Preheating induced homogeneity of the small heat shock protein from Methanococcus jannaschii

Small heat shock proteins usually exhibit increased chaperone-like activity either at high temperatures or after preheating. However, the activation mechanism is still unclear. In the current study, we investigated the preheating-activation process of Mj HSP16.5, using various biophysical methods. Although Mj HSP16.5 was reported to be the most monodispersed sHSPs, we found that the newly purified Mj HSP16.5 was actually heterogeneous. 85 degrees C-preheating could activate Mj HSP16.5 and turn it into a more compact homogeneous species at the same time. Different cooling rates after preheating did not change the activity of Mj HSP16.5, suggesting that the 85 degrees C-preheated Mj HSP16.5 is in the most active and also the most stable state. These results demonstrate that the activation process of Mj HSP16.5 might accompany a refolding process.     

Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A

Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level.     

C-terminal modulators of heat shock protein of 90 kDa (HSP90): State of development and modes of action

Cells constantly need to adopt to changing environmental conditions, maintaining homeostasis and proteostasis. Heat shock proteins are a diverse class of molecular chaperones that assist proteins in folding to prevent stress-induced misfolding and aggregation. The heat shock protein of 90 kDa (HSP90) is the most abundant heat shock protein. While basal expression of HSP90 is essential for cell survival, in many tumors elevated HSP90 levels are found, which is often associated with bad prognosis. Therefore, HSP90 has emerged as a major target in tumor therapy. The HSP90 machinery is very complex in that it involves large conformational changes during the chaperoning cycle and a variety of co-chaperones. At the same time, this complexity offers a plethora of possibilities to interfere with HSP90 function. The best characterized class of HSP90 modulators are competitive inhibitors targeting the N-terminal ATP-binding pocket. Nineteen compounds of this class entered clinical trials. However, due to severe adverse effects, including induction of the heat shock response, no N-terminal inhibitor has been approved by the FDA so far. As alternatives, compounds commonly referred to as “C-terminal inhibitors” have been developed, either as natural product-based analogues or by rational design, which employ multiple mechanisms to modulate HSP90 function, including modulation of the interaction with co-chaperones, induction of conformational changes that influence the chaperoning cycle, or inhibition of C-terminal dimerization. In this review, we summarize the current development state of characteristic C-terminal inhibitors, with an emphasis on their (proposed) molecular modes of action and binding sites.     

Extracellular heat shock protein HSP90β secreted by MG63 osteosarcoma cells inhibits activation of latent TGF-β1

Transforming growth factor-beta 1 (TGF-β1) is secreted as a latent complex, which consists of latency-associated peptide (LAP) and the mature ligand. The release of the mature ligand from LAP usually occurs through conformational change of the latent complex and is therefore considered to be the first step in the activation of the TGF-β signaling pathway. So far, factors such as heat, pH changes, and proteolytic cleavage are reportedly involved in this activation process, but the precise molecular mechanism is still far from clear. Identification and characterization of the cell surface proteins that bind to LAP are important to our understanding of the latent TGF-β activation process. In this study, we have identified heat shock protein 90 β (HSP90β) from the cell surface of the MG63 osteosarcoma cell line as a LAP binding protein. We have also found that MG63 cells secrete HSP90β into extracellular space which inhibits the activation of latent TGF-β1, and that there is a subsequent decrease in cell proliferation. TGF-β1-mediated stimulation of MG63 cells resulted in the increased cell surface expression of HSP90β. Thus, extracellular HSP90β is a negative regulator for the activation of latent TGF-β1 modulating TGF-β signaling in the extracellular domain.     

Two-dimensional crystallization of a small heat shock protein HSP16.3 on lipid layer

As a member of small heat shock proteins, HSP16.3 was identified as the major membrane-bound protein of Mycobacterium tuberculosis during stationary phase. Previous studies revealed that HSP16.3 was in a nonameric form in solution. Here, two-dimensional crystal of HSP16.3 molecules on lipid monolayer was obtained for the first time. The crystal exhibited p422 symmetry with lattice parameters a=b=90A, gamma=90 degrees. The projection map of untilted crystals showed that the basic unit of the crystal was a rod-like structure with two high-density regions. The three-dimensional map at 2.2 nm resolution revealed a rod-like structure with a dimension of 56A x 32A x 25A, similar to the dimeric forms of M. jannaschii HSP16.5 and wheat HSP16.9. Cross-linking experiments confirmed that HSP16.3 nonamers dissociated into dimers upon interaction with the positively charged lipid layer. Surface plasmon resonance measurements revealed that both electrostatic and hydrophobic forces involved in the formation of the 2D crystal on the lipid monolayer. These results provide a basis for further investigation on the unique dimeric structure of HSP16.3 and its functions in vivo.     

