The expression patterns of heat shock genes and proteins and their role during vertebrate's development

Highly evolutionary conserved heat shock proteins (HSPs) act as molecular chaperones in regulation of cellular homeostasis and promoting survival. Generally they are induced by a variety of stressors whose effect could be disastrous on the organism, but they are also widely constitutively expressed in the absence of stress. Varied HSP expressions seem to be very essential in the critical steps of embryonic and extra-embryonic structures formation and may correspond to cell movements, proliferation, morphogenesis and apoptosis, which occur during embryonic development. While our knowledge of detailed HSP expression patterns is in constant progress, their functions during embryonic development are not yet fully understood. In the paper, we review available data on HSP expression and discuss their role during vertebrate development.     

Immunomodulatory activity of extracellular heat shock proteins and their autoantibodies

HSP are groups of stress‐inducible proteins which contribute to quality control by assisting the correct folding of both nascent and denatured proteins, and promoting the degradation of unrecoverable denatured proteins. HSP also help to maintain cellular homeostasis and protect from cell death through a mechanism called thermotolerance. Cells subjected to mild stress induce HSP which then protect them against subsequent stress. However, in cells subjected to severe stress, HSP promote apoptosis. Besides these intracellular events, HSP also exist in extracellular fluids, and have been shown to contribute to immunomodulation. In innate immunity extracellular HSP, like various microbial substances, induce various proinflammatory cytokines. In acquired immunity they interact with antigenic polypeptides and assist in antigen presentation. The extracellular HSP are so‐called adjuvant. Release of HSP from cells is triggered by stress and trauma, and is thus regarded as an immunological “danger signal”. In addition, anti‐HSP autoantibodies are frequently found in patients with autoimmune diseases and inflammatory disorders, and these autoantibodies can modulate the “danger signal” triggered by extracellular HSP.     

Microbial small heat shock proteins and their use in biotechnology

Small heat shock proteins (sHsps) exist in almost all organisms. Most organisms have more than one sHsp, but their number can be as high as 65, as found in the eukaryote, Vitis vinifera. The function of sHsps is well-known; they confer thermotolerance to cellular cultures and proteins in cellular extracts during prolonged incubations at elevated temperatures. This demonstrates the ability of sHsps to protect cellular proteins, and to maintain cellular viability under conditions of intensive stress, such as heat shock or chemical treatments. sHsps have several properties that distinguish them from heat shock proteins (Hsps): they function as ATP-independent chaperones, require the flexible assembly and reassembly of oligomeric complex structures for their activation, and exhibit a wide range of substrate-binding capacities. Recent studies indicate that sHsps have important biological functions in thermostability, disaggregation, and proteolysis inhibition. These functions can be harnessed for various applications, including nanobiotechnology, proteomics, bioproduction, and bioseparation. In this review, we discuss the properties and diversity of microbial sHsps, as well as their potential uses in the biotechnology industry.     

Extreme thermotolerance and behavioral induction of 70-kDa heat shock proteins and their encoding genes in honey bees

Foraging honey bees frequently leave the hive to gather pollen and nectar for the colony. This period of their lives is marked by periodic extremes of body temperature, metabolic expenditure, and flight muscle activity. Following ecologically relevant episodes of hyperthermia between 33°C and 50°C, heat shock protein 70 (Hsp70) expression and hsp70/hsc70-4 activity in brains of nonflying laboratory-held bees increased by only two to three times baseline at temperatures 46–50°C. Induction was undetectable in thoracic–flight muscles. Yet, thorax hsp70 mRNA (but not hsc70-4 mRNA) levels were up to ten times higher in flight-capable hive bees and foraging bees compared to 1-day-old, flight-incapable bees, while brain hsp70/hsc70-4 mRNA levels were low and varied little among behavioral groups. These data suggest honey bee tissues, especially flight muscles, are extremely thermotolerant. Furthermore, Hsp70 expression in the thoraces of flight-capable bees is probably flight-induced by oxidative and mechanical damage to flight muscle proteins rather than temperature.     

