Chicken heat shock protein HSPB1 increases and interacts with αB-crystallin in aged skeletal muscle

International trading markets of meat require the animal’s age information to prevent cross-contamination of ineligible meat products. Individual livestock age is either evaluated from physiological features or verified by breeding history. However, it remains impossible to perform age verification on meat when a suspicion of error occurred in the importing country. To investigate an age-related protein in skeletal muscle of livestock, we compared protein expression among chicken pectoralis major of different ages. Results indicated that the level of expression of chicken HSPB1, one of the small heat shock proteins, was increased in aged muscles. On the other hand, other heat shock proteins, heat shock factors, and myosin heavy chain isoform did not change the expression levels in aged chicken muscle. In addition, we identified that αB-crystallin interacted with HSPB1 in aged chicken muscle. These results suggest that HSPB1 protein forms complexes with αB-crystallin in aged chicken muscle and suppose to become the candidate of age-related bio-marker for verifying the age of chicken meat.     

The small heat shock proteins αB-crystallin and Hsp27 suppress SOD1 aggregation in vitro

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease. The mechanism that underlies amyotrophic lateral sclerosis (ALS) pathology remains unclear, but protein inclusions are associated with all forms of the disease. Apart from pathogenic proteins, such as TDP-43 and SOD1, other proteins are associated with ALS inclusions including small heat shock proteins. However, whether small heat shock proteins have a direct effect on SOD1 aggregation remains unknown. In this study, we have examined the ability of small heat shock proteins αB-crystallin and Hsp27 to inhibit the aggregation of SOD1 in vitro. We show that these chaperone proteins suppress the increase in thioflavin T fluorescence associated with SOD1 aggregation, primarily through inhibiting aggregate growth, not the lag phase in which nuclei are formed. αB-crystallin forms high molecular mass complexes with SOD1 and binds directly to SOD1 aggregates. Our data are consistent with an overload of proteostasis systems being associated with pathology in ALS.     

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.     

The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. Proteostasis is preserved in the face of stress by a complex network of cellular machinery, including the small heat shock molecular chaperone proteins (sHsps), which act to inhibit the aggregation and deposition of misfolded protein intermediates. Despite this, the pathogenesis of several neurodegenerative diseases has been inextricably linked with the amyloid fibrillar aggregation and deposition of α-synuclein (α-syn). The sHsps are potent inhibitors of α-syn aggregation in vitro. However, the limited availability of a robust, cell-based model of α-syn aggregation has, thus far, restricted evaluation of sHsp efficacy in the cellular context. 相似文献   

Comparison of the homologous carboxy-terminal domain and tail of α-crystallin and small heat shock protein

The C-terminal domain and tail, which is the most conserved region of the -crystallin/small heat shock protein (HSP) family, was obtained from rat A-crystallin, bovine B-crystallin and mouse HSP25. All three domains have primarily -sheet conformation and less than 10% of -helix, like the proteins from which they are derived. Whereas the C-terminal part of A-crystallin forms dimers or tetramers, the corresponding regions of B-crystallin and HSP25 form larger aggregates. The heat-protective activity, recently described for the -crystallin/small HSP family, is not retained in the C-terminal domain and tail. In the course of this study some differences with the previously published sequence of HSP25 were observed, and a revision is proposed.Abbreviations A2Dt residues 64–173 of rat -crystallin - B2Dt residues 70–175 of bovine B-crystallin - bp base pair - HSP2Dt residues 92–209 of HSP25 - HSP(s) heat shock protein(s) - HSP25 mouse small HSP - PCR polymerase chain reaction - PMSF phenylmethylsulfonyl chloride - SDS sodium dodecyl sulfate; polyacrylamide - WSF water-soluble fraction     

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.     

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

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

Dynamical structure of αB-crystallin

The human small heat-shock protein αB-crystallin is an extremely difficult molecule to study, with its inherent structural dynamics posing unique challenges to all biophysical and structural biology techniques. Here we highlight how the polydispersity and quaternary dynamics of αB-crystallin are intrinsically inter-twined, and how this can impact on measurements of the oligomeric distribution. We show that, in spite of these difficulties, considerable understanding of the varied fluctuations αB-crystallin undergoes at equilibrium has emerged in the last few years. By reporting on data obtained from a variety of biophysical techniques, we demonstrate how the αB-crystallin solution ensemble is governed by molecular motions of varying amplitude and time-scales spanning several orders of magnitude. We describe how these diverse measurements are being used to construct an integrated view of the dynamical structure of αB-crystallin, and highlight areas that require further interrogation. With its study motivating the refinement of experimental techniques, and the development of new approaches to combine the hybrid datasets, we conclude that αB-crystallin continues to represent a paradigm for dynamical biology.     

