Human and yeast Hsp110 chaperones exhibit functional differences

Hsp110 proteins constitute a heterogeneous family of abundant molecular chaperones, related to the Hsp70 proteins and exclusively found in the cytosol of eukaryotic organisms. Hsp110 family members are described as efficient holdases, preventing the aggregation and assisting the refolding of heat-denatured model substrates in the presence of Hsp70 chaperones and their co-chaperones. To gain more insights into the mode of action of this protein family we compared two homologues representing two subtypes of Hsp110 proteins, S. cerevisiae Sse1 and H. sapiens Apg-2, in their structural and functional properties in vitro. In contrast to previous publications both proteins exhibited intrinsic ATPase activities, which only in the case of Sse1 could be stimulated by the Hsp40 co-chaperone Sis1. Similar to Hsp70 proteins ATP binding and hydrolysis induced conformational rearrangements in both Hsp110 proteins as detected by tryptophane fluorescence. However, nucleotide induced changes in the proteolytic digestion pattern were detected only for Sse1. Sse1 and Apg-2 thus show significant differences in their biochemical properties, which may relate to differences in their functional roles in vivo.     

Hsp105 but not Hsp70 family proteins suppress the aggregation of heat-denatured protein in the presence of ADP

Hsp105alpha and Hsp105beta are mammalian members of the Hsp105/110 family, a diverged subgroup of the Hsp70 family. Here, we show that Hsp105alpha and Hsp105beta bind non-native protein through the beta-sheet domain and suppress the aggregation of heat-denatured protein in the presence of ADP rather than ATP. In contrast, Hsc70/Hsp40 suppressed the aggregation of heat-denatured protein in the presence of ATP rather than ADP. Furthermore, the overexpression of Hsp105alpha but not Hsp70 in COS-7 cells rescued the inactivation of luciferase caused by ATP depletion. Thus, Hsp105/110 family proteins are suggested to function as a substitute for Hsp70 family proteins to suppress the aggregation of denatured proteins in cells under severe stress, in which the cellular ATP level decreases markedly.     

Characterization of stress sensitivity and chaperone activity of Hsp105 in mammalian cells

Hsp105 is a major mammalian heat shock protein that belongs to the Hsp105/110 family, a diverged subgroup of the Hsp70 family. Hsp105 not only protects the thermal aggregation of proteins, but also regulates the Hsc70 chaperone system in vitro. Recently, it has been shown that Hsp105/110 family members act as nucleotide exchange factors for cytosolic Hsp70s. However, the biological functions of Hsp105/110 family proteins still remain to be clarified. Here, we examined the function of Hsp105 in mammalian cells, and showed that the sensitivity to various stresses was enhanced in the Hsp105-deficient cells compared with that in control cells. In addition, we found that deficiency of Hsp105 impaired the refolding of heat-denatured luciferase in mammalian cells. In contrast, overexpression of Hsp105α enhanced the ability to recover heat-inactivated luciferase in mammalian cells. Thus, Hsp105 may play an important role in the refolding of denatured proteins and protection against stress-induced cell death in mammalian cells.     

Cleavage at the carboxyl-terminus of Ku80 during apoptosis in human Jurkat T cells

We have previously reported that the amount of Apg-2, an Hsp110 family protein, decreases during apoptosis in Jurkat T cells. Since we hypothesized that Apg-2 would be cleaved by caspase-3 during apoptosis, a cleavage-site-directed antibody was raised against the carboxyl-terminus of the Apg-2 fragment that appears after the cleavage by caspase-3. Although this antibody could not detect the Apg-2 fragment in apoptotic cells, three additional fragments were unexpectedly detected. Based on the results of microsequencing, one of these fragments was identified as Ku80. Ku80 is a nuclear protein and a component of DNA-dependent protein kinase (DNA-PK). In this study, we observed that Ku80 is cleaved at Asp-730 residue during apoptosis, and this cleavage occurs in the nucleus in the early apoptotic phase. Furthermore, Ku80 is distributed in the cytoplasm of nuclear fragmented apoptotic cells, although the cleaved fragment contains the nuclear-localization signal (NLS). Our study clearly shows that Ku80 is cleaved in the nucleus, and distributes in the cytoplasm during apoptosis.     

Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana

The Arabidopsis genome contains at least 18 genes encoding members of the 70-kilodalton heat shock protein (Hsp70) family, 14 in the DnaK subfamily and 4 in the Hsp110/SSE subfamily. While the Hsp70s are highly conserved, a phylogenetic analysis including all members of this family in Arabidopsis and in yeast indicates the homology of Hsp70s in the subgroups, such as those predicted to localize in the same subcellular compartment and those similar to the mammalian Hsp110 and Grp170. Gene structure and genome organization suggest duplication in the origin of some genes. The Arabidopsis hsp70s exhibit distinct expression profiles; representative genes of the subgroups are expressed at relatively high levels during specific developmental stages and under thermal stress.     

Distinguishing integral and receptor-bound heat shock protein 70 (Hsp70) on the cell surface by Hsp70-specific antibodies

Cell Stress & Chaperones journal has become a major outlet for papers and review articles about anti-heat shock protein (HSP) antibodies. In the last decade, it became evident that apart from their intracellular localization, members of the heat shock protein 90 (Hsp90; HSPC) and Hsp70 (HSPA) family are also found on the cell surface. In this review, we will focus on Hsp70 (HSPA1A), the major stress-inducible member of the human Hsp70 family. Depending on the cell type, the membrane association of Hsp70 comes in two forms. In tumor cells, Hsp70 appears to be integrated within the plasma membrane, whereas in non-malignantly transformed (herein termed normal) cells, Hsp70 is associated with cell surface receptors. This observation raises the question whether or not these two surface forms of Hsp70 in tumor and normal cells can be distinguished using Hsp70 specific antibodies. Presently a number of Hsp70 specific antibodies are commercially available. These antibodies were generated by immunizing mice either with recombinant or HeLa-derived human Hsp70 protein, parts of the Hsp70 protein, or with synthetic peptides. This review aims to characterize the binding of different anti-human Hsp70 antibodies and their capacity to distinguish between integrated and receptor-bound Hsp70 in tumor and normal cells.     

Therapeutic inducers of the HSP70/HSP110 protect mice against traumatic brain injury

Traumatic brain injury (TBI) induces severe harm and disability in many accident victims and combat‐related activities. The heat‐shock proteins Hsp70/Hsp110 protect cells against death and ischemic damage. In this study, we used mice deficient in Hsp110 or Hsp70 to examine their potential requirement following TBI. Data indicate that loss of Hsp110 or Hsp70 increases brain injury and death of neurons. One of the mechanisms underlying the increased cell death observed in the absence of Hsp110 and Hsp70 following TBI is the increased expression of reactive oxygen species‐induced p53 target genes Pig1, Pig8, and Pig12. To examine whether drugs that increase the levels of Hsp70/Hsp110 can protect cells against TBI, we subjected mice to TBI and administered Celastrol or BGP‐15. In contrast to Hsp110‐ or Hsp70i‐deficient mice that were not protected following TBI and Celastrol treatment, there was a significant improvement of wild‐type mice following administration of these drugs during the first week following TBI. In addition, assessment of neurological injury shows significant improvement in contextual and cued fear conditioning tests and beam balance in wild‐type mice that were treated with Celastrol or BGP‐15 following TBI compared to TBI‐treated mice. These studies indicate a significant role of Hsp70/Hsp110 in neuronal survival following TBI and the beneficial effects of Hsp70/Hsp110 inducers toward reducing the pathological consequences of TBI.

     

The mammalian disaggregase machinery: Hsp110 synergizes with Hsp70 and Hsp40 to catalyze protein disaggregation and reactivation in a cell-free system

