Molecular chaperones, stress resistance and development in Artemia franciscana

Embryos of the brine shrimp, Artemia franciscana, either develop directly into swimming larvae or are released from females as encysted gastrulae (cysts) which enter diapause, a reversible state of dormancy. Metabolic activity in diapause cysts is very low and these embryos are remarkably resistant to physiological stresses. Encysting embryos, but not those undergoing uninterrupted development, synthesize large amounts of two proteins, namely p26 and artemin. Cloning and sequencing demonstrated p26 is a small heat shock/alpha-crystallin protein while artemin has structural similarity to ferritin. p26 exhibits molecular chaperone activity in vitro, moves reversibly into nuclei during stress and confers thermotolerance on transformed organisms, suggesting critical roles in cyst development. The function of artemin is unknown. Encysted Artemia also contain an abundance of trehalose, a disaccharide capable of protecting embryos. Artemia represent a novel experimental system where the developmental functions of small heat shock/alpha-crystallin proteins and other stress response elements can be explored.     

Artemin is an RNA-binding protein with high thermal stability and potential RNA chaperone activity

Encysted embryos of the crustacean, Artemia franciscana, are among the most stress-resistant of all multicellular eukaryotes, due in part to massive amounts of p26, a small heat shock protein, that acts as a molecular chaperone. These embryos contain equally large amounts of another protein called artemin, of previously unknown function, that we report on here. Its thermal stability allows large-scale purification in about a day, using ammonium sulfate fractionation and incubation at 70 degrees C for 7 min, followed by gel filtration. The latter yields an artemin-RNA complex from which the pure protein, apo-artemin, was obtained by anion-exchange chromatography. We evaluated the possibility that artemin acts as a molecular chaperone for proteins, but obtained no evidence for that in vitro. The association of RNA with apo-artemin occurs at high temperatures and, although it is not yet clear whether artemin has a specific role as an RNA chaperone, it does bind non-polyadenylated RNAs which are then translated in vitro. Artemin-RNA is thermostable, some molecules resisting destruction after 30 min at 90 degrees C. The first order rate constant for denaturation and aggregation of artemin-RNA at 85 degrees C is 8.5 x 10(-3)min(-1), which compares well with other thermostable proteins of similar size ( approximately 500 kDa) such as the ferritins with which artemin has amino acid sequence similarity. The amount of artemin extracted from embryos that had been stored dry, under laboratory conditions, since 1951 is comparable to the amount in contemporary embryos, indicating its stability in situ, and supporting the in vitro heating studies.     

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.     

Ontogeny of low molecular weight stress protein p26 during early development of the brine shrimp, Artemia franciscana

Embryogenesis in the brine shrimp, Artemia sp., occurs by one of two pathways: (i) the direct, uninterrupted development of nauplius larvae within the female or (ii) the production of embryos that arrest development at the gastrula stage and enter diapause. Diapause embryos are released from females into the aqueous environment where they remain in diapause until activated by appropriate environmental cues and resume development. These encysted embryos possess at least one low molecular weight stress protein, which we refer to as p26 and which has been implicated previously in the stress response of activated embryos. We investigated the appearance of p26 in developing diapause embryos in utero and looked for its presence in embryos developing directly into nauplii. We found p26 to be specific to diapause-destined embryos; it was not detected in direct-developing embryos. We conclude that p26 is not required for the basic developmental program that produces the nauplius. In diapause-destined embryos, p26 was first detectable after 3 days of development, at which time the embryos were late gastrulae. This protein continues to increase in amount until the encysted embryos are released, approximately 5 days after fertilization. At the time of release almost all p26 is located in the low speed supernatant fraction, but as released embryos continue diapause, p26 transfers to the pelleted nuclear fraction in increasing amounts. Our working hypothesis views p26 as a molecular chaperone preventing protein denaturation and aggregation under conditions associated with metabolic arrest and other stressful states, which these encysted embryos encounter.     