Changes in the heat shock 70 kDa protein level in human neutrophils induced by heat shock

Changes in the content of constitutive and inducible proteins of the family of heat shock 70 kDa proteins (HSP70) caused by heat shock in human neutrophils, white blood cells with an atypically short lifespan, which provide a nonspecific defense of the organism against bacterial pathogens, have been studied. An analysis of the intracellular content of the constitutive and inducible HSP70 proteins by flow cytometry revealed a biphasic dynamics of changes in the protein level, which was characterized by an increase in the protein level immediately after heat shock followed by a decrease within 15–30 min after the termination of heat treatment. Because the inhibitor of protein synthesis cycloheximide did not change the dynamics profile, it was assumed that the increase in the HSP70 level is related not to the de novo synthesis of these proteins but to conformational changes of HSP70 molecules and an increased accessibility of some epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or the C-terminal substrate-binding domains of the protein, it was shown by cell immunofluorescence and flow cytometry that the heat shock-associated increase in the intracellular HSP70 level results from an increased efficiency of the binding of antibodies recognizing the substrate-binding domain. It was also demonstrated that the decrease in the intracellular HSP70 level after the heat shock, may be partially due to a release into the extracellular space of both the constitutive and inducible HSP70 proteins, which is regulated with the involvement of ABC-transporters.     

A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state.

The small heat shock proteins (sHSPs) recently have been reported to have molecular chaperone activity in vitro; however, the mechanism of this activity is poorly defined. We found that HSP18.1, a dodecameric sHSP from pea, prevented the aggregation of malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase heated to 45 degrees C. Under conditions in which HSP18.1 prevented aggregation of substrates, size-exclusion chromatography and electron microscopy revealed that denatured substrates coated the HSP18.1 dodecamers to form expanded complexes. SDS-PAGE of isolated complexes demonstrated that each HSP18.1 dodecamer can bind the equivalent of 12 MDH monomers, indicating that HSP18.1 has a large capacity for non-native substrates compared with other known molecular chaperones. Photoincorporation of the hydrophobic probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) into a conserved C-terminal region of HSP18.1 increased reversibly with increasing temperature, but was blocked by prior binding of MDH, suggesting that bis-ANS incorporates proximal to substrate binding regions and that substrate-HSP18.1 interactions are hydrophobic. We also show that heat-denatured firefly luciferase bound to HSP18.1, in contrast to heat-aggregated luciferase, can be reactivated in the presence of rabbit reticulocyte or wheat germ extracts in an ATP-dependent process. These data support a model in which sHSPs prevent protein aggregation and facilitate substrate refolding in conjunction with other molecular chaperones.     

辅分子伴侣调控热休克蛋白HSP90功能的研究

热休克蛋白90(HSP90)是一类ATPase依赖性蛋白,作为分子伴侣,可在辅分子伴侣协助下,通过自身构象改变,参与众多细胞的生物学事件,从而协助新合成蛋白的正确折叠、成功装配、功能稳定及异常蛋白的降解过程。HSP90功能的发挥依赖于辅分子伴侣及氨基末端结合的核苷酸。辅分子伴侣是一类可与分子伴侣(如,HSP90)结合并调节其功能的蛋白,通过参与ATPase循环从而调节HSP90分子伴侣的功能。近年来,辅分子伴侣的研究得到越来越多的关注,本文就辅分子伴侣调控HSP90功能的作用进行综述。     

Heat shock factor 1 is a key regulator of the stress response in Chlamydomonas

We report here on the characterization of heat shock factor 1 (HSF1), encoded by one of two HSF genes identified in the genome of Chlamydomonas reinhardtii. Chlamydomonas HSF1 shares features characteristic of class A HSFs of higher plants. HSF1 is weakly expressed under non-stress conditions and rapidly induced by heat shock. Heat shock also resulted in hyperphosphorylation of HSF1, and the extent of phosphorylation correlated with the degree of induction of heat shock genes, suggesting a role for phosphorylation in HSF1 activation. HSF1, like HSFs in yeasts, forms high-molecular-weight complexes, presumably trimers, under non-stress, stress and recovery conditions. Immunoprecipitation of HSF1 under these conditions led to the identification of cytosolic HSP70A as a protein constitutively interacting with HSF1. Strains in which HSF1 was strongly under-expressed by RNAi were highly sensitive to heat stress. 14C-labelling of nuclear-encoded proteins under heat stress revealed that synthesis of members of the HSP100, HSP90, HSP70, HSP60 and small HSP families in the HSF1-RNAi strains was dramatically reduced or completely abolished. This correlated with a complete loss of HSP gene induction at the RNA level. These data suggest that HSF1 is a key regulator of the stress response in Chlamydomonas.     

