Major Heat Shock Protein Research Articles

Decrease in Penicillin Susceptibility Due to Heat Shock Protein ClpL in Streptococcus pneumoniae

Antibiotic resistance and tolerance are increasing threats to global health as antibiotic-resistant bacteria can cause severe morbidity and mortality and can increase treatment cost 10-fold. Although several genes contributing to antibiotic tolerance among pneumococci have been identified, we report here that ClpL, a major heat shock protein, could modulate cell wall biosynthetic enzymes and lead to decreased penicillin susceptibility. On capsular type 1, 2, and 19 genetic backgrounds, mutants lacking ClpL were more susceptible to penicillin and had thinner cell walls than the parental strains, whereas a ClpL-overexpressing strain showed a higher resistance to penicillin and a thicker cell wall. Although exposure of Streptococcus pneumoniae D39 to penicillin inhibited expression of the major cell wall synthesis gene pbp2x, heat shock induced a ClpL-dependent increase in the mRNA levels and protein synthesized by pbp2x. Inducible ClpL expression correlated with PBP2x expression and penicillin susceptibility. Fractionation and electron micrograph data revealed that ClpL induced by heat shock is localized at the cell wall, and the ΔclpL showed significantly reduced net translocation of PBP2x into the cell wall. Moreover, coimmunoprecipitation with either ClpL or PBP2x antibody followed by reprobing with ClpL or PBP2x antibody showed an interaction between ClpL and PBP2x after heat stress. This interaction was confirmed by His tag pulldown assay with either ClpLHis₆ or PBP2xHis₆. Thus, ClpL stabilized pbp2x expression, interacted with PBP2x, and facilitated translocation of PBP2x, a key protein of cell wall synthesis process, contributing to the decrease of antibiotic susceptibility in S. pneumoniae.

The Heat Shock-binding Protein (HspBP1) Protects Cells against the Cytotoxic Action of the Tag7-Hsp70 Complex

Heat shock-binding protein HspBP1 is a member of the Hsp70 co-chaperone family. The interaction between HspBP1 and the ATPase domain of the major heat shock protein Hsp70 up-regulates nucleotide exchange and reduces the affinity between Hsp70 and the peptide in its peptide-binding site. Previously we have shown that Tag7 (also known as peptidoglycan recognition protein PGRP-S), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. This complex can be produced in cytotoxic lymphocytes and released during interaction with tumor cells. Here the effect of HspBP1 on the cytotoxic activity of the Tag7-Hsp70 complex was examined. HspBP1 could bind not only to Hsp70, but also to Tag7. This interaction eliminated the cytotoxic activity of Tag7-Hsp70 complex and decreased the ATP concentration required to dissociate Tag7 from the peptide-binding site of Hsp70. Moreover, HspBP1 inhibited the cytotoxic activity of the Tag7-Hsp70 complex secreted by lymphocytes. HspBP1 was detected in cytotoxic CD8+ lymphocytes. This protein was released simultaneously with Tag7-Hsp70 during interaction of these lymphocytes with tumor cells. The simultaneous secretion of the cytotoxic complex with its inhibitor could be a mechanism protecting normal cells from the cytotoxic effect of this complex.

Heat Shock Proteins in Association with Heat Tolerance in Grasses

The grass family Poaceae includes annual species cultivated as major grain crops and perennial species cultivated as forage or turf grasses. Heat stress is a primary factor limiting growth and productivity of cool-season grass species and is becoming a more significant problem in the context of global warming. Plants have developed various mechanisms in heat-stress adaptation, including changes in protein metabolism such as the induction of heat shock proteins (HSPs). This paper summarizes the structure and function of major HSPs, recent research progress on the association of HSPs with grass tolerance to heat stress, and incorporation of HSPs in heat-tolerant grass breeding.