Matrix metalloproteinase-3 induction in rat brain astrocytes: focus on the role of two AP-1 elements

Embryos of the crustacean, Artemia franciscana, undergo alternative developmental pathways, producing either larvae or encysted embryos (cysts). The cysts enter diapause, characterized by exceptionally high resistance to environmental stress, a condition thought to involve the sHSP (small heat-shock protein), p26. Subtractive hybridization has revealed another sHSP, termed ArHsp21, in diapause-destined Artemia embryos. ArHsp21 shares sequence similarity with p26 and sHSPs from other organisms, especially in the alpha-crystallin domain. ArHsp21 is the product of a single gene and its synthesis occurred exclusively in diapause-destined embryos. Specifically, ArHsp21 mRNA appeared 2 days post-fertilization, followed 1 day later by the protein, and then increased until embryo release at day 5. No ArHsp21 protein was detected in embryos developing directly into larvae, although there was a small amount of mRNA at 3 days post-fertilization. The protein was degraded during post-diapause development and had disappeared completely from second instar larvae. ArHsp21 formed large oligomers in encysted embryos and transformed bacteria. When purified from bacteria, ArHsp21 functioned as a molecular chaperone in vitro, preventing heat-induced aggregation of citrate synthase and reduction-driven denaturation of insulin. Sequence characteristics, synthesis patterns and functional properties demonstrate clearly that ArHsp21 is an sHSP able to chaperone other proteins and contribute to stress tolerance during diapause. As such, ArHsp21 would augment p26 chaperone activity and it may also possess novel activities that benefit Artemia embryos exposed to stress.     

Phosphorylation-dependent structural alterations in the small hsp30 chaperone are associated with cellular recovery

Small heat shock proteins (hsps) act as molecular chaperones by preventing the thermal aggregation and unfolding of cellular protein; however, the manner by which cells regulate chaperone activity remains unclear. In the present study, we examined the role of phosphorylation on the chaperone function of the Xenopus small hsp30. Both heat stress and sodium arsenite treatment in A6 cells resulted in a rapid activation of p38alpha and MAPKAPK-2. Surprisingly, the association of MAPKAPK-2 with hsp30 and its subsequent phosphorylation were more prevalent during recovery after heat stress. Treatment of A6 cells with SB203580, an inhibitor of the p38 MAP kinase pathway, resulted in a loss of hsp30 phosphorylation. Phosphorylation resulted in the formation of smaller multimeric hsp30 complexes and resulted in a significant loss of secondary structure. Consequently the phosphorylation-induced structural changes severely compromised the ability of hsp30 to prevent the heat-induced aggregation of citrate synthase and luciferase in vitro. We confirmed that the loss of chaperone activity was coincident with an attenuated binding of phosphorylated hsp30 with target proteins. Our data suggest that phosphorylation may be necessary to regulate the post-heat stress molecular chaperone activity of hsp30.     

The Small Heat Shock Protein p26 Aids Development of Encysting Artemia Embryos, Prevents Spontaneous Diapause Termination and Protects against Stress

Artemia franciscana embryos enter diapause as encysted gastrulae, a physiological state of metabolic dormancy and enhanced stress resistance. The objective of this study was to use RNAi to investigate the function of p26, an abundant, diapause-specific small heat shock protein, in the development and behavior of encysted Artemia embryos (cysts). RNAi methodology was developed where injection of Artemia females with dsRNA specifically eliminated p26 from cysts. p26 mRNA and protein knock down were, respectively, confirmed by RT-PCR and immuno-probing of western blots. ArHsp21 and ArHsp22, diapause-related small heat shock proteins in Artemia cysts sharing a conserved α-crystallin domain with p26, were unaffected by injection of females with dsRNA for p26, demonstrating the specificity of protein knock down. Elimination of p26 delayed cyst release from females demonstrating that this molecular chaperone influences the development of diapause-destined embryos. Although development was slowed the metabolic activities of cysts either containing or lacking p26 were similar. p26 inhibited diapause termination after prolonged incubation of cysts in sea water perhaps by a direct effect on termination or indirectly because p26 is necessary for the preservation of diapause maintenance. Cyst diapause was however, terminated by desiccation and freezing, a procedure leading to high mortality within cyst populations lacking p26 and indicating the protein is required for stress tolerance. Cysts lacking p26 were also less resistant to heat shock. This is the first in vivo study to show that knock down of a small heat shock protein slows the development of diapause-destined embryos, suggesting a role for p26 in the developmental process. The same small heat shock protein prevents spontaneous termination of diapause and provides stress protection to encysted embryos.     

