Heat shock proteins hsp32 and hsp70 as biomarkers of an early response?: In vitro induction of heat shock proteins after exposure of cell culture to carcinogenic compounds and their real mixtures

Heat shock proteins (hsp) are a highly conserved group of proteins that are synthesized as a response to different forms of stress (heat, toxic chemicals, diseases, non-physiological pH changes). Because of their high sensitivity to changes in the environment, these proteins were suggested as possible early biomarkers of exposure in ecotoxicological studies. The purpose of the present study was to check the suitability of hsp32 and hsp70 as biomarkers of in vitro exposure to environmentally relevant carcinogens: polycyclic aromatic hydrocarbons (PAHs), their nitro-derivates, aromatic amines, acrylonitrile (ACN) and the mixture of organic compounds adsorbed onto ambient airborne particles (extractable organic matter, EOM).The expression of hsp32 and hsp70 was studied in human diploid lung fibroblasts (HEL cells) and human monocytic leukaemia cells (THP-1 cells) incubated in vitro with different concentrations of dibenzo[a,l]pyrene (DB[a,l]P), 1-nitropyrene, (NP), 4-aminobiphenyl (ABP), ACN and EOM for different periods of time. The incubation of cells with DB[a,l]P, NP, ABP and EOM did not result in increased levels of hsp32 or hsp70, either in dose- or time-dependent manner. ACN induced the expression of hsp32 as well as hsp70 in HEL and THP-1 cells, which probably reflects its ability to induce oxidative stress. We conclude that hsp32 and hsp70 are not suitable biomarkers of an early exposure to PAHs, their nitro-derivates, aromatic amines or EOM under the conditions used.     

Effect of thymopentin on the production of cytokines, heat shock proteins, and NF-κB signaling proteins

In vivo effects of thymopentin, an active fragment of the naturally occurring thymic hormone thymopoietin, on the production of cytokines, nitric oxide, heat shock proteins, and signaling proteins NF-κB, phNF-κB, and IκB-α in lymphoid cells of male NMRI mice was studied. Activation of production of several cytokines (IL-1α, IL-2, IL-6, IL-10, and IFN-γ), nitric oxide, and heat shock proteins (HSP70 and HSP90) was observed in peritoneal macrophages and spleen lymphocytes of mice that received intraperitoneal injections of thymopentin (15μg per 100 g body weight). Thymopentin apparently produces stress-like rather than damaging effects. A probable action mechanism of this hormone is activation of the NF-κB signaling pathway, which is most pronounced at the NF-κB phosphorylation stage.     

Small heat shock proteins and α-crystallins: dynamic proteins with flexible functions

The small heat shock proteins (sHSPs) and the related α-crystallins (αCs) are virtually ubiquitous proteins that are strongly induced by a variety of stresses, but that also function constitutively in multiple cell types in many organisms. Extensive research has demonstrated that a majority of sHSPs and αCs can act as ATP-independent molecular chaperones by binding denaturing proteins and thereby protecting cells from damage due to irreversible protein aggregation. As a result of their diverse evolutionary history, their connection to inherited human diseases, and their novel protein dynamics, sHSPs and αCs are of significant interest to many areas of biology and biochemistry. However, it is increasingly clear that no single model is sufficient to describe the structure, function or mechanism of action of sHSPs and αCs. In this review, we discuss recent data that provide insight into the variety of structures of these proteins, their dynamic behavior, how they recognize substrates, and their many possible cellular roles.     

Inhibition of α-synuclein aggregation by small heat shock proteins

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.     

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.     

Heat shock proteins in toxicology: How close and how far?

The response to stress triggers activation of the genes involved in cell survival and/or cell death. Stress response is a ubiquitous feature of cells that is induced under stress conditions. As a part of this response a set of genes called stress genes are induced to synthesize a group of proteins called heat shock proteins (Hsps). The Hsps play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and by preventing their aggregation. Because of their sensitivity to even minor assaults, Hsps are suitable as an early warning bio-indicator of cellular hazard. Despite having enormous use in toxicology, the current state of knowledge in defining a mechanism of action or accurately predicting toxicity based on stress gene expression warrants further investigation. The goal of this review is to summarize current developments in the application of stress genes and their products ‘Hsps’ in toxicology with a brief discussion of the caveats. While focusing on hsp70 because of its higher conservation across the taxa and since it is one of the first to be induced under stress conditions, we will also discuss other members of the stress gene family.     

