Extended conformational states dominate the Hsp90 chaperone dynamics

The heat shock protein 90 (Hsp90) is a molecular chaperone central to client protein folding and maturation in eukaryotic cells. During its chaperone cycle, Hsp90 undergoes ATPase-coupled large-scale conformational changes between open and closed states, where the N-terminal and middle domains of the protein form a compact dimerized conformation. However, the molecular principles of the switching motion between the open and closed states remain poorly understood. Here we show by integrating atomistic and coarse-grained molecular simulations with small-angle X-ray scattering experiments and NMR spectroscopy data that Hsp90 exhibits rich conformational dynamics modulated by the charged linker, which connects the N-terminal with the middle domain of the protein. We show that the dissociation of these domains is crucial for the conformational flexibility of the open state, with the separation distance controlled by a β-sheet motif next to the linker region. Taken together, our results suggest that the conformational ensemble of Hsp90 comprises highly extended states, which could be functionally crucial for client processing.     

Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB.

The major cold shock protein from Bacillus subtilis (CspB) was overexpressed using the bacteriophage T7 RNA polymerase/promoter system and purified to apparent homogeneity from recombinant Escherichia coli cells. CspB was crystallized in two different forms using vapor diffusion methods. The first crystal form obtained with ammonium sulfate as precipitant belongs to the trigonal crystal system, space group P3(1)21 (P3(2)21) with unit cell dimensions a = b = 59.1 A and c = 46.4 A. The second crystal form is tetragonal, space group P4(1)2(1)2 (P4(3)2(1)2) with unit cell dimensions a = b = 56.9 A and c = 53.0 A. These crystals grow with polyethylene glycol 4000 as precipitant.     

Evolving paradigms on the interplay of mitochondrial Hsp70 chaperone system in cell survival and senescence

Abstract

The role of mitochondria within a cell has grown beyond being the prime source of cellular energy to one of the major signaling platforms. Recent evidence provides several insights into the crucial roles of mitochondrial chaperones in regulating the organellar response to external triggers. The mitochondrial Hsp70 (mtHsp70/Mortalin/Grp75) chaperone system plays a critical role in the maintenance of proteostasis balance in the organelle. Defects in mtHsp70 network result in attenuated protein transport and misfolding of polypeptides leading to mitochondrial dysfunction. The functions of Hsp70 are primarily governed by J-protein cochaperones. Although human mitochondria possess a single Hsp70, its multifunctionality is characterized by the presence of multiple specific J-proteins. Several studies have shown a potential association of Hsp70 and J-proteins with diverse pathological states that are not limited to their canonical role as chaperones. The role of mitochondrial Hsp70 and its co-chaperones in disease pathogenesis has not been critically reviewed in recent years. We evaluated some of the cellular interfaces where Hsp70 machinery associated with pathophysiological conditions, particularly in context of tumorigenesis and neurodegeneration. The mitochondrial Hsp70 machinery shows a variable localization and integrates multiple components of the cellular processes with varied phenotypic consequences. Although Hsp70 and J-proteins function synergistically in proteins folding, their precise involvement in pathological conditions is mainly idiosyncratic. This machinery is associated with a heterogeneous set of molecules during the progression of a disorder. However, the precise binding to the substrate for a specific physiological response under a disease subtype is still an undocumented area of analysis.     

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.     

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     

Structural and functional analysis of the Hsp70/Hsp40 chaperone system

As one of the most abundant and highly conserved molecular chaperones, the 70‐kDa heat shock proteins (Hsp70s) play a key role in maintaining cellular protein homeostasis (proteostasis), one of the most fundamental tasks for every living organism. In this role, Hsp70s are inextricably linked to many human diseases, most notably cancers and neurodegenerative diseases, and are increasingly recognized as important drug targets for developing novel therapeutics for these diseases. Hsp40s are a class of essential and universal partners for Hsp70s in almost all aspects of proteostasis. Thus, Hsp70s and Hsp40s together constitute one of the most important chaperone systems across all kingdoms of life. In recent years, we have witnessed significant progress in understanding the molecular mechanism of this chaperone system through structural and functional analysis. This review will focus on this recent progress, mainly from a structural perspective.     

