Structure and function of the molecular chaperone Hsp104 from yeast

The molecular chaperone Hsp104 plays a central role in the clearance of aggregates after heat shock and the propagation of yeast prions. Hsp104's disaggregation activity and prion propagation have been linked to its ability to resolubilize or remodel protein aggregates. However, Hsp104 has also the capacity to catalyze protein aggregation of some substrates at specific conditions. Hence, it is a molecular chaperone with two opposing activities with respect to protein aggregation. In yeast models of Huntington's disease, Hsp104 is required for the aggregation and toxicity of polyglutamine (polyQ), but the expression of Hsp104 in cellular and animal models of Huntington's and Parkinson's disease protects against polyQ and α‐synuclein toxicity. Therefore, elucidating the molecular determinants and mechanisms underlying the ability of Hsp104 to switch between these two activities is of critical importance for understanding its function and could provide insight into novel strategies aimed at preventing or reversing the formation of toxic protein aggregation in systemic and neurodegenerative protein misfolding diseases. Here, we present an overview of the current molecular models and hypotheses that have been proposed to explain the role of Hsp104 in modulating protein aggregation and prion propagation. The experimental approaches and the evidences presented so far in relation to these models are examined. Our primary objective is to offer a critical review that will inspire the use of novel techniques and the design of new experiments to proceed towards a qualitative and quantitative understanding of the molecular mechanisms underlying the multifunctional properties of Hsp104 in vivo. © 2009 Wiley Periodicals, Inc. Biopolymers 93:252–276, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Zuotin, a DnaJ molecular chaperone, stimulates cap-independent translation in yeast

A small inhibitor RNA (IRNA) isolated from yeast has previously been shown to efficiently block poliovirus and hepatitis C virus IRES-mediated translation by sequestering mammalian RNA-binding (transacting) factors that play important roles in cap-independent translation. Here we have investigated the IRNA-binding proteins that might be involved in cap-independent translation in the yeast Saccharomyces cerevisiae. We have identified Zuotin, a DnaJ chaperone protein similar to mammalian HSP-40 chaperone, which interacts strongly with IRNA. Using ZUO1-deleted S. cerevisiae, we demonstrate a preferential requirement of Zuo1p for cap-independent translation mediated by the 5' untranslated region of the yeast TFIID mRNA. Further studies using zuo1delta S. cerevisiae complemented with various Zuo1p mutants indicate that the DnaJ domain of Zuo1p, known to influence its interaction with HSP-70, significantly affects cap-independent translation. These results demonstrate for the first time a role for an established chaperone protein in cap-independent translation of a cellular mRNA.     

The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.     

Thermotolerance and molecular chaperone function of an SGT1-like protein from the psychrophilic yeast, <Emphasis Type="Italic">Glaciozyma antarctica</Emphasis>

The ability of eukaryotes to adapt to an extreme range of temperatures is critically important for survival. Although adaptation to extreme high temperatures is well understood, reflecting the action of molecular chaperones, it is unclear whether these molecules play a role in survival at extremely low temperatures. The recent genome sequencing of the yeast Glaciozyma antarctica, isolated from Antarctic sea ice near Casey Station, provides an opportunity to investigate the role of molecular chaperones in adaptation to cold temperatures. We isolated a G. antarctica homologue of small heat shock protein 20 (HSP20), GaSGT1, and observed that the GaSGT1 mRNA expression in G. antarctica was markedly increased following culture exposure at low temperatures. Additionally, we demonstrated that GaSGT1 overexpression in Escherichia coli protected these bacteria from exposure to both high and low temperatures, which are lethal for growth. The recombinant GaSGT1 retained up to 60 % of its native luciferase activity after exposure to luciferase-denaturing temperatures. These results suggest that GaSGT1 promotes cell thermotolerance and employs molecular chaperone-like activity toward temperature assaults.     

BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast

Although transiently associated with numerous newly synthesized proteins, BiP has not been shown to be an essential component directly linked to the folding and oligomerization of newly synthesized proteins in the endoplasmic reticulum. To determine whether it is needed as a molecular chaperone, we analyzed the maturation of an endogenous yeast glycoprotein, carboxypeptidase Y (CPY) in several yeast strains with temperature-sensitive mutations in BiP. These kar2 mutant strains have previously been found to be defective in translocation at the nonpermissive temperature (Vogel, J. P., L. M. Misra, and M. D. Rose, 1990. J. Cell Biol, 110:1885-1895). To circumvent the translocation block, we used DTT at permissive temperature to delay folding and intracellular transport. We then followed the maturation of the ER- retained CPY after shifting to the nonpermissive temperature and dilution of the DTT. Without the functional chaperone, CPY aggregated, failed to be oxidized, and remained in the ER. In contrast to wild-type cells, in which BiP binding was transient with no more than 10-15% of labeled CPY associated at any time, 30-100% of the CPY remained associated with BiP in the mutant strains. In a heterozygous diploid strain, CPY matured and exited the ER normally. Taken together, the results provide clear evidence that BiP plays a critical role as a molecular chaperone in CPY folding.     

