Comparison of the small heat shock proteins alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP in heart and skeletal muscle

Seven members of the small heat shock protein (sHSP) family are exceptional with respect to their constitutive high abundance in muscle tissue. It has been suggested that sHSPs displaying chaperone-like properties may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. In the present study five sHSPs (alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP) were investigated with respect to similarities and differences of their expression in heart and skeletal muscle under normal and ischemic conditions. In ischemic heart and skeletal muscle these five sHSPs translocated from cytosol to the Z-/I-area of myofibrils. Myofibrillar binding of all sHSPs was very tight and resisted for the most part extraction with 1 M NaSCN or 1 M urea. MKBP and HSP20 became extracted by 1 M NaSCN to a significant extent indicating that these two sHSPs may bind partially to actin-associated proteins which were completely extracted by this treatment. Ultrastructural localization of alphaB-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes alphaB-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of alphaB-crystallin in cardiomyocytes versus skeletal muscle fibers. Further differences of the properties of sHSPs could be observed regarding fiber type distribution of sHSPs. Thus sHSPs form a complex stress-response system in striated muscle tissue with some common as well as some distinct functions in different muscle types.     

Muscle develops a specific form of small heat shock protein complex composed of MKBP/HSPB2 and HSPB3 during myogenic differentiation

Previously, we identified a new mammalian sHSP, MKBP, as a myotonic dystrophy protein kinase-binding protein, and suggested its important role in muscle maintenance (Suzuki, A., Sugiyama, Y., Hayashi, Y., Nyu-i, N., Yoshida, M., Nonaka, I., Ishiura, S., Arahata, K., and Ohno, S. (1998) J. Cell Biol. 140, 1113-1124). In this paper, we develop the former work by performing extensive characterization of five of the six sHSPs so far identified, that is, HSP27, alphaB-crystallin, p20, MKBP/HSPB2, and HSPB3, omitting lens-specific alphaA-crystallin. Tissue distribution analysis revealed that although each sHSP shows differential constitutive expression in restricted tissues, tissues that express all five sHSPs are only muscle-related tissues. Especially, the expressions of HSPB3, identified for the first time as a 17-kDa protein in this paper, and MKBP/HSPB2 are distinctly specific to muscles. Moreover, these sHSPs form an oligomeric complex with an apparent molecular mass of 150 kDa that is completely independent of the oligomers formed by HSP27, alphaB-crystallin, and p20. The expressions of MKBP/HSPB2 and HSPB3 are induced during muscle differentiation under the control of MyoD, suggesting that the sHSP oligomer comprising MKBP/HSPB2 and HSPB3 represents an additional system closely related to muscle function. The functional divergence among sHSPs in different oligomers is also demonstrated in several ways: 1) an interaction with myotonic dystrophy protein kinase, which has been suggested to be important for the maintenance of myofibril integrity, was observed only for MKBP/HSPB2; 2) a myotube-specific association with actin bundles was observed for HSP27 and alphaB-crystallin, but not for MKBP/HSPB2; and 3) sHSPs whose mRNAs are induced by heat shock are alphaB-crystallin and HSP27. Taken together, the results suggest that muscle cells develop two kinds of stress response systems composed of diverged sHSP members, and that these systems work independently in muscle maintenance and differentiation.     

Interaction of human HSP22 (HSPB8) with other small heat shock proteins

Mammalian small heat shock proteins (sHSP) are abundant in muscles and are implicated in both muscle function and myopathies. Recently a new sHSP, HSP22 (HSPB8, H11), was identified in the human heart by its interaction with HSP27 (HSPB1). Using phylogenetic analysis we show that HSP22 is a true member of the sHSP superfamily. sHSPs interact with each other and form homo- and hetero-oligomeric complexes. The function of these complexes is poorly understood. Using gel filtration HPLC, the yeast two-hybrid method, immunoprecipitation, cross-linking, and fluorescence resonance energy transfer microscopy, we report that (i). HSP22 forms high molecular mass complexes in the heart, (ii). HSP22 interacts with itself, cvHSP (HSPB7), MKBP (HSPB2) and HSP27, and (iii). HSP22 has two binding domains (N- and C-terminal) that are specific for different binding partners. HSP22 homo-dimers are formed through N-N and N-C interactions, and HSP22-cvHSP hetero-dimers through C-C interaction. HSP22-MKBP and HSP22-HSP27 hetero-dimers involve the N and C termini of HSP22 and HSP27, respectively, but appear to require full-length protein as a binding partner.     