A domain in the N-terminal part of Hsp26 is essential for chaperone function and oligomerization

Small heat-shock proteins (Hsps) are ubiquitous molecular chaperones which prevent the unspecific aggregation of non-native proteins. For Hsp26, a cytosolic sHsp from of Saccharomyces cerevisiae, it has been shown that, at elevated temperatures, the 24 subunit complex dissociates into dimers. This dissociation is required for the efficient interaction with non-native proteins. Deletion analysis of the protein showed that the N-terminal half of Hsp26 (amino acid residues 1-95) is required for the assembly of the oligomer. Limited proteolysis in combination with mass spectrometry suggested that this region can be divided in two parts, an N-terminal segment including amino acid residues 1-30 and a second part ranging from residues 31-95. To analyze the structure and function of the N-terminal part of Hsp26 we created a deletion mutant lacking amino acid residues 1-30. We show that the oligomeric state and the structure, as determined by size exclusion chromatography and electron microscopy, corresponds to that of the Hsp26 wild-type protein. Furthermore, this truncated version of Hsp26 is active as a chaperone. However, in contrast to full length Hsp26, the truncated version dissociates at lower temperatures and complexes with non-native proteins are less stable than those found with wild-type Hsp26. Our results suggest that the N-terminal segment of Hsp26 is involved in both, oligomerization and chaperone function and that the second part of the N-terminal region (amino acid residues 31-95) is essential for both functions.     

Crystallization and preliminary x-ray diffraction studies of human procathepsin L

Human procathepsin L has been expressed in the yeast Pichia pastoris and its inactive (Cys25Ser) and unglycosylated (Thr110Ala) mutant purified, concentrated to 4 mg/ml, and crystallized by vapor diffusion against solution containing 1.4 M (Na, K)PO₄ buffer, pH 7.8. Crystal size was Increased by multiple macroseeding. The crystals are orthorhombic, of space group P2₁2₁2₁, with cell dimensions of a = 40.2 Å, b = 88.4 Å, and c = 94.9 Å. A 2.2 Å native data set was collected using synchrotron radiation. Although molecular replacement solution for the mature portion of the enzyme was easily found, the resulting maps could not be interpreted in the proregion. Heavy-atom derivative search is in progress. © 1996 Wiley-Liss, Inc.     

Induction of Hsp27 and Hsp32 Stress Proteins and Vimentin in Glial Cells of the Rat Hippocampus Following Hyperthermia

In response to stressful stimuli, cells respond by inducing a set of heat shock (stress) proteins (hsps) that play important roles in repair and protective mechanisms. The present study investigates the expression patterns of Hsp27 and Hsp32 in the adult rat hippocampus following whole body hyperthermia. A pronounced induction of these low-molecular-weight stress proteins was apparent in populations of glial cells such as astrocytes and microglia that were identified Using cell-specific markers (GFAP for astrocytes and the lectin GSA I-B4 for microglia). Hyperthermia also resulted in a robust induction of the intermediate filament protein, vimentin, in glial cells in the adult rat hippocampus. Interestingly, a rapid induction of both Hsp27 and vimentin was observed in the microvasculature, suggesting that hyperthermic stress may compromise the blood-brain barrier.     