Structural and functional roles for beta-strand 7 in the alpha-crystallin domain of p26, a polydisperse small heat shock protein from Artemia franciscana

Oviparous development in the extremophile crustacean, Artemia franciscana, generates encysted embryos which enter a profound state of dormancy, termed diapause. Encystment is marked by the synthesis of p26, a polydisperse small heat shock protein thought to protect embryos from stress. In order to elucidate structural/functional relationships within p26 and other polydisperse small heat shock proteins, and to better define the protein's role during diapause, amino acid substitutions R110G, F112R, R114A and Y116D were generated within the p26 alpha-crystallin domain by site-directed mutagenesis. These residues were chosen because they are highly conserved across species boundaries, and molecular modelling indicates that they are part of a key structural interface between dimers. The F112R mutation, which had the greatest impact on oligomerization, placed two charged residues at the p26 dimer-dimer interface, demonstrating the importance of beta-strand 7 in tetramer formation. All mutated versions of p26 were less able than wild-type p26 to confer thermotolerance on transformed bacteria and they exhibited diminished chaperone action in three in vitro assays; however, all variants retained protective activity. This apparent stability of p26 may, by prolonging effective chaperone life in vivo, enhance embryo stress resistance. All substitutions modified p26 intrinsic fluorescence, surface hydrophobicity and secondary structure, and the pronounced changes in variant R114A, as indicated by these physical measurements, correlated with the greatest loss of function. Although mutation R114A had the greatest effect on p26 chaperoning, it had the least on oligomerization. These results demonstrate that in contrast to many other small heat shock proteins, p26 effectiveness as a chaperone is independent of oligomerization. The results also reinforce the idea, occasioned by modelling, that R114 is removed slightly from dimer-dimer interfaces. Moreover, beta-strand 7 is shown to have an important role in oligomerization of p26, a function first proposed for this structural element upon crystallization of wheat Hsp16.9, a small heat shock protein with different quaternary structure.     

Trehalose is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum but not for maintenance of membrane traffic functions after severe heat stress

Saccharomyces cerevisiae cells grown at physiological temperature 24 degrees C require preconditioning at 37 degrees C to acquire tolerance towards brief exposure to 48-50 degrees C. During preconditioning, the cytosolic trehalose content increases remarkably and in the absence of trehalose synthesis yeast cannot acquire thermotolerance. It has been speculated that trehalose protects proteins and membranes under environmental stress conditions, but recently it was shown to assist the Hsp104 chaperone in refolding of heat-damaged proteins in the yeast cytosol. We have demonstrated that heat-denatured proteins residing in the endoplasmic reticulum (ER) also can be refolded once the cells are returned to physiological temperature. Unexpectedly, not only ER chaperones but also the cytosolic Hsp104 chaperone is required for conformational repair events in the ER lumen. Here we show that trehalose facilitates refolding of glycoproteins in the ER after severe heat stress. In the absence of Tps1p, a subunit of trehalose synthase, refolding of heat-damaged glycoproteins to bioactive and secretion-competent forms failed or was retarded. In contrast, membrane traffic operated many hours after severe heat stress even in the absence of the TPS1 gene, demonstrating that trehalose had no role in thermoprotection of membranes engaged in vesicular traffic. However, cytosolic proteins were aggregated and protein synthesis abolished, resulting finally in cell death.     

Functional analysis of a small heat shock/alpha-crystallin protein from Artemia franciscana. Oligomerization and thermotolerance.