Heat shock protein Hsp72 plays an essential role in Her2-induced mammary tumorigenesis

The major heat shock protein Hsp72 is expressed at elevated levels in many human cancers and its expression correlates with tumor progression. Here we investigated the role of Hsp72 in Her2 oncogene-induced neoplastic transformation and tumorigenesis. Expression of Her2 in untransformed MCF10A mammary epithelial cells caused transformation, as judged by foci formation in culture and tumorigenesis in xenografts. However, expression of Her2 in Hsp72-depleted cells failed to induce transformation. The anti-tumorigenic effects of Hsp72 downregulation were associated with cellular senescence due to accumulation of p21 and depletion of survivin. Accordingly, either knockdown of p21 or expression of survivin reversed this senescence process. Further, we developed an animal model of Hsp72-dependent breast cancer associated with expression of Her2. Knockout of Hsp72 almost completely suppressed tumorigenesis in the MMTVneu breast cancer mouse model. In young Hsp72 KO mice, expression of Her2 instead of mammary tissue hyperplasia led to suppression of duct development and blocked alveolar budding. These effects were due to massive cell senescence in mammary tissue, which was associated with upregulation of p21 and downregulation of survivin. Therefore Hsp72 plays an essential role in Her2-induced tumorigenesis by regulating oncogene-induced senescence pathways.

The heat shock factor family and adaptation to proteotoxic stress

The heat shock response was originally characterized as the induction of a set of major heat shock proteins encoded by heat shock genes. Because heat shock proteins act as molecular chaperones that facilitate protein folding and suppress protein aggregation, this response plays a major role in maintaining protein homeostasis. The heat shock response is regulated mainly at the level of transcription by heat shock factors (HSFs) in eukaryotes. HSF1 is a master regulator of the heat shock genes in mammalian cells, as is HSF3 in avian cells. HSFs play a significant role in suppressing protein misfolding in cells and in ameliorating the progression of Caenorhabditis elegans, Drosophila and mouse models of protein-misfolding disorders, by inducing the expression of heat shock genes. Recently, numerous HSF target genes were identified, such as the classical heat shock genes and other heat-inducible genes, called nonclassical heat shock genes in this study. Importance of the expression of the nonclassical heat shock genes was evidenced by the fact that mouse HSF3 and chicken HSF1 play a substantial role in the protection of cells from heat shock without inducing classical heat shock genes. Furthermore, HSF2 and HSF4, as well as HSF1, shown to have roles in development, were also revealed to be necessary for the expression of certain nonclassical heat shock genes. Thus, the heat shock response regulated by the HSF family should consist of the induction of classical as well as of nonclassical heat shock genes, both of which might be required to maintain protein homeostasis.

Heat shock factor 1 ameliorates proteotoxicity in cooperation with the transcription factor NFAT

Heat shock transcription factor 1 (HSF1) is an important regulator of protein homeostasis (proteostasis) by controlling the expression of major heat shock proteins (Hsps) that facilitate protein folding. However, it is unclear whether other proteostasis pathways are mediated by HSF1. Here, we identified novel targets of HSF1 in mammalian cells, which suppress the aggregation of polyglutamine (polyQ) protein. Among them, we show that one of the nuclear factor of activated T cells (NFAT) proteins, NFATc2, significantly inhibits polyQ aggregation in cells and is required for HSF1-mediated suppression of polyQ aggregation. NFAT deficiency accelerated disease progression including aggregation of a mutant polyQ-huntingtin protein and shortening of lifespan in R6/2 Huntington's disease mice. Furthermore, we found that HSF1 and NFAT cooperatively induce the expression of the scaffold protein PDZK3 and αB-crystallin, which facilitate the degradation of polyQ protein. These results show the first mechanistic basis for the observation that HSF1 has a much more profound effect on proteostasis than individual Hsp or combination of different Hsps, and suggest a new pathway for ameliorating protein-misfolding diseases.

Gene expression profiles of cytosolic heat shock proteins Hsp70 and Hsp90 from symbiotic dinoflagellates in response to thermal stress: possible implications for coral bleaching