苹果蠹蛾热激蛋白Hsp90基因的克隆及热胁迫下的表达分析

世界检疫性害虫苹果蠹蛾Cydia pomonella是一种温度耐受可塑性很高的物种。本研究针对温度波动可能导致其耐热性增强的科学问题, 采用生测法鉴定了苹果蠹蛾实验种群的高温耐受阈值, 采用同源克隆、 RACE和实时荧光定量PCR (RT-qPCR)等方法研究了苹果蠹蛾热激蛋白Hsp90基因的应激表达对耐热性的重要作用。高温耐受阈值研究结果表明, 苹果蠹蛾实验种群的死亡率随温度的升高和时间的延长显著性升高, 1-5龄幼虫分别经50℃和52℃高温处理2, 5和10 min后, 3龄幼虫耐热性最差, 5龄幼虫最强。50℃和52℃分别处理10 min和5 min均可导致1-4龄幼虫全部死亡, 而5龄幼虫在这两种处理下仍有25.0%和11.1%的存活率。以35℃处理的5龄雌幼虫为材料克隆苹果蠹蛾Hsp90基因全长cDNA, 结果显示该基因全长为2 470 bp, 完整开放阅读框为2 148 bp, 共编码716个氨基酸, 预测分子量为82.07 kDa, 命名为Cphsp90 (GenBank登录号JN624775)。该基因编码的氨基酸序列与亚洲玉米螟Ostrinia furnacalis和甘蓝夜蛾Mamestra brassicae等昆虫的Hsp90的氨基酸序列一致性高达96%, 表明了Hsp90家族的保守特性。Cphsp90 mRNA的相对表达量在32~44℃高温胁迫下随温度的升高而显著增高, 证实Cphsp90是诱导型热激基因, 且mRNA相对表达量与胁迫程度正相关。Cphsp90基因的表达还具有组织特异性, 35℃处理幼虫的表皮中Cphsp90相对表达量显著高于血淋巴、 脂肪体和中肠, 应激响应最为活跃。与未经温热预处理的昆虫相比, 35℃温热预处理3 h后的5龄幼虫在40, 45和50℃更高的温度胁迫下, Cphsp90 mRNA达到最高表达量所需要的胁迫温度有所提升, 由未经预热处理的40℃处理10 min提高到45℃处理10 min, 这与温热预处理会增强5龄幼虫耐热性的现象相符, 表明Cphsp90基因的响应表达在苹果蠹蛾耐热性及其可塑性过程中发挥重要的作用。     

Expression of the protein chaperone, clusterin, in spinal cord cells constitutively and following cellular stress, and upregulation by treatment with Hsp90 inhibitor

Clusterin, a protein chaperone found at high levels in physiological fluids, is expressed in nervous tissue and upregulated in several neurological diseases. To assess relevance to amyotrophic lateral sclerosis (ALS) and other motor neuron disorders, clusterin expression was evaluated using long-term dissociated cultures of murine spinal cord and SOD1^G93A transgenic mice, a model of familial ALS. Motor neurons and astrocytes constitutively expressed nuclear and cytoplasmic forms of clusterin, and secreted clusterin accumulated in culture media. Although clusterin can be stress inducible, heat shock failed to increase levels in these neural cell compartments despite robust upregulation of stress-inducible Hsp70 (HspA1) in non-neuronal cells. In common with HSPs, clusterin was upregulated by treatment with the Hsp90 inhibitor, geldanamycin, and thus could contribute to the neuroprotection previously identified for such compounds in disease models. Clusterin expression was not altered in cultured motor neurons expressing SOD1^G93A by gene transfer or in presymptomatic SOD1^G93A transgenic mice; however, clusterin immunolabeling was weakly increased in lumbar spinal cord of overtly symptomatic mice. More striking, mutant SOD1 inclusions, a pathological hallmark, were strongly labeled by anti-clusterin. Since secreted, as well as intracellular, mutant SOD1 contributes to toxicity, the extracellular chaperoning property of clusterin could be important for folding and clearance of SOD1 and other misfolded proteins in the extracellular space. Evaluation of chaperone-based therapies should include evaluation of clusterin as well as HSPs, using experimental models that replicate the control mechanisms operant in the cells and tissue of interest.     