The emerging role of HSP20 as a multifunctional protective agent

The small heat shock proteins (sHSPs) are a highly conserved family of molecular chaperones that are ubiquitously expressed throughout nature. They are transiently upregulated in many tissue types following stressful stimuli. Recently, one member of the sHSP family, HSP20 (HspB6), has been shown to be highly effective as a protective mediator against a number of debilitating pathological conditions, including cardiac hypertrophy and Alzheimer's disease. Hsp20 is also an important modulator of vital physiological processes, such as smooth muscle relaxation and cardiac contractility. This review focuses on the molecular mechanisms employed by HSP20 that allow it to act as an innate protector in the context of cardiovascular and neurological diseases. Emerging evidence for a possible role as an anti-cancer agent is also presented.     

The Small Heat-Shock Proteins HSPB2 and HSPB3 Form Well-defined Heterooligomers in a Unique 3 to 1 Subunit Ratio

Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from Escherichia coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintaining a strict 3:1 HSPB2/HSPB3 subunit ratio. These complexes are thermally stable up to 40 °C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or αB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could associate efficiently with HSP20. Taken altogether, this study provides evidence that, despite the high level of sequence homology within the sHSP family the biochemical properties of the HSPB2/B3 complex are distinctly different from those of other sHSPs, indicating that the HSPB2/B3 assembly is likely to possess cellular functions other than those of its family members.     

Regulation of the levels of small heat-shock proteins during differentiation of C2C12 cells

Levels of the small heat-shock proteins (sHSPs) HSP27 and alphaB-crystallin during differentiation of mouse C2C12 cells were determined using specific immunoassays. Increases of these proteins were about 3-fold and 10-fold, respectively. Under the same conditions, however, the level of HSP70 in C2C12 cells barely increased, indicating selective accumulation of HSP27 and alphaB-crystallin with differentiation. While expression of mRNA for alphaB-crystallin was also markedly increased and that for HSP27 was but to a lesser extent, mRNA for HSP70 could barely be detected during differentiation. Activation of the heat-shock factor was not observed, in contrast to the case with heat-stressed undifferentiated cells. Various inhibitors of protein kinases affected the differentiation and the associated increase of sHSPs. Rapamycin, an inhibitor of p70 S6 kinase, completely inhibited the differentiation and suppressed the accumulation of HSP27 and alphaB-crystallin. SB203580, an inhibitor of p38 MAP kinase, also inhibited differentiation, but the accumulation of alphaB-crystallin was rather enhanced. PD98059, an inhibitor of MAP kinase kinase, significantly increased expression of a differentiation marker for muscle cells, creatine kinase M isozyme, as well as accumulation of alphaB-crystallin. These results suggest that accumulation of sHSPs during differentiation of C2C12 cells is regulated in a complex manner.     

小菜蛾热休克蛋白基因的鉴定及其表达模式分析

热休克蛋白(heat shock protein, HSP)在昆虫应对外界胁迫刺激时起着重要作用。为了系统研究小菜蛾Plutella xylostella HSP基因家族, 根据家蚕的HSP蛋白序列, 采用本地Blast程序对小菜蛾全基因组数据库进行同源序列检索, 从小菜蛾基因组数据库中鉴定了25个HSP基因, 包括2个HSP90、 8个HSP70和15个sHSP（small heat shock protein, sHSP）基因。小菜蛾、 家蚕Bombyx mori、 黑腹果蝇Drosophila melanogaster和赤拟谷盗Tribolium castaneum的HSP系统进化分析显示, 昆虫的小分子量热休克蛋白sHSP具有很强的种属特异性, HSP70家族的保守性比sHSP强。小菜蛾HSP基因表达模式分析显示, 与敏感品系对比, 抗性品系（抗毒死蜱和抗氟虫氰品系）中HSP基因具有不同的表达模式。小菜蛾1, 2和3龄幼虫HSP基因表达模式较为接近, 而与4龄幼虫中的表达模式相差较大; 4龄幼虫和蛹中的表达模式相近; 雌成虫和雄成虫中的表达模式显著不同, 与果蝇精子形成有关的两个热休克蛋白HSP23和HSP27基因［分别为CCG003980.1 (Px23.5)和CCG005412.2 (Px27.5)］, 在小菜蛾雄成虫中的表达量显著高于雌成虫。研究结果表明小菜蛾HSP基因不仅在杀虫剂抗性、 发育分化, 甚至在生殖上均可能起着重要的作用。本研究为深入研究小菜蛾HSP与生长发育、 抗逆行为的相互关系奠定了基础。     