Protein disulfide isomerase serves as a molecular chaperone to maintain estrogen receptor alpha structure and function

The effects of the steroid hormone 17beta-estradiol are mediated through its interaction with the nuclear estrogen receptor (ER). Upon binding 17beta-estradiol, the ER initiates changes in gene expression through its interaction with specific DNA sequences, estrogen response elements (EREs), and recruits coregulatory proteins that influence gene expression. To better understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus ERE. One of these proteins, protein disulfide isomerase (PDI), has two distinct functions: acting as a molecular chaperone to maintain properly folded proteins and regulating the redox state of proteins by catalyzing the thiol-disulfide exchange reaction through two thioredoxin-like domains. Using a battery of biochemical and molecular techniques, we have demonstrated that PDI colocalizes with ERalpha in MCF-7 nuclei, alters ERalpha conformation, enhances the ERalpha-ERE interaction in the absence and presence of an oxidizing agent, influences the ability of ERalpha to mediate changes in gene expression, and associates with promoter regions of two endogenous estrogen-responsive genes. Our studies suggest that PDI plays a critical role in estrogen responsiveness by functioning as a molecular chaperone and assisting the receptor in differentially regulating target gene expression.     

HSP47 as a collagen-specific molecular chaperone: function and expression in normal mouse development

A large family of molecular chaperones can be divided into two major groups: general chaperone and substrate-specific chaperone. HSP47 is a collagen-specific molecular chaperone residing in the endoplasmic reticulum (ER). Recent studies revealed that HSP47 is essential molecular chaperone for mouse development and is essential for collagen molecular maturation in the ER. In the absence of HSP47, collagen microfibril formation and basement membrane formation are impaired in mouse embryos because the failure in the molecular maturation of types I and IV collagens, respectively. The tissue-specific expression of HSP47 is always correlated with that of various types of collagens and closely related with the collagen-related diseases including fibrosis in various organs. The importance of HSP47 in the therapeutic strategy for fibrotic diseases as well as for a marker of collagen-related autoimmune diseases will also be discussed.     

Development of yeast molecular display systems focused on therapeutic proteins, enzymes, and foods: functional analysis of proteins and its application to bioconversion

Molecular display systems using yeast have been developed for industrial, medical, pharmaceutical, and biological studies. Although several host cells are available to construct a molecular display system, the yeast Saccharomyces cerevisiae is a well-established and convenient organism in eukaryotes. A wide variety of prokaryotic and eukaryotic proteins have been displayed on yeast cell surfaces. In addition, functional analyses and applications to bioconversion have been performed on the cell surface, and cells are conveniently engineered by molecular display systems. In this review, we focus on the yeast molecular display system with regard to therapeutic proteins, several enzymes, and food ingredients. In addition, recent patents on molecular display using yeast cell for production of those compounds, screening technology and related techniques are introduced. Development of devices for functional analysis of created and modified proteins in the yeast display system is also described.     

Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H+ symporter

Candida intermedia PYCC 4715 was previously shown to grow well on xylose and to transport this sugar by two different transport systems: high-capacity and low-affinity facilitated diffusion and a high-affinity xylose-proton symporter, both of which accept glucose as a substrate. Here we report the isolation of genes encoding both transporters, designated GXF1 (glucose/xylose facilitator 1) and GXS1 (glucose/xylose symporter 1) respectively. Although GXF1 was isolated by functional complementation of an HXT-null (where Hxt refers to hexose transporters) Saccharomyces cerevisiae strain, isolation of the GXS1 cDNA required partial purification and micro-sequencing of the transporter, identified by its relative abundance in cells grown on low xylose concentrations. Both genes were expressed in S. cerevisiae and the kinetic parameters of glucose and xylose transport were determined. Gxs1 is the first yeast xylose/glucose-H+ symporter to be characterized at the molecular level. Comparison of its amino acid sequence with available sequence data revealed the existence of a family of putative monosaccharide-H+ symporters encompassing proteins from several yeasts and filamentous fungi.     

Role of glutathione peroxidase against oxidative stress in yeast: phenotypic character of lipid hydroperoxide-sensitive mutants derived from Hansenula mrakii

Wild type cells of Hansenula mrakii IFO 0895 were highly resistant to the oxidative stress caused by lipid hydroperoxide. The resistance was due to a glutathione peroxidase (GSHPx) which was induced when the yeast was cultured in a medium containing lipid hydroperoxide (Inoue, Y. et al., Agric. Biol. Chem., 54, 3289–3293, 1990). In order to investigate the role of GSHPx, two mutants sensitive to lipid hydroperoxide were isolated. The phenotypes of the mutants were temperature-dependent; i.e., the mutants could grow at 28°C, but not at 35°C in the presence of lipid hydroperoxide. The mutants failed to induce the GSHPx at 35°C. However, the enzyme induced at 28°C and prepared from both mutants was stable after incubation at 37°C for 1 h.     