The small heat shock proteins and their role in human disease

Small heat shock proteins (sHSPs) function as molecular chaperones, preventing stress induced aggregation of partially denatured proteins and promoting their return to native conformations when favorable conditions pertain. Sequence similarity between sHSPs resides predominately in an internal stretch of residues termed the alpha-crystallin domain, a region usually flanked by two extensions. The poorly conserved N-terminal extension influences oligomer construction and chaperone activity, whereas the flexible C-terminal extension stabilizes quaternary structure and enhances protein/substrate complex solubility. sHSP polypeptides assemble into dynamic oligomers which undergo subunit exchange and they bind a wide range of cellular substrates. As molecular chaperones, the sHSPs protect protein structure and activity, thereby preventing disease, but they may contribute to cell malfunction when perturbed. For example, sHSPs prevent cataract in the mammalian lens and guard against ischemic and reperfusion injury due to heart attack and stroke. On the other hand, mutated sHSPs are implicated in diseases such as desmin-related myopathy and they have an uncertain relationship to neurological disorders including Parkinson's and Alzheimer's disease. This review explores the involvement of sHSPs in disease and their potential for therapeutic intervention.     

Chromosomal location of genes controlling heat shock proteins in hexaploid wheat

Summary The low molecular weight heat shock protein (HSP) profiles of the hexaploid wheat cultivar Chinese Spring and its ditelosomic series were characterized by isoelectric focusing polyacrylamide gel electrophoresis of denatured in vivo radiolabeled proteins. Comparisons of the ditelosomics (DTs) to the euploid Chinese Spring enabled the assignment of genes controlling 9 of the 13 targeted HSPs to seven chromosome arms. There did not appear to be a genome-specific action in the regulation of expression of these HSPs. There did appear to be a higher frequency of controlling genes within homoeologous DT lines 3, 4 and 7. Significant variation in protein quantity was evident among the DT lines for some HSPs, while other HSPs were remarkably stable in their expression across all DTs examined. The results are useful in identifying specific DT lines for the investigation of HSP functions in hexaploid wheat.     

The evolution of heat shock genes and expression patterns of heat shock proteins in the species from temperature contrasting habitats

Heat shock genes are the most evolutionarily ancient among the systems responsible for adaptation of organisms to a harsh environment. The encoded proteins (heat shock proteins, Hsps) represent the most important factors of adaptation to adverse environmental conditions. They serve as molecular chaperones, providing protein folding and preventing aggregation of damaged cellular proteins. Structural analysis of the heat shock genes in individuals from both phylogenetically close and very distant taxa made it possible to reveal the basic trends of the heat shock gene organization in the context of adaptation to extreme conditions. Using different model objects and nonmodel species from natural populations, it was demonstrated that modulation of the Hsps expression during adaptation to different environmental conditions could be achieved by changing the number and structural organization of heat shock genes in the genome, as well as the structure of their promoters. It was demonstrated that thermotolerant species were usually characterized by elevated levels of Hsps under normal temperature or by the increase in the synthesis of these proteins in response to heat shock. Analysis of the heat shock genes in phylogenetically distant organisms is of great interest because, on one hand, it contributes to the understanding of the molecular mechanisms of evolution of adaptogenes and, on the other hand, sheds the light on the role of different Hsps families in the development of thermotolerance and the resistance to other stress factors.     

Methylglyoxal and small heat shock proteins

Methylglyoxal is a highly reactive dicarbonyl compound formed during glucose metabolism and able to modify phospholipids, nucleic acids, and proteins belonging to the so-called dicarbonyl proteome. Small heat shock proteins participating in protection of the cell against different unfavorable conditions can be modified by methylglyoxal. The probability of methylglyoxal modification is increased in the case of distortion of glucose metabolism (diabetes), in the case of utilization of glycolysis as the main source of energy (malignancy), and/or at low rate of modified protein turnover. We have analyzed data on modification of small heat shock protein HspB1 in different tumors and under distortion of carbohydrate metabolism. Data on the effect of methylglyoxal modification on stability, chaperone-like activity, and antiapoptotic activity of HspB1 were analyzed. We discuss data on methylglyoxal modifications of lens α-crystallins. The mutual dependence and mutual effects of methylglyoxal modification and other posttranslational modifications of lens crystallins are analyzed. We conclude that although there is no doubt that the small heat shock proteins undergo methylglyoxal modification, the physiological significance of this process remains enigmatic, and new experimental approaches should be developed for understanding how this type of modification affects functioning of small heat shock proteins in the cell.     