真菌HSP30的研究概况

热休克蛋白30是小分子热休克蛋白(small heat shock proteins,sHSPs)中的一种,也是真菌中研究最广泛的小分子热休克蛋白。多种真菌编码热休克蛋白的基因序列已经被克隆和检测,HSP30的研究主要集中在应激水平下的表达和转录水平的调控,HSP30在应激反应中的合成机制仍不是很清楚,综述了它的研究概况以及应用前景。     

Small heat-shock protein structures reveal a continuum from symmetric to variable assemblies

The small heat-shock proteins (sHSPs) form a diverse family of proteins that are produced in all organisms. They function as chaperone-like proteins in that they bind unfolded polypeptides and prevent uncontrolled protein aggregation. Here, we present parallel cryo-electron microscopy studies of five different sHSP assemblies: Methanococcus jannaschii HSP16.5, human alphaB-crystallin, human HSP27, bovine native alpha-crystallin, and the complex of alphaB-crystallin and unfolded alpha-lactalbumin. Gel-filtration chromatography indicated that HSP16.5 is the most monodisperse, while HSP27 and the alpha-crystallin assemblies are more polydisperse. Particle images revealed a similar trend showing mostly regular and symmetric assemblies for HSP16.5 particles and the most irregular assemblies with a wide range of diameters for HSP27. A symmetry test on the particle images indicated stronger octahedral symmetry for HSP16.5 than for HSP27 or the alpha-crystallin assemblies. A single particle reconstruction of HSP16.5, based on 5772 particle images with imposed octahedral symmetry, resulted in a structure that closely matched the crystal structure. In addition, the cryo-EM reconstruction revealed internal density presumably corresponding to the flexible 32 N-terminal residues that were not observed in the crystal structure. The N termini were found to partially fill the central cavity making it unlikely that HSP16.5 sequesters denatured proteins in the cavity. A reconstruction calculated without imposed symmetry confirmed the presence of at least loose octahedral symmetry for HSP16.5 in contrast to the other sHSPs examined, which displayed no clear overall symmetry. Asymmetric reconstructions for the alpha-crystallin assemblies, with an additional mass selection step during image processing, resulted in lower resolution structures. We interpret the alpha-crystallin reconstructions to be average representations of variable assemblies and suggest that the resolutions achieved indicate the degree of variability. Quaternary structural information derived from cryo-electron microscopy is related to recent EPR studies of the alpha-crystallin domain fold and dimer interface of alphaA-crystallin.     

Roles for alphaB-crystallin and HSPB2 in protecting the myocardium from ischemia-reperfusion-induced damage in a KO mouse model

Overexpression studies have shown that the small heat shock proteins (sHSP) protect the myocardium from ischemia-reperfusion (I/R)-induced damage. However, gene deletion studies are necessary to demonstrate whether sHSPs are required for protection. The genes for alphaB-crystallin (alphaBC) and HSPB2, two sHSPs that are expressed in high levels in the heart, are in close proximity to one another; as a result, both genes were disrupted in a recently generated knockout (KO) mouse line. The alphaBC/HSPB2 KO mouse line is currently the only model that features disruption of sHSPs normally expressed in the heart. Accordingly, we examined the cardiac morphology, function, and response to I/R-induced stress in alphaBC-HSPB2 KO mice. Initial gross, light microscopic and echocardiographic characterization showed that the morphological and functional properties of hearts from adult KO mice were indistinguishable from age-matched wild-type (WT) mice. Electron microscopy showed that, compared with WT mouse hearts, KO mouse heart sarcomeres were relatively normal. Isolated perfused KO mouse hearts displayed normal contractility; however, when compared with WT, after I/R, KO mouse hearts exhibited a twofold reduction in contractile recovery, as well as increased necrosis and apoptosis. Additionally, when compared with WT, KO mouse hearts exhibited 43% less reduced glutathione, which is known to protect from I/R-induced damage. Thus, whereas neither alphaBC nor HSPB2 is essential for myocardial development and function under nonstressful conditions, one or both are required for maximal functional recovery and protection from I/R-induced necrosis and apoptosis.     

