gel electrophoresis to studies of DNA structure and function.
The interaction of lambda phage cro repressor with double-stranded non-specific DNA has been investigated by monitoring the quenching of its intrinsic tyrosyl fluorescence. The McGhee & von Hippel (1974) analysis of the binding of cro repressor to DNA showed that cro repressor undergoes structural variations in the ionic strength range from 0.04 to 0.18m-KCl. Under these salt conditions, the excluded binding site size of cro repressor on the DNA lattice changes from three to four base-pairs (6 to 8 nucleotides) at the lower ionic strengths, to seven to eight base-pairs (14 to 16 nucleotides) at the higher ionic strength. Quaternary structure variation, which does not cause the excluded site size variation, was also noted at low ionic strengths. Evidence is presented to indicate that cro repressor binds only one side of the DNA helix, such that cro repressor covers a stretch of 14 to 16 nucleotides along one side of the helix in the presence of 0.2 m-salt. Under conditions where the cro repressor structure is constant, approximately nine ion-pairs are formed in the cro repressor-non-specific DNA complex. These results are in agreement with the model proposed by Anderson et al. (1981).
At normal temperatures, Hsp90 is one of the most abundant proteins in the cytosol of various eucaryotic cells. Upon heat shock, the level of Hsp90 is increased even more, suggesting that it is important for helping cells to survive under these conditions. However, studies so far have been almost exclusively concerned with the function of Hsp90 under non-stress conditions, and therefore only little is known about the role of Hsp90 during heat shock. As a model for heat shock in vitro, we have monitored the inactivation and subsequent aggregation of dimeric citrate synthase (CS) at elevated temperatures. Hsp90 effectively “stabilized” CS under conditions where the enzyme is normally inactivated and finally aggregates very rapidly. A kinetic dissection of the unfolding pathway of CS succeeded in revealing two intermediates which form and subsequently undergo irreversible aggregation reactions. Hsp90 apparently interacts transiently with these highly structured early unfolding intermediates. Binding and subsequent release of the intermediates favorably influences the kinetic partitioning between two competing processes, the further unfolding of CS and the productive refolding to the native state. As a consequence, CS is effectively stabilized in the presence of Hsp90. The significance of this interaction is especially evident in the suppression of aggregation, the major end result of thermal unfolding events in vivo and in vitro. These effects, which are ATP-independent, seem to be a general function of members of the Hsp90 family, since yeast and bovine Hsp90 as well as the Hsp90 homologue from Escherichia coli gave similar results. It seems likely that this function also reflects the role of Hsp90 under heat shock conditions in vivo. We therefore propose that members of the Hsp90 family convey thermotolerance by transiently binding to highly structured early unfolding intermediates, thereby preventing their irreversible aggregation and stabilizing the active species.
The objective of this study is to quantify the contributions of cations, anions and water to stability and specificity of the interaction of lac repressor (lac R) protein with the strong-binding symmetric lac, operator (Osym) DNA site. To this end, binding constants Kobs and their power dependences on univalent salt (MX) concentration ( ) have been determined for the interactions of lac R with Osym operator and with non-operator DNA using filter binding and DNA cellulose chromatography, respectively.
A simple inexpensive sensitive protease assay was developed using soluble fluorescein isothiocyanate (FITC)-labeled casein. Casein was reacted with FITC to form the fluorescein thiocarbamoyl derivative. This substrate is cleaved by trypsin, chymotrypsin, elastase, subtilisin, and thermolysin in a linear time-dependent manner. These enzymes can be measured in the nanogram and subnanogram range using this assay. The assay is reproducible, has a low blank, and uses casein, which resembles natural substrates of most proteases.
Binding parameters for the interaction of lac repressor with non-operator DNA have been determined using a sedimentation velocity technique. Analytical ultracentrifugation is used to separate DNA-protein complexes from unbound protein, thereby permitting direct optical determination of the concentration of free protein as a function of input DNA and repressor concentrations. The method yields absolute values for the association constant (K) for proteins binding nonspecifically to nucleic acid lattices; the binding site size (n), and the binding cooperativity parameter (ω) can also be estimated by this approach. Values of K for the binding of repressor to non-operator DNA have been determined under a variety of solvent conditions. At 0.15 M Na+, 20°C, pH 7.5, the average value of K for repressor binding to native phage λ DNA is 2.4 × 105 M-1 (DNA concentration in base pairs, protein in repressor tetramers). Binding is very ionic strength dependent; we find that δ log K/δ log [Na+] ≃ -10. Analysis of the ionic strength dependence by the method of Record, M. T., Jr., Lohman, T. M., and de Haseth, P. ((1976), J. Mol. Biol. 107, 145) indicates the formation of ∼ 11 ion-pair bonds between DNA phosphates and basic amino acid residues per repressor tetramer bound. Repressor binding is moderately pH dependent, the value of K decreasing as the pH increases from 7.2 to 7.8. Thus one or more histidine residues may be involved in the binding. The binding affinity also decreases with increasing temperature, and is somewhat increased in 30% glycerol. Under comparable ionic conditions binding to poly[d(A-T)] is ca. sixfold tighter than to phage λ (or calf thymus) DNA. It is shown directly that the binding of inducer (IPTG) to repressor does not change the repressor-DNA association constant. However "core" repressor, produced by brief digestion of the protein with trypsin to remove the N-terminal 58 residue peptide from each subunit, does not bind to DNA. The repressor-DNA binding data are consistent with a site size (n) of about 12 base pairs covered per repressor molecule bound (assuming binding to only one side of the double-helical DNA lattice), and show that binding involves no protein-protein cooperativity (ω = 1).
Some recent modifications of the protein assay by the method of Lowry, Rosebrough, Farr, and Randall (1951, J. Biol. Chem.193, 265–275) have been reexamined and altered to provide a consolidated method which is simple, rapid, objective, and more generally applicable. A DOC-TCA protein precipitation technique provides for rapid quantitative recovery of soluble and membrane proteins from interfering substances even in very dilute solutions (< 1 μg/ml of protein). SDS is added to alleviate possible nonionic and cationic detergent and lipid interferences, and to provide mild conditions for rapid denaturation of membrane and proteolipid proteins. A simple method based on a linear log-log protein standard curve is presented to permit rapid and totally objective protein analysis using small programmable calculators. The new modification compared favorably with the original method of Lowry et al.
We have investigated the nonspecific interaction of lac repressor protein with DNA by a quantitative application of DNA-cellulose chromatography (deHaseth, P.L., et al. (1977), Biochemistry 16 (third of five papers in a series in this issue)). The observed association constant for the interaction, KRD obsd, is a sensitive function of ion concentrations and pH. Application of binding theory to interpret these effects gives the results that 11 +/- 2 monovalent ions are released in the interaction and two groups on repressor must be protonated for repressor to bind to DNA. We argue that much of the ion release results from the displacement of cations from the DNA, and estimate on this basis that 12 +/- 2 phosphates are involved in ionic interactions with the protein. Ion release drives the protonation reaction and the overall repressor-DNA interaction. The major role of low molecular weight ions in the repressor-DNA interaction suggests that ion concentration changes must be considered in discussing mechanisms of control of gene expression.