Hsp90 chaperones have an energetic hot‐spot for binding inhibitors

Although Hsp90‐family chaperones have been extensively targeted with ATP‐competitive inhibitors, it is unknown whether high affinity is achieved from a few highly stabilizing contacts or from many weaker contacts within the ATP‐binding pocket. A large‐scale analysis of Hsp90α:inhibitor structures shows that inhibitor hydrogen‐bonding to a conserved aspartate (D93 in Hsp90α) stands out as most universal among Hsp90 inhibitors. Here we show that the D93 region makes a dominant energetic contribution to inhibitor binding for both cytosolic and organelle‐specific Hsp90 paralogs. For inhibitors in the resorcinol family, the D93:inhibitor hydrogen‐bond is pH‐dependent because the associated inhibitor hydroxyl group is titratable, rationalizing a linked‐protonation event previously observed by the Matulis group. The inhibitor hydroxyl group pK_a associated with the D93 hydrogen‐bond is therefore critical for optimizing the affinity of resorcinol derivatives, and we demonstrate that spectrophotometric measurements can determine this pK_a value. Quantifying the energetic contribution of the D93 hotspot is best achieved with the mitochondrial Hsp90 paralog, yielding 3–6 kcal/mol of stabilization (35–60% of the total binding energy) for a diverse set of inhibitors. The Hsp90 Asp93?Asn substitution has long been known to abolish nucleotide binding, yet puzzlingly, native sequences of structurally similar ATPases, such as Topoisomerasese II, have an asparagine at this same crucial site. While aspartate and asparagine sidechains can both act as hydrogen bond acceptors, we show that a steric clash prevents the Hsp90 Asp93?Asn sidechain from adopting the necessary rotamer, whereas this steric restriction is absent in Topoisomerasese II.     

Interdomain interactions dictate the function of the Candida albicans Hsp110 protein Msi3

Heat shock proteins of 110 kDa (Hsp110s), a unique class of molecular chaperones, are essential for maintaining protein homeostasis. Hsp110s exhibit a strong chaperone activity preventing protein aggregation (the “holdase” activity) and also function as the major nucleotide-exchange factor (NEF) for Hsp70 chaperones. Hsp110s contain two functional domains: a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). ATP binding is essential for Hsp110 function and results in close contacts between the NBD and SBD. However, the molecular mechanism of this ATP-induced allosteric coupling remains poorly defined. In this study, we carried out biochemical analysis on Msi3, the sole Hsp110 in Candida albicans, to dissect the unique allosteric coupling of Hsp110s using three mutations affecting the domain–domain interface. All the mutations abolished both the in vivo and in vitro functions of Msi3. While the ATP-bound state was disrupted in all mutants, only mutation of the NBD-SBDβ interfaces showed significant ATPase activity, suggesting that the full-length Hsp110s have an ATPase that is mainly suppressed by NBD-SBDβ contacts. Moreover, the high-affinity ATP-binding unexpectedly appears to require these NBD-SBD contacts. Remarkably, the “holdase” activity was largely intact for all mutants tested while NEF activity was mostly compromised, although both activities strictly depended on the ATP-bound state, indicating different requirements for these two activities. Stable peptide substrate binding to Msi3 led to dissociation of the NBD-SBD contacts and compromised interactions with Hsp70. Taken together, our data demonstrate that the exceptionally strong NBD-SBD contacts in Hsp110s dictate the unique allosteric coupling and biochemical activities.     

Further in vivo studies on the role of the molecular chaperone, Hsp93, in plastid protein import