Oviparously developing embryos of the brine shrimp, Artemia franciscana, synthesize abundant quantities of a small heat shock/alpha-crystallin protein, termed p26. Wild-type p26 functions as a molecular chaperone in vitro and is thought to help encysted Artemia embryos survive severe physiological stress encountered during diapause and anoxia. Full-length and truncated p26 cDNA derivatives were generated by PCR amplification of p26-3-6-3, then cloned in either pET21(+) or pRSETC and expressed in Escherichia coli BL21(DE3). All constructs gave a polypeptide detectable on Western blots with either p26 specific antibody, or with antibody to the His(6) epitope tag encoded by pRSETC. Full-length p26 in cell-free extracts of E. coli was about equal in mass to that found in Artemia embryos, but p26 lacking N- and C-terminal residues remained either as monomers or small multimers. All p26 constructs conferred thermotolerance on transformed E. coli, although not all formed oligomers, and cells expressing N-terminal truncated derivatives of p26 were more heat resistant than bacteria expressing p26 with C-terminal deletions. The C-terminal extension of p26 is seemingly more important for thermotolerance than is the N-terminus, and p26 protects E. coli against heat shock when oligomer size and protein concentration are low. The findings have important implications for understanding the functional mechanisms of small heat shock/alpha-crystallin proteins.     

A small heat-shock protein, p26, from the crustacean Artemia protects mammalian cells (Cos-1) against oxidative damage

A small heat-shock protein (p26) purified from stress-resistant embryos of the crustacean, Artemia franciscana, was introduced into cultured cells of green monkey kidney (Cos-1) using the BioPORTER delivery system. Cells containing p26 exhibited impressive resistance to hydrogen peroxide compared to controls. Introduction of the disaccharide trehalose did not provide protection against oxidative damage, but enhanced substantially the protective performance of p26 when both were present. These studies extend previous research on the protective role played by p26 in cells exposed to various forms of stress, presumably through its ability to function as a molecular chaperone.     

Characterization of novel sequence motifs within N- and C-terminal extensions of p26, a small heat shock protein from Artemia franciscana

The small heat shock proteins function as molecular chaperones, an activity often requiring reversible oligomerization and which protects against irreversible protein denaturation. An abundantly produced small heat shock protein termed p26 is thought to contribute to the remarkable stress resistance exhibited by encysted embryos of the crustacean, Artemia franciscana. Three novel sequence motifs termed G, R and TS were individually deleted from p26 by site-directed mutagenesis. G encompasses residues G8-G29, a glycine-enriched region, and R includes residues R36-R45, an arginine-enhanced sequence, both in the amino terminus. TS, composed of residues T169-T186, resides in the carboxy-extension and is augmented in threonine and serine. Deletion of R had more influence than removal of G on p26 oligomerization and chaperoning, the latter determined by thermotolerance induction in Escherichia coli, protection of insulin and citrate synthase from dithiothreitol- and heat-induced aggregation, respectively, and preservation of citrate synthase activity upon heating. Oligomerization of the TS and R variants was similar, but the TS deletion was slightly more effective than R as a chaperone. The extent of p26 structural perturbation introduced by internal deletions, including modification of intrinsic fluorescence, 1-anilino-8-naphthalene-sulphonate binding and secondary structure, paralleled reductions in oligomerization and chaperoning. Three-dimensional modeling of p26 based on wheat Hsp16.9 crystal structure indicated many similarities between the two proteins, including peptide loops associated with secondary structure elements. Loop 1 of p26 was deleted in the G variant with minimal effect on oligomerization and chaperoning, whereas loop 3, containing beta-strand 6 was smaller than the corresponding loop in Hsp16.9, which may influence p26 function.     

Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation.