Unicellular photosynthetic dinoflagellates of the genus Symbiodinium are the most common endosymbionts of reef-building scleractinian corals, living in a symbiotic partnership known to be highly susceptible to environmental changes such as hyperthermic stress. In this study, we identified members of two major heat shock proteins (HSPs) families, Hsp70 and Hsp90, in Symbiodinium sp. (clade C) with full-length sequences that showed the highest similarity and evolutionary relationship with other known HSPs from dinoflagellate protists. Regulation of HSPs gene expression was examined in samples of the scleractinian coral Acropora millepora subjected to elevated temperatures progressively over 18h (fast) and 120h (gradual thermal stress). Moderate to severe heat stress at 26°C and 29°C (+3°C and +6°C above average sea temperature) resulted in an increase in algal Hsp70 gene expression from 39% to 57%, while extreme heat stress (+9°C) reduced Hsp70 transcript abundance by 60% (after 18h) and 70% (after 120h). Elevated temperatures decreased an Hsp90 expression under both rapid and gradual heat stress scenarios. Comparable Hsp70 and Hsp90 gene expression patterns were observed in Symbiodinium cultures and in hospite, indicating their independent regulation from the host. Differential gene expression profiles observed for Hsp70 and Hsp90 suggests diverse roles of these molecular chaperones during heat stress response. Reduced expression of the Hsp90 gene under heat stress can indicate a reduced role in inhibiting the heat shock transcription factor which may lead to activation of heat-inducible genes and heat acclimation.

Structural Disorder within the Measles Virus Nucleoprotein and Phosphoprotein

In this review, we summarize the main experimental data showing the abundance of structural disorder within the measles virus (MeV) nucleoprotein (N) and phosphoprotein (P), and focus on the molecular mechanisms governing the disorder-to-order transition of the intrinsically disordered C-terminal domain of MeV N (N(TAIL)) upon binding to the C-terminal X domain of P (XD). The functional implications of structural disorder are discussed in light of the ability of disordered regions to establish a complex molecular partnership, thereby leading to a variety of biological effects, including tethering of the polymerase complex onto the nucleocapsid template, stimulation of viral transcription and replication, and virus assembly. We also discuss the ability of N(TAIL) to establish interactions with additional cellular co-factors, including the major inducible heat shock protein, which can modulate the strength of the N(TAIL)-XD interaction. Taking into account the promiscuity that typifies disordered regions, we propose that the main functional advantage of the abundance of disorder within viruses would reside in pleiotropy and genetic compaction, where a single gene would encode a single (regulatory) protein product able to establish multiple interactions via its disordered regions, and hence to exert multiple concomitant biological effects.

Melatonin induces pro-apoptotic signaling pathway in human pancreatic carcinoma cells (PANC-1)

Pancreatic cancer is a highly lethal disease with a poor prognosis for long-term survival rate at all stages of invasiveness. It responds poorly to radio- and chemotherapy because the tumor cells are resistant to apoptosis. Melatonin has been reported to inhibit pancreatic cancer growth in experimental studies in animals but the effect of melatonin on cultured human pancreatic carcinoma cells has not been tested. Moreover, we have recently shown that melatonin stimulates production of two major anti-apoptotic heat shock proteins, HSP27 and HSP 90, in pancreatic carcinoma cells. This study investigated the changes in intrinsic pathway of apoptosis at the mitochondrial level and cascade of caspases in human pancreatic carcinoma cells (PANC-1) cells subjected to melatonin and/or luzindole. Melatonin (10⁻⁸ -10⁻¹² m), the nonselective melatonin receptor antagonist, luzindole (10⁻⁸ -10⁻¹² m) or a combination of both agents were added to PANC-1 cell cultures. Cells were harvested, and the cytoplasmic proteins were isolated after 24 and 48 hr of incubation and analyzed employing co-immunoprecipitation and western blot. Administration of melatonin to the PANC-1 cells resulted in the stimulation of Bcl-2/Bax and caspase-9 proteins levels. The strongest signal of these pro-apoptotic factors was observed at the low concentration (10⁻¹² m) of melatonin. Pretreatment with luzindole alone and prior to the addition of melatonin reversed the stimulatory effect of this indoloamine on Bcl-2/Bax and caspase-9 proteins expression in PANC-1 cells. This is the first study to demonstrate a pro-apoptotic effect of low (physiological) concentration of melatonin on the pancreatic carcinoma cells. In conclusion, melatonin induced pro-apoptotic pathways in human pancreatic carcinoma, probably by interaction with the Mel-1 A/B receptors.