The HSP90 complex of plants

Heat shock protein 90 (HSP90) is a highly conserved and essential molecular chaperone involved in maturation and activation of signaling proteins in eukaryotes. HSP90 operates as a dimer in a conformational cycle driven by ATP binding and hydrolysis. HSP90 often functions together with co-chaperones that regulate the conformational cycle and/or load a substrate "client" protein onto HSP90. In plants, immune sensing NLR (nucleotide-binding domain and leucine-rich repeat containing) proteins are among the few known client proteins of HSP90. In the process of chaperoning NLR proteins, co-chaperones, RAR1 and SGT1 function together with HSP90. Recent structural and functional analyses indicate that RAR1 dynamically controls conformational changes of the HSP90 dimer, allowing SGT1 to bridge the interaction between NLR proteins and HSP90. Here, we discuss the regulation of NLR proteins by HSP90 upon interaction with RAR1 and SGT1, emphasizing the recent progress in our understanding of the structure and function of the complex. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

The chloroplast DnaJ homolog CDJ1 of Chlamydomonas reinhardtii is part of a multichaperone complex containing HSP70B, CGE1, and HSP90C

We report on the molecular and biochemical characterization of CDJ1, one of three zinc-finger-containing J-domain proteins encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that CDJ1 is a plastidic protein. In the chloroplast, CDJ1 was localized to the soluble stroma fraction, but also to thylakoids and to low density membranes. Although the CDJ1 gene was strongly heat shock inducible, CDJ1 protein levels increased only slightly during heat shock. Cellular CDJ1 concentrations were close to those of heat shock protein 70B (HSP70B), the major HSP70 in the Chlamydomonas chloroplast. CDJ1 complemented the temperature-sensitive phenotype of an Escherichia coli mutant lacking its dnaJ gene and interacted with E. coli DnaK, hence classifying it as a bona fide DnaJ protein. In soluble cell extracts, CDJ1 was found to organize into stable dimers and into complexes of high molecular mass. Immunoprecipitation experiments revealed that CDJ1 forms common complexes with plastidic HSP90C, HSP70B, and CGE1. In blue native-polyacrylamide gel electrophoresis, all four (co)chaperones migrated at 40% to 90% higher apparent than calculated molecular masses, indicating that greatest care must be taken when molecular masses of protein complexes are estimated from their migration relative to standard native marker proteins. Immunoprecipitation experiments from size-fractioned soluble cell extracts suggested that HSP90C and HSP70B exist as preformed complex that is joined by CDJ1. In summary, CDJ1 and CGE1 are novel cohort proteins of the chloroplast HSP90-HSP70 multichaperone complex. As HSP70B, CDJ1, and CGE1 are derived from the endosymbiont, whereas HSP90C is of eukaryotic origin, we observe in the chloroplast the interaction of two chaperone systems of distinct evolutionary origin.     

Pressure activation of the chaperone function of small heat shock proteins.

Small heat shock proteins play an important role in the stress response of cells and in several other cellular functions. They possess chaperone-like activity; i.e. they can bind and protect damaged proteins from aggregation and maintain them in a folding-competent state. Two members of this family were investigated in this work: bovine alpha-crystallin and heat shock protein (HSP)16.5 from the thermophilic archaebacteria Methanococcus jannaschii. We reported earlier the enhancement of chaperone potency of alpha-crystallin by high pressure. We now report the completion of the work with results on HSP16.5. The chaperone potency of both proteins can be enhanced significantly by applying high pressure. Evidence by light scattering, Fourier transform infrared (FT-IR) and tryptophan fluorescence experiments show that while the secondary and tertiary structure of these proteins are not influenced by high pressure, their quatemary structure becomes affected: H bonds between subunits are weakened or broken, tryptophan environments become more polar, oligomers dissociate to some extent. We conclude that the oligomeric structure of both proteins is loosened, resulting in stronger dynamics and in more accessible hydrophobic surfaces. These properties lead to increased chaperone potency.     

Cisplatin differently affects amino terminal and carboxyl terminal domains of HSP90

The 90-kDa heat shock protein (HSP90) is a molecular chaperone that assists in the folding and assembly of proteins in the cytosol. We previously demonstrated that the antineoplastic reagent, cisplatin, inhibits the aggregation prevention activity of mammalian HSP90. We now show that cisplatin binds both the amino terminal and carboxyl terminal domains of the human HSP90 and differently affects these two domains. Cisplatin blocks the aggregation prevention activity of HSP90C, but not HSP90N. In contrast, cisplatin induces a conformational change in HSP90N, but not HSP90C. These results indicate that cisplatin modulates the HSP90 activities through two different mechanisms using the two distinct binding sites of the HSP90 molecule.     