Small heat shock proteins (sHsps) are a conserved and ubiquitous protein family. Their ability to convey thermoresistance suggests their participation in protecting the native conformation of proteins. However, the underlying functional principles of their protective properties and their role in concert with other chaperone families remain enigmatic. Here, we analysed the influence of Hsp25 on the inactivation and subsequent aggregation of a model protein, citrate synthase (CS), under heat shock conditions in vitro. We show that stable binding of several non-native CS molecules to one Hsp25 oligomer leads to an accumulation of CS unfolding intermediates, which are protected from irreversible aggregation. Furthermore, a number of different proteins which bind to Hsp25 can be isolated from heat-shocked extracts of cells. Under permissive folding conditions, CS can be released from Hsp25 and, in cooperation with Hsp70, an ATP-dependent chaperone, the native state can be restored. Taken together, our findings allow us to integrate sHsps functionally in the cellular chaperone system operating under heat shock conditions. The task of sHsps in this context is to efficiently trap a large number of unfolding proteins in a folding-competent state and thus create a reservoir of non-native proteins for an extended period of time, allowing refolding after restoration of physiological conditions in cooperation with other chaperones.     

Oligomerization, chaperone activity, and nuclear localization of p26, a small heat shock protein from Artemia franciscana

Artemia franciscana embryos undergo encystment, developmental arrest and diapause, the last characterized by profound metabolic dormancy and extreme stress resistance. Encysted embryos contain an abundant small heat shock protein termed p26, a molecular chaperone that undoubtedly has an important role in development. To understand better the role of p26 in Artemia embryos, the structural and functional characteristics of full-length and truncated p26 expressed in Escherichia coli and COS-1 cells were determined. p26 chaperone activity declined with increasing truncation of the protein, and those deletions with the greatest adverse effect on protection of citrate synthase during thermal stress had the most influence on oligomerization. When produced in either prokaryotic or eukaryotic cells the p26 alpha-crystallin domain consisting of amino acid residues 61-152 existed predominantly as monomers, and p26 variants lacking the amino-terminal domain but with intact carboxyl-terminal extensions were mainly monomers and dimers. The amino terminus was, therefore, required for efficient dimer formation. Assembly of higher order oligomers was enhanced by the carboxyl-terminal extension, although removing the 10 carboxyl-terminal residues had relatively little effect on oligomerization and chaperoning. Full-length and carboxyl-terminal truncated p26 resided in the cytoplasm of transfected COS-1 cells; however, variants missing the complete amino-terminal domain and existing predominantly as monomers/dimers entered the nuclei. A mechanism whereby oligomer disassembly assisted entry of p26 into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei during development and stress. However, when examined in Artemia, the p26 oligomer size was unchanged under conditions that allowed movement into nuclei, suggesting a process more complex than just oligomer dissociation.     

Hydroxybutyrate prevents protein aggregation in the halotolerant bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. CT13 under abiotic stress

Polyhydroxybutyrate (PHB), a typical carbon and energy storage compound, is widely found in Bacteria and Archae domains. This polymer is produced in response to conditions of physiological stress. PHB is composed of repeating units of β-hydroxybutyrate (R-3HB). It has been previously shown that R-3HB functions as an osmolyte in extremophile strains. In this study, Pseudomonas sp. CT13, a halotolerant bacterium, and its PHB synthase-minus mutant (phaC) were used to analyze the chaperone role of R-3HB. The production of this compound was found to be essential to salt stress resistance and positively correlated with salt concentration, suggesting that PHB monomer acts as a compatible solute in Pseudomonas sp. CT13. R-3HB accumulation was also associated with the prevention of protein aggregation under combined salt and thermal stresses in Pseudomonas sp. CT13. Physiological concentrations of R-3HB efficiently reduced citrate synthase (CS) aggregation and stabilized the enzymatic activities of CS during thermal stress. Docking analysis of the CS/R-3HB interaction predicted the stability of this complex under physiological concentrations of R-3HB. Thus, in vivo, in vitro and in silico analyses suggest that R-3HB can act as a chemical chaperone.     