Three-dimensional structure of α-crystallin domain dimers of human small heat shock proteins HSPB1 and HSPB6

Small heat shock proteins (sHSPs) are a family of evolutionary conserved ATP-independent chaperones. These proteins share a common architecture defined by a signature α-crystallin domain (ACD) flanked by highly variable N- and C-terminal extensions. The ACD, which has an immunoglobulin-like fold, plays an important role in sHSP assembly. This domain mediates dimer formation of individual protomers, which then may assemble into larger oligomers. In vertebrate sHSPs, the dimer interface is formed by the symmetrical antiparallel pairing of two β-strands (β7), generating an extended β-sheet on one face of the ACD dimer. Recent structural studies of isolated ACDs from a number of vertebrate sHSPs suggest a variability in the register of the β7/β7 strand interface, which may, in part, give rise to the polydispersity often associated with the full-length proteins. To further analyze the structure of ACD dimers, we have employed a combination of X-ray crystallography and solution small-angle X-ray scattering (SAXS) to study the ACD-containing fragments of human HSPB1 (HSP27) and HSPB6 (HSP20). Unexpectedly, the obtained crystal structure of the HSPB1 fragment does not reveal the typical β7/β7 dimers but, rather, hexamers formed by an asymmetric contact between the β4 and the β7 strands from adjacent ACDs. Nevertheless, in solution, both ACDs form stable dimers via the symmetric antiparallel interaction of β7 strands. Using SAXS, we show that it is possible to discriminate between different putative registers of the β7/β7 interface, with the results indicating that, under physiological conditions, there is only a single register of the strands for both proteins.     

Comparison of the small heat shock proteins αB-crystallin, MKBP, HSP25, HSP20, and cvHSP in heart and skeletal muscle

Seven members of the small heat shock protein (sHSP) family are exceptional with respect to their constitutive high abundance in muscle tissue. It has been suggested that sHSPs displaying chaperone-like properties may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. In the present study five sHSPs (B-crystallin, MKBP, HSP25, HSP20, and cvHSP) were investigated with respect to similarities and differences of their expression in heart and skeletal muscle under normal and ischemic conditions. In ischemic heart and skeletal muscle these five sHSPs translocated from cytosol to the Z-/I-area of myofibrils. Myofibrillar binding of all sHSPs was very tight and resisted for the most part extraction with 1 M NaSCN or 1 M urea. MKBP and HSP20 became extracted by 1 M NaSCN to a significant extent indicating that these two sHSPs may bind partially to actin-associated proteins which were completely extracted by this treatment. Ultrastructural localization of B-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes B-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of B-crystallin in cardiomyocytes versus skeletal muscle fibers. Further differences of the properties of sHSPs could be observed regarding fiber type distribution of sHSPs. Thus sHSPs form a complex stress–response system in striated muscle tissue with some common as well as some distinct functions in different muscle types.     

The N-terminal arm of small heat shock proteins is important for both chaperone activity and substrate specificity

Small heat shock proteins (sHSPs) are a ubiquitous class of molecular chaperones that interacts with substrates to prevent their irreversible insolubilization during denaturation. How sHSPs interact with substrates remains poorly defined. To investigate the role of the conserved C-terminal alpha-crystallin domain versus the variable N-terminal arm in substrate interactions, we compared two closely related dodecameric plant sHSPs, Hsp18.1 and Hsp16.9, and four chimeras of these two sHSPs, in which all or part of the N-terminal arm was switched. The efficiency of substrate protection and formation of sHSP-substrate complexes by these sHSPs with three different model substrates, firefly luciferase, citrate synthase, and malate dehydrogenase (MDH) provide new insights into sHSP/substrate interactions. Results indicate that different substrates have varying affinities for different domains of the sHSP. For luciferase and citrate synthase, the efficiency of substrate protection was determined by the identity of the N-terminal arm in the chimeric proteins. In contrast, for MDH, efficient protection clearly required interactions with the alpha-crystallin domain in addition to the N-terminal arm. Furthermore, we show that sHSP-substrate complexes with varying stability and composition can protect substrate equally, and substrate protection is not correlated with sHSP oligomeric stability for all substrates. Protection of MDH by the dimeric chimera composed of the Hsp16.9 N-terminal arm and Hsp18.1 alpha-crystallin domain supports the model that a dimeric form of the sHSP can bind and protect substrate. In total, results demonstrate that sHSP-substrate interactions are complex, likely involve multiple sites on the sHSP, and vary depending on substrate.     

Analytical assays of human HSP27 and thermal-stress survival of Escherichia coli cells that overexpress it

HSP27 is a small heat-shock protein (sHSP). Such proteins are produced in all organisms. These small HSPs exhibit chaperone-like activity that can bind to unfolded polypeptides and prevent uncontrolled protein aggregation in vitro. Cellular anti-apoptosis function and enhanced cell survival are correlated with increased expression of HSPs. This study presents a thermal-stress survival model for cells using the Escherichia coli expression system for which human HSP27, a recombinant protein, is inducible. Results show that E. coli cells overexpressing human HSP27 have enhanced tolerance to 50 degrees C thermal stress.     