Characterisation of chloroplast heat shock proteins in young leaves of C4 monocotyledons

In vivo radiolabeling of chloroplast proteins in grain sorghum (Sorghum bicolor L. cv. Texas 610) leaves and their separation by one-dimensional electrophoresis revealed at least 6 heat shock proteins (HSPs) between 24 and 94 kDa. of which the 24 kDa protein was the most prominent. All of these chloroplast heat shock proteins were found exclusively in the stroma. The 24 kDa heat shock protein, upon closer examination using two-dimensional electrophoresis proved to be two similarly-sized heat shock polypeptides with identical molecular masses and level of radiolahel incorporation, hut slightly different in isoeiectric points, suggesting isomers. Separation of stromal heat shock proteins synthesised in two other C₄ monocotyledons ( Punicum miliaceum L. and Umchloa panictrides L.) revealed similar putative isomers. each of 24 kDa. Several other, previously unidentified, heat shock proteins between 22 and 38 kDa were also observed in all three species. In P. miliaceum. the most prominent HSP was the pair of 24 kDa proteins, whereas in U. panicoides. it was a group of 35 to 38 kDa HSPs that was most abundant. In vivo chlorophyll fluorescence measurements showed that no sustained impairment to photosynthetic efficiency had occurred for each species after the heat stress regime. However, when cytoplasmic protein synthesis was inhibited during the high temperature treatment, a dramatic decrease was observed in photosynthetic efficiency, suggesting a possible protective role for chloroplast heat shock proteins. It was also shown that a single chloroplast HSP complex of around 380 kDa was observed in the stroma of both 5. bicolor and P. miliaceum leaves in vivo. This was in contrast to the smaller HSP complex (200–265 kDa) observed in previous studies on chloroplast heat shock proteins in C_j species.     

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.     

Targeted heat activation of HSP promoters in the skin of mammalian animals and humans

The use of highly inducible HSP promoters for exerting spatial and/or temporal control over the expression of therapeutic transgenes has long been discussed. Localized and time-limited induction of the heat shock response may potentially also be of medical interest. However, such applications would require targeted delivery of heat doses capable of activating HSP promoters in tissues or organs of interest. Accessible areas, including the skin and tissues immediately underneath it, may be most readily targeted. A few applications for heat-directed or heat-controlled therapy in the skin might involve expression of proteins to restore or protect normal skin function, protein antigens for vaccination/immunotherapy, vaccine viruses or even systemically active proteins, e.g., cytokines and chemokines. A review of the literature relating to localized heat activation of HSP promoters and HSP genes in the skin revealed that a multitude of different technologies has been explored in small animal models. In contrast, we uncovered few publications that examine HSP promoter activation in human skin. None of these publications has a therapeutic focus. We present herein two, clinically relevant, developments of heating technologies that effectively activate HSP promoters in targeted regions of human skin. The first development advances a system that is capable of reliably activating HSP promoters in human scalp, in particular in hair follicles. The second development outlines a simple, robust, and inexpensive methodology for locally activating HSP promoters in small, defined skin areas.     

Restoring HSP70 deficiencies improves glucose tolerance in diabetic monkeys

We evaluated heat shock protein 70 (HSP70) changes in diabetes mellitus (DM) in a nonhuman primate model. To this end, two studies were conducted in DM vervet monkeys. 1) Normal control and streptozotocin-induced DM monkeys (Stz-DM) that were differentiated into moderately or poorly controlled DM by judicious insulin administration were evaluated. Liver was collected at 4, 8, 12, 16, and 20 wk after streptozotocin, exposed to ex vivo heat shock at 42°C, and immunoblotted for heat shock factor 1 (HSF1), HSP70, and phosphorylated HSF1. 2) Spontaneous DM monkeys that were not pharmacologically induced were included in a crossover study of the HSP70-inducing drug geranylgeranylacetone (GGA). GGA at 20 mg/kg was given for 14 days with a 6-wk washout period. Glucose tolerance testing and plasma and muscle HSP70 were the primary outcome measurements. In Stz-DM, hyperglycemia reduced hepatic HSP70 in a dose-dependent fashion. HSF1 was increased in livers of monkeys with Stz-DM, but responses to ex vivo heat shock were impaired vs. normal monkeys. Activation of HSF1 appears to be important, because the phosphorylation change with heat stress was nearly perfectly correlated with HSP70 increases. Impaired HSF1 activation was also seen in Stz-DM after chronic hyperglycemia (>12 wk). In naturally occurring DM, increased circulating HSP70 resulted in significantly improved glucose tolerance and significant, positive trends in other measurements of insulin resistance. No change in muscle HSP70 content was observed. We conclude that increasing HSP70, potentially through targeting hyperglycemia-related deficits in HSF1 induction and activation in the liver, is a potent and viable strategy to improve glucose tolerance.