Plant heat-shock proteins: A mini review

Plants as sessile organisms are exposed to persistently changing stress factors. The primary stresses such as drought, salinity, cold and hot temperatures and chemicals are interconnected in their effects on plants. These factors cause damage to the plant cell and lead to secondary stresses such as osmotic and oxidative stresses. Plants cannot avoid the exposure to these factors but adapt morphologically and physiologically by some other mechanisms. Almost all stresses induce the production of a group of proteins called heat-shock proteins (Hsps) or stress-induced proteins. The induction of transcription of these proteins is a common phenomenon in all living things. These proteins are grouped in plants into five classes according to their approximate molecular weight: (1) Hsp100, (2) Hsp90, (3) Hsp70, (4) Hsp60 and (5) small heat-shock proteins (sHsps). Higher plants have at least 20 sHsps and there might be 40 kinds of these sHsps in one plant species. It is believed that this diversification of these proteins reflects an adaptation to tolerate the heat stress. Transcription of heat-shock protein genes is controlled by regulatory proteins called heat stress transcription factors (Hsfs). Plants show at least 21 Hsfs with each one having its role in regulation, but they also cooperate in all phases of periodical heat stress responses (triggering, maintenance and recovery). There are more than 52 plant species (including crop ones) that have been genetically engineered for different traits such as yield, herbicide and insecticide resistance and some metabolic changes.In conclusion, major heat-shock proteins have some kind of related roles in solving the problem of misfolding and aggregation, as well as their role as chaperones.

Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70

Hsp105α and Hsp105β are major heat shock proteins in mammalian cells and belong to the HSP105/110 family. Hsp105α is expressed constitutively in the cytoplasm of cells, while Hsp105β, an alternatively spliced form of Hsp105α, is expressed specifically in the nucleus of cells during mild heat shock. Here, we show that not only Hsp105β but also Hsp105α accumulated in the nucleus of cells following the expression of enhanced green fluorescent protein with a pathological length polyQ tract (EGFP-polyQ97) and suppressed the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Mutants of Hsp105α and Hsp105β with changes in the nuclear localization signal sequences, which localized exclusively in the cytoplasm with or without the expression of EGFP-polyQ97, did not suppress the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Furthermore, Hsp70 was induced by the co-expression of Hsp105α and EGFP-polyQ97, and the knockdown of Hsp70 reduced the inhibitory effect of Hsp105α and Hsp105β on the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. These observations suggested that Hsp105α and Hsp105β suppressed the expanded polyQ tract-induced protein aggregation and apoptosis through the induction of Hsp70.

Major heat shock protein Hsp72 controls oncogene‐induced senescence

Various heat shock proteins, including Hsp72, are strongly upregulated in cancers, but their significance for tumor emergence and growth is poorly understood. Here we review recent data from several labs to indicate that Hsps, including Hsp72, are critical for growth of transformed but not normal cells. By manipulating expression and activity of Hsp72 and several oncogenes, it was shown that Hsp72 suppresses oncogene-induced senescence, thus allowing proliferation of cancer cells. Importantly, Hsp72 is able to suppress both p53-dependent and p53-independent senescence pathways. We propose that targeting Hsp72 may be a promising approach toward development of novel cancer therapies.

The influence of pH on heat stress response by probiotic Lactobacillus plantarum LP-Onlly

The influence of pH on the heat stress response of a human-derived probiotic strain of Lactobacillus plantarum, LP-Onlly, was investigated. The heat resistance of mid-log- and stationary-phase cells that were heat-adapted (45°C) in cultures that were or were not pH adjusted (pH 4.3–7.0) was evaluated at a lethal temperature of 55°C. The results demonstrated that the heat resistance of heat-adapted stationary-phase cells increased sharply as the pH for heat adaptation in cultures was adjusted from pH 4.3 (non-adjusted pH) to pH 7.0 step by step (pH 5.0, 5.5, 6.0, 6.5, and 7.0); however, the mid-log-phase cells did not show the similar pH-related trend. The optimal adjusted pH for heat adaptation in cultures was found to be 6.5, with stationary-phase cells that were heat-adapted at pH 6.5 exhibiting a 3.4-log-cycle increase in heat resistance and 6-fold greater storage stability than controls. Real-time reverse transcription PCR analysis of five major heat shock protein genes (dnak, dnaJ, grpE, groES, and groEL) revealed that the heat-inductive expression of these genes after heat adaptation was up-regulated in stationary-phase cells when the pH in cultures for heat adaptation was adjusted from 4.3 (non-adjusted pH) to 6.5. Nevertheless, the transcription levels of these genes after heat adaptation was down-regulated in mid-log-phase cells when the pH in cultures for heat adaptation was shifted from 6.0 (non-adjusted pH) to 6.5.