Changes in oligomerization are essential for the chaperone activity of a small heat shock protein in vivo and in vitro

The ability of small heat shock proteins (sHSPs) to prevent thermal aggregation of other proteins may require disassembly and reassembly of sHSP oligomers. We investigated the role of changes in sHSP oligomerization by studying a mutant with reduced oligomeric stability. In HSP16.6, the single sHSP in the cyanobacterium Synechocystis sp. PCC 6803, the mutation L66A causes oligomer instability and reduced chaperone activity in vitro. Because thermotolerance of Synechocystis depends on HSP16.6, a phenotype that is enhanced in a deltaClpB1 strain, the effect of mutations can also be assayed in vivo. L66A causes severe defects in thermotolerance, suggesting that oligomeric stability of sHSPs is required for cellular function. This hypothesis was supported by a selection for intragenic suppressors of L66A, which identified mutations that stabilize oligomers of both L66A and wild-type HSP16.6. Analysis of both over- and under-oligomerizing mutants suggests that sHSPs must disassemble before they can release substrates. Furthermore, the suppressor mutations not only restore in vivo activity to L66A, they also ameliorate chaperone defects in vitro, and thus provide the first direct evidence for a chaperone function of an sHSP in cellular thermotolerance.     

MKBP, a Novel Member of the Small Heat Shock Protein Family, Binds and Activates the Myotonic Dystrophy Protein Kinase

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as αB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by αB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.     

Proline isomerization in the C-terminal region of HSP27

In mammals, small heat-shock proteins (sHSPs) typically assemble into interconverting, polydisperse oligomers. The dynamic exchange of sHSP oligomers is regulated, at least in part, by molecular interactions between the α-crystallin domain and the C-terminal region (CTR). Here we report solution-state nuclear magnetic resonance (NMR) spectroscopy investigations of the conformation and dynamics of the disordered and flexible CTR of human HSP27, a systemically expressed sHSP. We observed multiple NMR signals for residues in the vicinity of proline 194, and we determined that, while all observed forms are highly disordered, the extra resonances arise from cis-trans peptidyl-prolyl isomerization about the G193-P194 peptide bond. The cis-P194 state is populated to near 15% at physiological temperatures, and, although both cis- and trans-P194 forms of the CTR are flexible and dynamic, both states show a residual but differing tendency to adopt β-strand conformations. In NMR spectra of an isolated CTR peptide, we observed similar evidence for isomerization involving proline 182, found within the IPI/V motif. Collectively, these data indicate a potential role for cis-trans proline isomerization in regulating the oligomerization of sHSPs.     

The role of the heat shock proteins (HSP70 and sHSP) in the thermotolerance of freshwater amphipods from contrasting habitats

The aim of this study was to clarify the significance of HSP70 and sHSP for thermotolerance in freshwater amphipods. We compared four amphipod species from different freshwater habitats and biogeographical regions (Central Europe vs. Lake Baikal). Test individuals were exposed to thermal stress generated by a water temperature of 25 °C. The thermotolerance of the species, determined by median lethal time (LT50), followed in decreasing order by Gmelinoides fasciatus, Echinogammarus berilloni, Gammarus pulex, Eulimnogammarus verrucosus. HSP70 and sHSP base level concentrations for the species were determined at control (i.e. non-stress) conditions. For HSP70, the base levels were positively correlated to the species' thermotolerances. For sHSP, however, only thermotolerant G. fasciatus showed a high level. Thermal stress at 25 °C water temperature caused a deferred onset of HSP70 and sHSP expression followed by a subsequent offset, delineating a unimodal response curve. The time lag to the expression onset of HSP70 was shorter in the thermosensitive species, compared to thermotolerant ones. Conversely, the time span until the maximum level of HSP70 was variable, not showing a dependence on the thermotolerance properties of the species. The peak concentration in G. pulex was distinctly higher than in the other species, whereas E. verrucosus did not develop a well-defined response maximum at all. In sHSP, the temporal pattern of expression was even more variable than in HSP70. However, the thermosensitive species E. verrucosus showed a time lag of expression onset significantly shorter than the other species and thermotolerant G. fasciatus developed the most pronounced response maximum. Basing on these results, the cellular response to thermal stress in amphipods is more consistently reflected by HSP70, compared to sHSP.