High levels of acute phase proteins and soluble 70 kDa heat shock proteins are independent and additive risk factors for mortality in colorectal cancer

Recently, we reported that high soluble Hsp70 (sHsp70) level was a significant predictor of mortality during an almost 3-year-long follow-up period in patients with colorectal cancer. This association was the strongest in the group of <70-year-old female patients as well as in those who were in a less advanced stage of the disease at baseline. According to these observations, measurement of the serum level of sHsp70 is a useful, stage-independent prognostic marker in colorectal cancer, especially in patients without distant metastasis. Since many literature data indicated that measurement of C-reactive protein (CRP) and other acute phase proteins (APPs) may also be suitable for predicting the mortality of patients with colorectal cancer, it seemed reasonable to study whether the effect of sHsp70 and other APPs are related or independent. In order to answer this question, we measured the concentrations of CRP as well as of other complement-related APPs (C1 inhibitor, C3, and C9) along with that of the MASP-2 complement component in the sera of 175 patients with colorectal cancer and known levels of sHsp70, which have been used in our previous study. High (above median) levels of CRP, C1 esterase inhibitor (C1-INH), and sHsp70 were found to be independently associated with poor patient survival, whereas no such association was observed with the other proteins tested. According to the adjusted Cox proportional hazards analysis, the additive effect of high sHsp70, CRP, and C1-INH levels on the survival of patients exceeded that of high sHsp70 alone, with a hazard ratio (HR) of 2.83 (1.13–70.9). In some subgroups of patients, such as in females [HR 4.80 (1.07–21.60)] or in ≤70-year-old patients [HR 11.53 (2.78–47.70)], even greater differences were obtained. These findings indicate that the clinical mortality–prediction value of combined measurements of sHsp70, CRP, and C1-INH with inexpensive methods can be very high, especially in specific subgroups of patients with colorectal cancer.     

Heat shock proteins in animal neoplasms and human tumours—a comparison

Heat shock proteins (HSPs) are implicated in all phases of cancer from proliferation, impaired apoptosis and sustained angiogenesis to invasion and metastasis. The presence of abnormal HSP levels in several human tumours suggests that these proteins could be used as diagnostic and/or prognostic markers, whilst the direct correlation between HSP expression and drug resistance in neoplastic tissues means they could also be used to predict cancer response to specific treatment. HSPs have also been successfully targeted in clinical trials modifying their expression or chaperone activity. Preliminary studies in veterinary medicine have also demonstrated the presence of altered HSP expression in neoplasms, and the study of carcinogenesis and the role of HSPs in animal models will surely be an additional source of information for clinical cancer research.     

Molecular characterization of heat shock proteins 90 (HSP83?) and 70 in tropical strains of Bombyx mori

Following the concept of whole organism, we have extracted total protein from the Bombyx mori for the identification and analysis of HSPs. Expression of 90 kDa HSP in first, second and third instars, 84 kDa in fourth instar and 90‐, 84‐, 62‐, 60‐, 52‐ and 33‐kDa HSPs in fifth instar larvae of tropical polyvoltine and bivoltine silkworm strains were obvious. Further, we have combined single and 2‐DE with MALDI‐TOF for analysis of BmHSPs. Ninety kilodalton band excised from 1‐DE gel was identified as HSP83 by MALDI‐TOF‐MS. The immunoblot analysis confirmed the expression of HSP90 in all the instars larvae of B. mori. Heat shock‐induced protein spots were excised from 2‐DE gels for MALDI‐TOF‐MS analysis. The Mascot search results are for HSP68, HSC70‐1 and HSP70Ba in Pure Mysore, and major HSP70Bbb, HSP68, HSC‐3 and HSP83 in NB₄D₂. Multiple sequence alignment explicit the variations in amino acid sequence between Pure Mysore and NB₄D₂. Notably, the PMF of spot 2 matched the coding sequence of B. mori and its gene annotation was determined on chromosome 9. With this novel approach, expression of BmHSP90 was confirmed in all the instars and uncovered isoforms of BmHSP70, which provided unequivocal insight to analyze and understand the biological significance in B. mori.     