Oligomerization and chaperone activity of a plant 2-Cys peroxiredoxin in response to oxidative stress

Plant 2-Cys peroxiredoxins (2-Cys Prxs) have been reported to localize to chloroplasts and perform antioxidative roles during plant development and photosynthesis. In this study, we identified that, in addition to the well-known function of thioredoxin (Trx)-dependent peroxidase, the plant 2-Cys Prx in Chinese cabbage 2-Cys Prx1, designated C2C-Prx1, also behaves as a molecular chaperone under oxidative stress conditions, like the yeast and mammalian 2-Cys Prxs. By the chaperone function of C2C-Prx1, the protein efficiently prevented the denaturation of citrate synthase and insulin from heat shock and dithiothreitol (DTT)-induced chemical stresses. Also, the protein structure of C2C-Prx1 was shown to have discretely sized multiple structures, whose molecular sizes were in the diverse ranges of low molecular weight (LMW) proteins to high molecular weight (HMW) protein complexes. The dual functions of C2C-Prx1 acting as a peroxidase and as a molecular chaperone are alternatively switched by heat shock and oxidative stresses, accompanying with its structural changes. The peroxidase function predominates in the lower MW forms, but the chaperone function predominates in the higher MW complexes. The precise regulation of C2C-Prx1 structures and functions may play a pivotal role in the protection of plant chloroplasts from photo-oxidative stress.     

Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

Molecular chaperones in aqueous‐organic mixtures can broaden the utility of biocatalysis by stabilizing enzymes in denaturing conditions. We have designed a self‐renaturing enzyme‐chaperone chimera consisting of penicillin amidase and a thermophilic chaperonin that functions in aqueous‐organic mixtures. The flexible linker separating the enzyme and chaperone domains was optimized and the design was extended to incorporate a chitin binding domain to facilitate immobilization of the chimera to a chitin support. The initial specific activity of penicillin amidase was not compromised by the enzyme‐chaperone fusion or by immobilization. The total turnover number of immobilized chimera for amoxicillin synthesis in aqueous‐methanol mixtures was 2.8 times higher after 95 h than the total turnover number of the immobilized penicillin amidase lacking a chaperone domain. Similarly, in 32% methanol the soluble chimera was active for over three times longer than the enzyme alone. This approach could easily be extended to other enzyme systems. Biotechnol. Bioeng. 2009;102: 1316–1322. © 2009 Wiley Periodicals, Inc.     

Divergence of function in the thioredoxin fold suprafamily: evidence for evolution of peroxiredoxins from a thioredoxin-like ancestor

The thioredoxin fold is found in proteins that serve a wide variety of functions. Among these are peroxiredoxins, which catalyze the reduction of hydrogen peroxide and alkyl peroxides. Although the common structural fold shared by thioredoxins and peroxiredoxins suggests the possibility that they have evolved from a common progenitor, it has been difficult to examine this hypothesis in depth because pairwise sequence identities between proteins in these two superfamilies are statistically insignificant. Using the Shotgun program, we have found that sequences of reductases involved in maturation of cytochromes in certain bacteria bridge the sequences of thioredoxins and peroxiredoxins. Analysis of motifs found in a divergent set of thioredoxins, cytochrome maturation proteins, and peroxiredoxins provides further support for an evolutionary relationship between these proteins. Within the conserved motifs are specific residues that are characteristic of individual protein classes, and therefore are likely to be involved in the specific functions of those classes. We have used this information, in combination with existing structural and functional information, to gain new insight into the structure-function relationships in these proteins and to construct a model for the emergence of peroxiredoxins from a thioredoxin-like ancestor.     

ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.

Hsp90 is an abundant molecular chaperone essential to the establishment of many cellular regulation and signal transduction systems, but remains one of the least well described chaperones. The biochemical mechanism of protein folding by Hsp90 is poorly understood, and the direct involvement of ATP has been particularly contentious. Here we demonstrate in vitro an inherent ATPase activity in both yeast Hsp90 and the Escherichia coli homologue HtpG, which is sensitive to inhibition by the Hsp90-specific antibiotic geldanamycin. Mutations of residues implicated in ATP binding and hydrolysis by structural studies abolish this ATPase activity in vitro and disrupt Hsp90 function in vivo. These results show that Hsp90 is directly ATP dependent in vivo, and suggest an ATP-coupled chaperone cycle for Hsp90-mediated protein folding.