Comparisons of stress proteins and soluble carbohydrate in encysted embryos of Artemia franciscana and two species of Parartemia

We compared stress proteins (p26, artemin, hsp70) and alcohol-soluble carbohydrates (ASC) in cysts of Artemia franciscana and two as yet un-named species populations of Parartemia, the brine shrimp endemic to Australia. The small stress proteins and molecular chaperones, p26 and artemin, previously thought to be restricted to Artemia, and present in very large amounts in its encysted embryos (cysts), were also detected by western blotting in Parartemia cysts, even though roughly 85-100 million years have passed since these genera diverged. We interpret this finding as further evidence for the adaptive importance of these proteins in coping with the severe stresses these encysted embryos endure. As expected, hsp70 was present in all three groups of cysts, but apparently at somewhat lower concentrations in those of Parartemia. Based on measurements of ASC we propose that the disaccharide trehalose, critical for desiccation tolerance in many animal cells, has probably also been maintained in the metabolic repertoire of Parartemia whose cysts have well developed tolerance to severe desiccation.     

Cloning and expression analysis of p26 gene in Artemia sinica

The protein p26 is a small heat shock protein that functions as a molecular chaperone to protect embryos by preventing irreversible protein damage during embryonic development. A 542 bp fragment of the p26 gene was cloned and sequenced. The fragment encoded 174 amino acid residues and the amino acid sequence contained the α-crystallin domain. Phylogenetic analysis showed that eight Artemia populations were divided into four major groups. Artemia sinica (YC) belonged to the East Asia bisexual group. Expression of the p26 gene at different developmental stages ofA. sinica was quantified using real-time quantitative polymerase chain reaction followed by cloning and sequencing. The relationship between the quantity of p26 gene expression and embryonic development was analyzed. The results indicated that massive amounts of p26 were expressed during the development of A. sinica. At the developmental stage of 0 h, A. sinica expressed the highest level of p26. As development proceeded, expression levels of the p26 gene reduced significantly. There was a small quantity of p26 gene expression at the developmental stages of 16 h and 24 h. We concluded that p26 might be involved in protecting the embryo from physiological stress during embryonic development.     

The translation initiation factor, PeIF5B, from Pisum sativum displays chaperone activity

We earlier documented the structural and functional characterization of PeIF5B factor from Pisum sativum that shows strong homology to the universal translation initiation factor eIF5B (Rasheedi et al., 2007, 2010 [12] and [13]). We now show that PeIF5B is an unusually thermo-stable protein resisting temperatures up to 95 °C. PeIF5B prevents thermal aggregation of heat labile proteins, such as citrate synthase (CS) and NdeI, under heat stress or chemical denaturation conditions and promotes their functional folding. It also prevents the aggregation of DTT induced insulin reduction. GTP appears to stimulate PeIF5B-mediated chaperone activity. In-vivo, PeIF5B over expression significantly enhances, the viability of Escherichia coli cells after heat stress (50 °C). These observations lead us to conclude that PeIF5B, in addition to its role in protein translation, has chaperone like activity and could be likely involved in protein folding and protection from stress.     

Structural and functional implications of C-terminal regions of alpha-synuclein

Aggregation of alpha-synuclein is thought to play a major role in the pathogenesis of Parkinson's disease (PD), which is characterized by the presence of intracytoplasmic Lewy bodies (LB) in the brain. alpha-Synuclein and its deletion mutants are largely unfolded proteins with random coil structures as revealed by CD spectra, fluorescence spectra, gel filtration chromatography, and ultracentrifugation. On the basis of its highly unfolded and flexible conformation, we have investigated the chaperone-like activity of alpha-synuclein in vitro. In our experiments, alpha-synuclein inhibited the aggregation of model substrates and protected the catalytic activity of alcohol dehydrogenase and rhodanese during heat stress. In addition, alpha-synuclein inhibited the initial aggregation of reduced/denatured lysozyme on the refolding pathway. Interestingly, deletion of the C-terminal regions led to the abolishment of chaperone activity, although largely unstructured conformations are maintained. Moreover, alpha-synuclein could inhibit the aggregation of various Escherichia coli cellular proteins during heat stress, and C-terminal deletion mutants could not provide any protection to these cellular proteins. Results with synthetic C-terminal peptides and C-terminal deletion mutants suggest that the second acidic repeat, (125)YEMPSEEGYQDYEPEA(140), is important for the chaperone activity of alpha-synuclein, and C-terminal deletion leads to the facilitated aggregation with the elimination of chaperone activity.