Bacteria, fungi, protozoa, chromista and plants all harbor homologues of Hsp104, a AAA+ ATPase that collaborates with Hsp70 and Hsp40 to promote protein disaggregation and reactivation. Curiously, however, metazoa do not possess an Hsp104 homologue. Thus, whether animal cells renature large protein aggregates has long remained unclear. Here, it is established that mammalian cytosol prepared from different sources possesses a potent, ATP-dependent protein disaggregase and reactivation activity, which can be accelerated and stimulated by Hsp104. This activity did not require the AAA+ ATPase, p97. Rather, mammalian Hsp110 (Apg-2), Hsp70 (Hsc70 or Hsp70) and Hsp40 (Hdj1) were necessary and sufficient to slowly dissolve large disordered aggregates and recover natively folded protein. This slow disaggregase activity was conserved to yeast Hsp110 (Sse1), Hsp70 (Ssa1) and Hsp40 (Sis1 or Ydj1). Hsp110 must engage substrate, engage Hsp70, promote nucleotide exchange on Hsp70, and hydrolyze ATP to promote disaggregation of disordered aggregates. Similarly, Hsp70 must engage substrate and Hsp110, and hydrolyze ATP for protein disaggregation. Hsp40 must harbor a functional J domain to promote protein disaggregation, but the J domain alone is insufficient. Optimal disaggregase activity is achieved when the Hsp40 can stimulate the ATPase activity of Hsp110 and Hsp70. Finally, Hsp110, Hsp70 and Hsp40 fail to rapidly remodel amyloid forms of the yeast prion protein, Sup35, or the Parkinson's disease protein, alpha-synuclein. However, Hsp110, Hsp70 and Hsp40 enhanced the activity of Hsp104 against these amyloid substrates. Taken together, these findings suggest that Hsp110 fulfils a subset of Hsp104 activities in mammals. Moreover, they suggest that Hsp104 can collaborate with the mammalian disaggregase machinery to rapidly remodel amyloid conformers.     

Characterization of Hsp70 binding and nucleotide exchange by the yeast Hsp110 chaperone Sse1

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related Hsp110 molecular chaperone family. Both mammalian Hsp110 and the Sse proteins functionally interact with cognate cytosolic Hsp70s as nucleotide exchange factors. We demonstrate here that Sse1 forms high-affinity (Kd approximately 10-8 M) heterodimeric complexes with both yeast Ssa and mammalian Hsp70 chaperones and that binding of ATP to Sse1 is required for binding to Hsp70s. Sse1.Hsp70 heterodimerization confers resistance to exogenously added protease, indicative of conformational changes in Sse1 resulting in a more compact structure. The nucleotide binding domains of both Sse1/2 and the Hsp70s dictate interaction specificity and are sufficient for mediating heterodimerization with no discernible contribution from the peptide binding domains. In support of a strongly conserved functional interaction between Hsp110 and Hsp70, Sse1 is shown to associate with and promote nucleotide exchange on human Hsp70. Nucleotide exchange activity by Sse1 is physiologically significant, as deletion of both SSE1 and the Ssa ATPase stimulatory protein YDJ1 is synthetically lethal. The Hsp110 family must therefore be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.     

Zebrafish heat shock protein a4 genes in the intestinal epithelium are up-regulated during inflammation

A number of heat shock proteins (HSPs), including Hsp70 and Hsp110, function as molecular chaperones within intestinal epithelial cells that line the mammalian digestive system. HSPs confer cellular protection against environmental stress induced by chemical toxins or pathogens. There is interest in how members of this protein family might influence the progression of inflammatory bowel disease. Using the zebrafish model system, we report the expression of the duplicated hspa4 genes within the intestinal epithelium. The hspa4 genes belong to the Hsp110 family. We show that under inflammatory stress conditions within the gut, expression of these genes is up-regulated in a similar manner to that previously observed for mammalian Hsp70. Because of the amenability of the zebrafish to whole-animal screening protocols, the hspa4 genes could be used as effective read-outs for genetic, chemical and environmental factors that might influence intestinal inflammation.     

Heteromeric complexes of heat shock protein 70 (HSP70) family members,including Hsp70B′, in differentiated human neuronal cells

Human neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis have been termed “protein misfolding disorders.” Upregulation of heat shock proteins that target misfolded aggregation-prone proteins has been proposed as a potential therapeutic strategy to counter neurodegenerative disorders. The heat shock protein 70 (HSP70) family is well characterized for its cytoprotective effects against cell death and has been implicated in neuroprotection by overexpression studies. HSP70 family members exhibit sequence and structural conservation. The significance of the multiplicity of HSP70 proteins is unknown. In this study, coimmunoprecipitation was employed to determine if association of HSP70 family members occurs, including Hsp70B′ which is present in the human genome but not in mouse and rat. Heteromeric complexes of Hsp70B′, Hsp70, and Hsc70 were detected in differentiated human SH-SY5Y neuronal cells. Hsp70B′ also formed complexes with Hsp40 suggesting a common co-chaperone for HSP70 family members.     