In Arabidopsis, Hsp93 is encoded by two genes, atHSP93-V and atHSP93-III. We identified two T-DNA mutants for atHSP93-III: one being a partial 'knockdown' (hsp93-III-1) and the other a complete 'knockout' (hsp93-III-2). Homozygotes for both mutants were indistinguishable from wild type. We crossed each mutant to an atHSP93-V knockout, and identified double mutants with strongly chlorotic phenotypes. This implied redundancy, which was confirmed by the complementation of mildly chlorotic hsp93-V plants by atHSP93-III over-expression. While the hsp93-V hsp93-III-1 mutant was doubly homozygous, the second double mutant was heterozygous for hsp93-III-2 (genotype: hsp93-V/hsp93-V; +/hsp93-III-2). Attempts to identify an hsp93-V hsp93-III-2 double homozygote were unsuccessful, indicating that the Hsp93 pool is essential for viability. Consistently, siliques of the second double mutant contained aborted seeds (because of a block in the zygote-embryo transition) and failed ovules (because of a moderate defect in female gametophytes). Double-mutant plants were chlorophyll-deficient, contained under-developed chloroplasts, and exhibited stunted growth. In import assays using a chimeric pre-protein (plastocyanin transit peptide fused to dihydrofolate reductase; PC-DHFR), a clear defect was observed in hsp93-V hsp93-III-1 chloroplasts. Interestingly, while denaturation or stabilization of the DHFR moiety had a strong effect on import efficiency in the wild type, no such effects were observed with double-mutant (or tic40) chloroplasts. This indicated that pre-protein unfolding is not rate-limiting for import into mutant chloroplasts, and suggested that (unlike the situation in mitochondria) the inner membrane import machinery does not contribute to pre-protein unfolding at the organellar surface.     

Extracellular HspBP1 and Hsp72 synergistically activate epidermal growth factor receptor

Background information. Heat‐inducible Hsp72 is the founding member of the Hsp70 (heat shock proteins of 70 kDa) family of molecular chaperones. It is localized primarily in cytoplasm and nucleus but is also found extracellularly. The source of e‐Hsp72 (extracellular Hsp72) is not precisely identified and may not be the same in every situation. A number of studies demonstrated that e‐Hsp72 plays an important role in cell survival, tumour rejection and immune response. However, currently little is known about regulation of e‐Hsp72 function. In cells, Hsp72 is controlled by co‐chaperones. An abundant co‐chaperone, HspBP1 (Hsp72‐binding protein 1) was found extracellularly in the serum. In the present study we analysed the secretion and function of e‐HspBP1 (extracellular HspBP1). Results. A431 human squamous carcinoma cells accumulated Hsp72 and HspBP1 in chromogranin A‐positive granules following heat stress or in the presence of U73122, an inhibitor of phospholipase C. Following these treatments, A431 cells also increased the secretion of both proteins into the culture medium. The secreted e‐Hsp72 and e‐HspBP1 were co‐immunoprecipitated from the conditioned medium. Purified recombinant HspBP1 augmented e‐Hsp72‐mediated phosphorylation of EGFR (epidermal growth factor receptor) and its down‐stream targets, ERK1 (extracellular signal‐regulated kinase 1) and ERK2 in a concentration‐dependent manner. Finally, a HspBP1 N‐terminal domain deletion mutant and boiled recombinant HspBP1 did not affect the e‐Hsp72‐mediated activity. Conclusions. Heat stress and PLC (phospholipase C) inhibition result in the enhanced secretion of both Hsp72 and HspBP1. In an extracellular environment, the two chaperones interact both physically and functionally, leading to the activation of th EGFR—ERK1/2 signalling pathway. However, the magnitude of EGFR activation depends on the e‐HspBP1/e‐Hsp72 ratio in the medium. Extracellular chaperone‐mediated activation of EGFR can provide a survival advantage to cells under heat shock and other stresses.     

Purification,crystallization, and preliminary X-ray diffraction studies of tRNA-guanine transglycosylase from Zymomonas mobilis

The tRNA modifying enzyme tRNA–guanine transglycosylase (Tgt) catalyzes the exchange of guanine in the first position of the anticodon with the queuine precursor 7-aminomethyl-7-deazaguanine. Tgt from Zymomonas mobilis has been purified by crystallization and further recrystallized to obtain single crystals suitable for x-ray diffraction studies. Crystals were grown by vapor diffusion/gel crystallization methods using PEG 8,000 as precipitant. Macroseeding techniques were employed to produce large single crystals. The crystals of Tgt belong to the monoclinic space group C2 with cell constants a = 92.1 Å, b = 65.1 Å, c = 71.9 Å, and β = 97.5°, and contain one molecule per asymmetric unit. A complete diffraction data set from one native crystal has been obtained at 1.85 Å resolution.