Structure and properties of small heat shock proteins (sHsp) and their interaction with cytoskeleton proteins

The modern classification of small heat shock proteins (sHsp) is presented and peculiarities of their primary structure and the mechanism of formation of oligomeric complexes are described. Data on phosphorylation of sHsp by different protein kinases are presented and the effect of phosphorylation on oligomeric state and chaperone activity of sHsp is discussed. Intracellular location of sHsp under normal and stress conditions is described and it is emphasized that under certain condition sHsp interact with different elements of cytoskeleton. The literature concerning the effect of sHsp on polymerization of actin in vitro is analyzed. An attempt is made to compare effects of sHsp on polymerization of actin in vitro with the results obtained on living cells under normal conditions and after heat shock or hormone action. The literature concerning possible effects of sHsp on cell motility is also analyzed.     

Nuclear localization and the heat shock proteins

The highly conserved heat shock proteins (HSP) belong to a subset of cellular proteins that localize to the nucleus. HSPs are atypical nuclear proteins in that they localize to the nucleus selectively, rather than invariably. Nuclear localization of HSPs is associated with cell stress and cell growth. This aspect of HSPs is highly conserved with nuclear localization occurring in response to a wide variety of cell stresses. Nuclear localization is likely important for at least some of the heat shock proteins’ protective functions; little is known about the function of the heat shock proteins in the nucleus. Nuclear localization is signalled by the presence of a basic nuclear localization sequence (NLS) within a protein. Though most is known about HSP 72’s nuclear localization, the NLS(s) has not been definitively identified for any of the heat shock proteins. Likely more is involved than presence of a NLS; since the heat shock proteins only localize to the nucleus under selective conditions, nuclear localization must be regulated. HSPs also function as chaperons of nuclear transport, facilitating the movement of other macromolecules across the nuclear membrane. The mechanisms involved in chaperoning of proteins by HSPs into the nucleus are still being identified.     

Heat shock proteins and effects of heat shock in plants

Soybean seedlings when exposed to a heat shock respond in a manner very similar to that exhibited by cultured cells, and reported earlier [2]. Maximum synthesis of heat shock proteins (HSPs) occurs at 40C. The heat shock response is maintained for a relatively short time under continuous high temperature. After 2.5 hr at 40 C the synthesis of HSPs decreases reaching a very low level by 6 hr. The HSPs synthesized by cultured cells and seedlings are identical and there is a large degree of similarity in HSPs synthesized between the taxonomically widely separated species, soybean and corn. Storage protein synthesis in the developing soybean embryo is not inhibited but is actually stimulated during a heat shock, unlike most other non-HSPs, whose synthesis is greatly reduced. Seedlings respond differently to a gradual increase in temperature than they do a sudden heat shock. There is an upward shift of several degrees in the temperature at which maximum protein synthesis occurs and before it begins to be inhibited. In addition, there appears to be a protection of normal protein synthesis from heat shock inhibition when the temperature increase is gradual. An additional function of the heat shock phenomenon might be the protection of seedlings from death caused by extreme heat stress. The heat shock response appears to have relevance to plants in the field.     