Is heat shock protein re-induction during tolerance related to the stressor-specific induction of heat shock proteins?

The existence of stressor-specific induction programs of heat shock proteins (hsps) leads us to analyze the possible occurrence of a stressor-specific tolerance induced by either heat shock, arsenite, or cadmium. As a measure of this tolerance re-induction of hsps was studied. In this paper, we tested whether the refractory state is either valid for each specific hsp (implying independent regulation of every member of the heat shock protein family) or extends from small subsets of the hsp-family to even larger groups of proteins (indicating a more common denominator in their regulation). (Re-)induction of hsps does not seem to be regulated at the level of each individual hsp since differences in induced synthesis of hsps between two stressor conditions are not supplemented systematically upon the sequential application of the two stressors. The most notable example in this respect is hsp60. A pretreatment with cadmium, which hardly induces synthesis of this hsp, does induce a tolerance to (re)-induction by heat shock, which normally induces hsp60. This suggests the existence of a more common denominator regulating the coordinate expression of at least some hsps. From our data we conclude that the degree, but not the pattern, of hsp re-induction is influenced by the type of stressor used in the pretreatment. The pattern of hsps induced by a secondary applied stressor still shows most of its stressor-specificity and seems to be independent of any pretreatment. The possible implications of stressor-specificity are discussed. © 1996 Wiley-Liss, Inc.     

Stat1 mediates an auto-regulation of hsp90β gene in heat shock response

We have reported earlier that a heat shock element in the first intron of human hsp90β gene (iHSE) acts as an intronic enhancer to bind the heat shock factor (HSF1) and activates hsp90β gene under heat shock. Here, we show that, in addition to the HSF1, Stat1 phosphorylation is indispensable in the event. We show that Jak2, a Janus kinase specifically associated with the β subunit of IFNγ receptor, and PKCε? an isoform of the atypical PKC family, are the two dominant kinases responsible for the heat shock induced phosphorylation on Y701 and S727 of Stat1. However, the activation of these kinases under heat shock requires the association of chaperone proteins of the Hsp90 family, in particular, the Hsp90β under heat shock. Furthermore, Brg1, an ATPase subunit of the SWI/SNF chromatin remodeling complex is likely recruited by HSF1 and Stat1 at the iHSE under heat shock. Brg1 further confers an open chromatin conformation at the promoter region that is pivotal to the heat shock induced fully activation of the hsp90β gene in Jurkat cells. This is a novel example of how multiple activation steps occur under heat shock, first on the kinases and then the Stat1 and the SWI/SNF chromatin remodeling complex that follows to conduct an auto-regulation based fully activation of the gene.     

Actin cytoskeleton and small heat shock proteins: how do they interact?

Actin and small heat shock proteins (sHsps) are ubiquitous and multifaceted proteins that exist in 2 reversible forms, monomers and multimers, ie, the microfilament of the cytoskeleton and oligomers of the sHsps, generally, supposed to be in a spherical and hollow form. Two situations are described in the literature, where the properties of actin are modulated by sHsps; the actin polymerization is inhibited in vitro by some sHsps acting as capping proteins, and the actin cytoskeleton is protected by some sHsps against the disruption induced by various stressful conditions. We propose that a direct actin-sHsp interaction occurs to inhibit actin polymerization and to participate in the in vivo regulation of actin filament dynamics. Protection of the actin cytoskeleton would result from an F-actin-sHsp interaction in which microfilaments would be coated by small oligomers of phosphorylated sHsps. Both proteins share common structural motives suggesting direct binding sites, but they remain to be demonstrated. Some sHsps would behave with the actin cytoskeleton as actin-binding proteins capable of either capping a microfilament when present as a nonphosphorylated monomer or stabilizing and protecting the microfilament when organized in small, phosphorylated oligomers.     