Human heat shock protein 105/110 kDa (Hsp105/110) regulates biogenesis and quality control of misfolded cystic fibrosis transmembrane conductance regulator at multiple levels

Heat shock protein 105/110-kDa (Hsp105/110), a member of the Hsp70 super family of molecular chaperones, serves as a nucleotide exchange factor for Hsc70, independently prevents the aggregation of misfolded proteins, and functionally relates to Hsp90. We investigated the roles of human Hsp105α, the constitutively expressed isoform, in the biogenesis and quality control of the cystic fibrosis transmembrane conductance regulator (CFTR). In the endoplasmic reticulum (ER), Hsp105 facilitates CFTR quality control at an early stage in its biosynthesis but promotes CFTR post-translational folding. Deletion of Phe-508 (ΔF508), the most prevalent mutation causing cystic fibrosis, interferes with de novo folding of CFTR, impairing its export from the ER and accelerating its clearance in the ER and post-Golgi compartments. We show that Hsp105 preferentially associates with and stabilizes ΔF508 CFTR at both levels. Introduction of the Hsp105 substrate binding domain potently increases the steady state level of ΔF508 CFTR by reducing its early-stage degradation. This in turn dramatically enhances ΔF508 CFTR cell surface functional expression in cystic fibrosis airway epithelial cells. Although other Hsc70 nucleotide exchange factors such as HspBP1 and BAG-2 inhibit CFTR post-translational degradation in the ER through cochaperone CHIP, Hsp105 has a primary role promoting CFTR quality control at an earlier stage. The Hsp105-mediated multilevel regulation of ΔF508 CFTR folding and quality control provides new opportunities to understand how chaperone machinery regulates the homeostasis and functional expression of misfolded proteins in the cell. Future studies in this direction will inform therapeutics development for cystic fibrosis and other protein misfolding diseases.     

The <Emphasis Type="Italic">Chlamydomonas</Emphasis> genome reveals its secrets: chaperone genes and the potential roles of their gene products in the chloroplast

The first draft of the Chlamydomonas nuclear genome was searched for genes potentially encoding members of the five major chaperone families, Hsp100/Clp, Hsp90, Hsp70, Hsp60, the small heat shock proteins, and the Hsp70 and Cpn60 co-chaperones GrpE and Cpn10/20, respectively. This search yielded 34 potential (co-)chaperone genes, among them those 8 that have been reported earlier inChlamydomonas. These 34 genes encode all the (co-)chaperones that have been expected for the different compartments and organelles from genome searches in Arabidopsis, where 74 genes have been described to encode basically the same set of (co-)chaperones. Genome data from Arabidopsis and Chlamydomonas on the five major chaperone families are compared and discussed, with particular emphasis on chloroplast chaperones.     

The metazoan protein disaggregase and amyloid depolymerase system

A baffling aspect of metazoan proteostasis is the lack of an Hsp104 ortholog that rapidly disaggregates and reactivates misfolded polypeptides trapped in stress induced disordered aggregates, preamyloid oligomers, or amyloid fibrils. By contrast, in bacteria, protozoa, chromista, fungi, and plants, Hsp104 orthologs are highly conserved and confer huge selective advantages in stress tolerance. Moreover, in fungi, the amyloid remodeling activity of Hsp104 has enabled deployment of prions for various beneficial modalities. Thus, a longstanding conundrum has remained unanswered: how do metazoan cells renature aggregated proteins or resolve amyloid fibrils without Hsp104? Here, we highlight recent advances that unveil the metazoan protein-disaggregase machinery, comprising Hsp110, Hsp70, and Hsp40, which synergize to dissolve disordered aggregates, but are unable to rapidly solubilize stable amyloid fibrils. However, Hsp110, Hsp70, and Hsp40 exploit the slow monomer exchange dynamics of amyloid, and can slowly depolymerize amyloid fibrils from their ends in a manner that is stimulated by small heat shock proteins. Upregulation of this system could have key therapeutic applications in various protein-misfolding disorders. Intriguingly, yeast Hsp104 can interface with metazoan Hsp110, Hsp70, and Hsp40 to rapidly eliminate disease associated amyloid. Thus, metazoan proteostasis is receptive to augmentation with exogenous disaggregases, which opens a number of therapeutic opportunities.     