Basophil-specific and post-translational silencing of a major endoplasmic reticulum heat shock protein gp96 (grp94, HSP90b1) for TLR and integrin (80.7)

Abstract Basophil has been implicated in anti-parasite defense, allergy and in polarizing TH2 response. However, little is known about its biological significance in vivo, due to lack of basic understanding of basophil developmental/functional cues. As an essential chaperone for multiple Toll-like receptors (TLRs) and integrins in the endoplasmic reticulum (ER), gp96 (grp94, HSP90b1) also participates in general protein homeostasis and in the ER unfolded protein response (UPR) to ensure cell survival during stress. No nucleic cells in mammals have been described to be naturally devoid of gp96. By genetically ablating gp96, we report herein that gp96 is dispensable for basophil development in vivo. Moreover, we found that gp96 was post-translationally cleaved in primary basophils leading to accumulation of a N-terminal ~50 kDa fragment of gp96 and up-regulation of several components of the UPR pathway. The alteration of gp96 was unique to basophils and was not observed in any other cell types including mast cells. Furthermore, we found that deletion of C-terminal domain of gp96 resulted in loss of its chaperone function. Our study revealed a remarkable biochemical event of gp96 silencing in basophils, arguing for the potential importance of shutting down gp96 post-translationally in regulating basophil development and function.

A systems biological approach towards understanding the dynamics of major heat shock proteins on recombinant protein production

A systems biological approach towards understanding the dynamics of major heat shock proteins on recombinant protein production

Heat shock proteins and exercise: a primer

Heat shock proteins (HSPs) are, in general, prosurvival molecules within the cellular environment, and the overexpression of even just 1 family of HSPs can lead to protection against and improvements after a variety of stressors. Not surprisingly, a fertile area of study has grown out of efforts to exploit the innate biologic behaviour of HSPs. Exercise, because of the inherent physiologic stresses associated with it, is but 1 stimulus that can result in a robust increase in various HSPs in several tissues, not the least of which happen to be the heart and skeletal muscle. The purpose of this review is to introduce the reader to the major HSP families, the control of their expression, and some of their biologic functions, specifically with respect to the influence of exercise. Moreover, as the first in a series of reviews from a common symposium, we will briefly introduce the concepts presented by the other authors, which include the effects of different exercise paradigms on skeletal muscle HSPs in the adult and aged systems, HSPs as regulators of inflammation, and the ion channel stabilizing effects of HSPs.

Mammalian 105 kDa heat shock family proteins suppress hydrogen peroxide‐induced apoptosis through a p38 MAPK‐dependent mitochondrial pathway in HeLa cells

Hsp105alpha and Hsp105beta are major heat shock proteins in mammalian cells that belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105alpha has opposite effects on stress-induced apoptosis depending on the cell type. However, it is not fully understood how Hsp105 regulates stress-induced apoptosis. In this study, we examined how Hsp105alpha and Hsp105beta regulate H2O2-induced apoptosis by using HeLa cells in which expression of Hsp105alpha or Hsp105beta was regulated using doxycycline. Overexpression of Hsp105alpha and Hsp105beta suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria in H2O2-treated cells. Furthermore, both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) were activated by treatment with H2O2, and the activation of both kinases was suppressed by overexpression of Hsp105alpha and Hsp105beta. However, H2O2-induced apoptosis was suppressed by treatment with a potent inhibitor of p38 MAPK, SB202190, but not a JNK inhibitor, SP600125. These findings suggest that Hsp105alpha and Hsp105beta suppress H2O2-induced apoptosis by suppression of p38 MAPK signaling, one of the essential pathways for apoptosis.