Cytokine function of heat shock proteins

Extensive work in the last 10 years has suggested that heat shock proteins (HSPs) may be potent activators of the innate immune system. It has been reported that Hsp60, Hsp70, Hsp90, and gp96 are capable of inducing the production of proinflammatory cytokines by the monocyte-macrophage system and the activation and maturation of dendritic cells (antigen-presenting cells) in a manner similar to the effects of lipopolysaccharide (LPS) and bacterial lipoprotein, e.g., via CD14/Toll-like receptor2 (TLR2) and CD14/TLR4 receptor complex-mediated signal transduction pathways. However, recent evidence suggests that the reported cytokine effects of HSPs may be due to the contaminating LPS and LPS-associated molecules. The reasons for previous failure to recognize the contaminant(s) as being responsible for the reported HSP cytokine effects include failure to use highly purified, low-LPS preparations of HSPs; failure to recognize the heat sensitivity of LPS; and failure to consider contaminant(s) other than LPS. Thus it is essential that efforts should be directed to conclusively determine whether the reported HSP cytokine effects are due to HSPs or to contaminant(s) present in the HSP preparations before further exploring the implication and therapeutic potential of the putative cytokine function of HSPs. tumor necrosis factor-; lipopolysaccharide; macrophages; innate immune system     

Interaction between heat shock proteins and antimicrobial peptides

Drosocin, pyrrhocoricin, and apidaecin, representing the short (18-20 amino acid residues) proline-rich antibacterial peptide family, originally isolated from insects, were shown to act on a target bacterial protein in a stereospecific manner. Native pyrrhocoricin and one of its analogues designed for this purpose protect mice from bacterial challenge and, therefore, may represent alternatives to existing antimicrobial drugs. Furthermore, this mode of action can be a basis for the design of a completely novel set of antibacterial compounds, peptidic or peptidomimetic, if the interacting bacterial biopolymers are known. Recently, apidaecin was shown to enter Escherichia coli and subsequently kill bacteria through sequential interactions with diverse target macromolecules. In this paper report, we used biotin- and fluorescein-labeled pyrrhocoricin, drosocin, and apidaecin analogues to identify biopolymers that bind to these peptides and are potentially involved in the above-mentioned multistep killing process. Through use of a biotin-labeled pyrrhocoricin analogue, we isolated two interacting proteins from E. coli. According to mass spectrometry, Western blot, and fluorescence polarization, the short, proline-rich peptides bound to DnaK, the 70-kDa bacterial heat shock protein, both in solution and on the solid-phase. GroEL, the 60-kDa chaperonin, also bound in solution. Control experiments with an unrelated labeled peptide showed that while binding to DnaK was specific for the antibacterial peptides, binding to GroEL was not specific for these insect sequences. The killing of bacteria and DnaK binding are related events, as an inactive pyrrhocoricin analogue made of all-D-amino acids failed to bind. The pharmaceutical potential of the insect antibacterial peptides is underscored by the fact that pyrrhocoricin did not bind to Hsp70, the human equivalent of DnaK. Competition assay with unlabeled pyrrhocoricin indicated differences in GroEL and DnaK binding and a probable two-site interaction with DnaK. In addition, all three antibacterial peptides strongly interacted with two bacterial lipopolysaccharide (LPS) preparations in solution, indicating that the initial step of the bacterial killing cascade proceeds through LPS-mediated cell entry.     

Molecular chaperones and heat shock proteins in atherosclerosis

In response to stress stimuli, mammalian cells activate an ancient signaling pathway leading to the transient expression of heat shock proteins (HSPs). HSPs are a family of proteins serving as molecular chaperones that prevent the formation of nonspecific protein aggregates and assist proteins in the acquisition of their native structures. Physiologically, HSPs play a protective role in the homeostasis of the vessel wall but have an impact on immunoinflammatory processes in pathological conditions involved in the development of atherosclerosis. For instance, some members of HSPs have been shown to have immunoregulatory properties and modification of innate and adaptive response to HSPs, and can protect the vessel wall from the disease. On the other hand, a high degree of sequence homology between microbial and mammalian HSPs, due to evolutionary conservation, carries a risk of misdirected autoimmunity against HSPs expressed on the stressed cells of vascular endothelium. Furthermore, HSPs and anti-HSP antibodies have been shown to elicit production of proinflammatory cytokines. Potential therapeutic use of HSP in prevention of atherosclerosis involves achieving optimal balance between protective and immunogenic effects of HSPs and in the progress of research on vaccination. In this review, we update the progress of studies on HSPs and the integrity of the vessel wall, discuss the mechanism by which HSPs exert their role in the disease development, and highlight the potential clinic translation in the research field.