棉花粉蚧热休克蛋白基因的鉴定

热休克蛋白(heat shock proteins,Hsps)是生物体或细胞受到热胁迫后新合成的一类遗传上高度保守的蛋白,在昆虫应对外界环境因子胁迫时起着重要作用。为了系统研究棉花粉蚧Phenacoccus solenopsis Hsp基因家族,对棉花粉蚧转录组基因注释信息进行分析、获得目标序列,并应用NCBI上Blast X等软件进行比对、共鉴定出24条热激蛋白(Hsp)基因,包括3个Hsp90、8个Hsp70、2个Hsp60和11个s Hsp(small heat shock protein,s Hsp)基因。对棉花粉蚧与模式昆虫家蚕Bombyx mori、黑腹果蝇Drosophila melanogaster、赤拟谷盗Tribolium castaneum系统进化关系分析显示,昆虫的小分子量热休克蛋白s Hsp具有很强的种属特异性,Hsp70家族的保守性比s Hsp强。棉花粉蚧热激蛋白基因的鉴定为深入研究该虫Hsp与生长发育、抗逆境的相互关系奠定了基础。     

Prokaryotic chaperones support yeast prions and thermotolerance and define disaggregation machinery interactions

Saccharomyces cerevisiae Hsp104 and Escherichia coli ClpB are Hsp100 family AAA+ chaperones that provide stress tolerance by cooperating with Hsp70 and Hsp40 to solubilize aggregated protein. Hsp104 also remodels amyloid in vitro and promotes propagation of amyloid prions in yeast, but ClpB does neither, leading to a view that Hsp104 evolved these activities. Although biochemical analyses identified disaggregation machinery components required for resolubilizing proteins, interactions among these components required for in vivo functions are not clearly defined. We express prokaryotic chaperones in yeast to address these issues and find ClpB supports both prion propagation and thermotolerance in yeast if it is modified to interact with yeast Hsp70 or if E. coli Hsp70 and its cognate nucleotide exchange factor (NEF) are present. Our findings show prion propagation and thermotolerance in yeast minimally require cooperation of species-specific Hsp100, Hsp70, and NEF with yeast Hsp40. The functions of this machinery in prion propagation were directed primarily by Hsp40 Sis1p, while thermotolerance relied mainly on Hsp40 Ydj1p. Our results define cooperative interactions among these components that are specific or interchangeable across life kingdoms and imply Hsp100 family disaggregases possess intrinsic amyloid remodeling activity.     

The structural and functional diversity of Hsp70 proteins from Plasmodium falciparum

It is becoming increasingly apparent that heat shock proteins play an important role in the survival of Plasmodium falciparum against temperature changes associated with its passage from the cold-blooded mosquito vector to the warm-blooded human host. Interest in understanding the possible role of P. falciparum Hsp70s in the life cycle of the parasite has led to the identification of six HSP70 genes. Although most research attention has focused primarily on one of the cytosolic Hsp70s (PfHsp70-1) and its endoplasmic reticulum homolog (PfHsp70-2), further functional insights could be inferred from the structural motifs exhibited by the rest of the Hsp70 family members of P. falciparum. There is increasing evidence that suggests that PfHsp70-1 could play an important role in the life cycle of P. falciparum both as a chaperone and immunogen. In addition, P. falciparum Hsp70s and Hsp40 partners are implicated in the intracellular and extracellular trafficking of proteins. This review summarizes data emerging from studies on the chaperone role of P. falciparum Hsp70s, taking advantage of inferences gleaned from their structures and information on their cellular localization. The possible associations between P. falciparum Hsp70s with their cochaperone partners as well as other chaperones and proteins are discussed.