Heat shock proteins in health and disease: therapeutic targets or therapeutic agents?

For many years, heat shock or stress proteins have been regarded as intracellular molecules that have a range of housekeeping and cytoprotective functions, only being released into the extracellular environment in pathological situations such as necrotic cell death. However, evidence is now accumulating to indicate that, under certain circumstances, these proteins can be released from cells in the absence of cellular necrosis, and that extracellular heat shock proteins have a range of immunoregulatory activities. The capacity of heat shock proteins to induce pro-inflammatory responses, together with the phylogenetic similarity between prokaryotic and eukaryotic heat shock proteins, has led to the proposition that these proteins provide a link between infection and autoimmune disease. Indeed, both elevated levels of antibodies to heat shock proteins and an enhanced immune reactivity to heat shock proteins have been noted in a variety of pathogenic disease states. However, further evaluation of heat shock protein reactivity in autoimmune disease and after transplantation has shown that, rather than promoting disease, reactivity to self-heat shock proteins can downregulate the disease process. It might be that self-reactivity to heat shock proteins is a physiological response that regulates the development and progression of pro-inflammatory immunity to these ubiquitously expressed molecules. The evolving evidence that heat shock proteins are present in the extracellular environment, that reactivity to heat shock proteins does not necessarily reflect adverse, pro-inflammatory responses and that the promotion of reactivity to self-heat shock proteins can downregulate pathogenic processes all suggest a potential role for heat shock proteins as therapeutic agents, rather than as therapeutic targets.     

Oligomerization and toxicity of Aβ fusion proteins

This study has found that the Maltose binding protein Aβ42 fusion protein (MBP-Aβ42) forms soluble oligomers while the shorter MBP-Aβ16 fusion and control MBP did not. MBP-Aβ42, but neither MBP-Aβ16 nor control MBP, was toxic in a dose-dependent manner in both yeast and primary cortical neuronal cells. This study demonstrates the potential utility of MBP-Aβ42 as a reagent for drug screening assays in yeast and neuronal cell cultures and as a candidate for further Aβ42 characterization.     

Do heat shock proteins provide protection against freezing?

Yeast cells were frozen by plunging directly into liquid nitrogen (LN2) after exposure at 43 degrees C. Both the cells frozen without prior exposure to heat shock and those treated with cycloheximide showed almost 100% loss of viability during freezing and thawing. Heat exposure prior to freezing and thawing significantly increased the cell viability. This increase in cell viability was associated with the induction of heat shock protein synthesis, which was detected by gel electrophoresis. This protein may act by stabilizing the macromolecules and by increasing the hydrophobic interactions.     

Hydrophilicity and antigenicity of proteins — A case study of myoglobin and hemoglobin

Hydrophilicity index is used to locate antigenic determinants on two related groups of proteins-myoglobin and hemoglobin. The data on 41 species (including 34 mammals) of myoglobin show that average hydrophilicity for the complete myoglobin molecules as well as the average hydrophilicity for all hydrophilic regions put together seem to remain constant; the variation in the size and location of the antigenic determinants in these species is very small indicating that the antigenic sites are not shifted during evolution. In the case of both the proteins there is a good agreement between the antigenic sites picked up by using hydrophilicity index and the experimentally determined antigenic sites. The data on 56 species of hemoglobin α-chains and 44 species of hemoglobinβ-chains showed that although there are few sites on hemoglobin which have remained invariant during evolution, there is a significant variation in other sites in terms of either a splitting of a site, or a drastic change in the hydrophilicity values and/or a length of the site. Comparison of the hydrophilicity data on these two groups of proteins suggests that hemoglobins which perform a variety of functions as compared to myoglobins are evolving faster than myoglobins supporting the contention of earlier workers.