α-crystallin-type heat-shock protein from mycoplasma Acholeplasma laidlawii (Mollicutes)

An increased amount of major heat-shock proteins (HSPs) after heat treatment has been revealed in Acholeplasma laidlawii cells grown in liquid culture, with the quantity of small HSPs, known as P17, being enhanced by hundreds times. The P17 protein was isolated and identified as an α-crystallin-type HSP (α-HSP) by sequencing the N-terminal 15 amino acids of the P17 polypeptide chain followed by finding the corresponding open reading frame (ORF) in the completely sequenced genome of A. laidlawii PG 8A. A computer-based search for homologous ORFs in the genomes of all 14 species of the Mycoplasmataceae family (mycoplasmas themselves) that have been completely sequenced to date yields a negative result. However, among the representatives of the Mollicutes (mycoplasma) class, genes encoding α-HSPs were found in two phytoplasma species (Phytoplasmataceae family) and the acholeplasma examined (Acholeplasmataceae family). It is supposed that the presence or absence of α-HSPs in microorganisms might be related to their inhabitancy; representatives of Acholeplasmataceae and Phytoplasmataceae families mostly reside in plant tissues, which is in contrast to the majority of the Mycoplasmataceae family, which lives in animal and human tissues, i.e., use ecological niches with relatively constant temperature.

RNA polymerase II-mediated transcription at active loci does not require histone H3S10 phosphorylation in Drosophila.

JIL-1 is the major kinase controlling the phosphorylation state of histone H3S10 at interphase in Drosophila. In this study, we used three different commercially available histone H3S10 phosphorylation antibodies, as well as an acid-free polytene chromosome squash protocol that preserves the antigenicity of the histone H3S10 phospho-epitope, to examine the role of histone H3S10 phosphorylation in transcription under both heat shock and non-heat shock conditions. We show that there is no redistribution or upregulation of JIL-1 or histone H3S10 phosphorylation at transcriptionally active puffs in such polytene squash preparations after heat shock treatment. Furthermore, we provide evidence that heat shock-induced puffs in JIL-1 null mutant backgrounds are strongly labeled by antibody to the elongating form of RNA polymerase II (Pol IIoser2), indicating that Pol IIoser2 is actively involved in heat shock-induced transcription in the absence of histone H3S10 phosphorylation. This is supported by the finding that there is no change in the levels of Pol IIoser2 in JIL-1 null mutant backgrounds compared with wild type. mRNA from the six genes that encode the major heat shock protein in Drosophila, Hsp70, is transcribed at robust levels in JIL-1 null mutants, as directly demonstrated by qRT-PCR. Taken together, these data are inconsistent with the model that Pol II-dependent transcription at active loci requires JIL-1-mediated histone H3S10 phosphorylation, and instead support a model in which transcriptional defects in the absence of histone H3S10 phosphorylation are a result of structural alterations of chromatin.

Intracellular and extracellular expression of the major inducible 70kDa heat shock protein in experimental ischemia-reperfusion injury of the spinal cord

Inflammatory responses exacerbate ischemia-reperfusion (IR) injury of spinal cord, although understanding of mediators is incomplete. The major inducible 70kDa heat shock protein (hsp70) is induced by ischemia and extracellular hsp70 (e-hsp70) can modulate inflammatory responses, but there is no published information regarding e-hsp70 levels in the cerebrospinal fluid (CSF) or serum as part of any neurological disease state save trauma. The present work addresses this deficiency by examining e-hsp70 in serum and CSF of dogs in an experimental model of spinal cord IR injury. IR injury of spinal cord caused hind limb paraplegia within 2–3 h that was correlated to lumbosacral poliomalacia with T cell infiltrates at 3 d post-ischemia. In this context, we showed a 5.2-fold elevation of e-hsp70 in CSF that was induced by ischemia and was sustained for the following 3 d observation interval. Plasma e-hsp70 levels were unaffected by IR injury, indicating e-hsp70 release from within the central nervous system. A putative source of this e-hsp70 was ependymal cells in the ischemic penumbra, based upon elevated i-hsp70 levels detected within these cells. Results warrant further investigation of e-hsp70's potential to modulate spinal cord IR injury.