Fas ligation and tumor necrosis factor α activation of murine astrocytes promote heat shock factor-1 activation and heat shock protein expression leading to chemokine induction and cell survival

Death-inducing ligands tumor necrosis factor alpha (TNFα) and Fas ligand (FasL) do not kill cultured astrocytes; instead they induce a variety of chemokines including macrophage-inflammatory protein-1α/CC chemokine ligand 3 (CCL3), monocyte chemoattractant protein-1 (CC CCL-2), macrophage-inflammatory protein-2/CXC chemokine ligand 2 (CXCL2, a murine homologue of interleukin 8), and interferon-induced protein of 10 kDa (CXCL10). Induction is enhanced by protein synthesis inhibition suggesting the existence of endogenous inhibitors. ERK, NF-κB, heat shock factor-1 (HSF-1) and heat shock proteins were examined for their possible roles in signal transduction. Inhibition of ERK activation by PD98059 partially inhibited expression of all but FasL-induced CXCL10. Although inhibition of NF-κB DNA binding inhibited chemokine induction, PD98059 did not inhibit TNFα-induced NF-κB DNA binding suggesting that ERK serves an NF-κB-independent pathway. Heat shock itself induced astrocytic chemokine expression; both TNFα and FasL induced HSF-1 DNA binding and Hsp72 production; and Hsp72-induced chemokine expression. Inhibition of either HSF-1 binding with quercetin or heat shock protein synthesis with KNK437 compromised chemokine induction without compromising cell survival. These data suggest that the induction of heat shock proteins via HSF-1 contribute to the TNFα- and FasL-induced expression of chemokines in astrocytes.     

Concentration dependence of chaperone-like activities of α-crystallin, αB-crystallin and proline

Chaperone-like activities of α-crystallin, αB-crystallin and proline were studied using a test system based on aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle. The biphasic character of the dependence of the initial rate of aggregation (v(agg)) of UV-irradiated Phb on the concentration of α-crystallin or αB-crystallin is indicative of the existence of two types of chaperone-protein substrate complexes differing significantly in affinity between the components of the complex. The dependence of v(agg) on the proline concentration is sigmoid (Hill coefficient is equal to 1.6) suggesting that the positive cooperative interactions between the proline molecules bound on the surface of the protein particles occur. When studying the combined suppressive action of α-crystallin and proline on aggregation of UV-irradiated Phb, a slight antagonism between proline used at a fixed concentration (0.15M) and α-crystallin was observed. At higher concentration of proline (0.5M) each chaperone acts independent of one another.     

Small heat shock proteins HSP27 (HspB1), αB-crystallin (HspB5) and HSP22 (HspB8) as regulators of cell death

Hsp27, αB-crystallin and HSP22 are ubiquitous small heat shock proteins (sHsp) whose expression is induced in response to a wide variety of unfavorable physiological and environmental conditions. These sHsp protect cells from otherwise lethal conditions mainly by their involvement in cell death pathways such as necrosis, apoptosis or autophagy. At a molecular level, the mechanisms accounting for sHsp functions in cell death are (1) prevention of denatured proteins aggregation, (2) regulation of caspase activity, (3) regulation of the intracellular redox state, (4) function in actin polymerization and cytoskeleton integrity and (5) proteasome-mediated degradation of selected proteins. In cancer cells, these sHsp are often overexpressed and associated with increased tumorigenicity, cancer cells metastatic potential and resistance to chemotherapy. Altogether, these properties suggest that Hsp27, αB-crystallin and Hsp22 are appropriate targets for modulating cell death pathways. In the present, we briefly review recent reports showing molecular evidence of cell death regulation by these sHsp and co-chaperones. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.     

Heat shock factor binds to heat shock elements upstream of heat shock protein 70a and Samui genes to confer transcriptional activity in Bombyx mori diapause eggs exposed to 5°C

To understand the molecular mechanisms of how 5 °C-incubation activates mRNA expression of Hsp70a and Samui genes in Bombyx mori diapause eggs, we first searched the 5′-upstream regions of the Hsp70a and Samui genes for heat shock elements (HSEs) and found two regions [Hsp70aHSE-1 (−95 to −58) and -2 (−145 to −121), and SamuiHSE-1 (−84 to −55) and -2 (−304 to −290)] corresponding to HSEs (repeats of nGAAn and/or nTTCn). We cloned four cDNAs encoding heat shock factor (HSF)-a2 (627 amino acids), -b (685 aa), -c (682 aa) and -d (705 aa), which were produced by alternative splicing. When we exposed diapause eggs to 5 °C beginning at 2 day post-oviposition to break diapause, HSFd mRNA only increased after chilling for 6–8 days, a pattern very similar to those of Hsp70a and Samui mRNAs. To examine further whether HSFd binds to the respective HSEs, we carried out a gel shift assay using HSFd protein expressed in a cell-free system and the isolated HSEs; migration of the respective digoxigenin(DIG)-labeled HSE-1 and -2 of Hsp70a and Samui was retarded by addition of HSFd; the retarded bands disappeared after addition of the corresponding unlabeled HSE-1 and -2 as competitors, but were not affected by addition of the respective mutated unlabeled HSE-1 and -2. These results indicated that HSFd protein binds to the respective HSEs and may activate mRNA expression of Hsp70a and Samui genes upon exposure of diapause eggs to 5 °C.     

Functional validation of hydrophobic adaptation to physiological temperature in the small heat shock protein αA-crystallin

Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish (V62T, C143S, T147V) confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution.     

Cell biological roles of αB-crystallin

αB-crystallin, also called HspB5, is a molecular chaperone able to interact with unfolding proteins. By interacting, it inhibits further unfolding, thereby preventing protein aggregation and allowing ATP-dependent chaperones to refold the proteins. αB-crystallin belongs to the family of small heat-shock proteins (sHsps), which in humans consists of 10 different members. The protein forms large oligomeric complexes, containing up to 40 or more subunits, which in vivo consist of heterooligomeric complexes formed by a mixture of αB-crystallin and other sHsps. αB-crystallin is highly expressed in the lens and to a lesser extent in several other tissues, among which heart, skeletal muscle and brain. αB-crystallin plays a role in several cellular processes, such as signal transduction, protein degradation, stabilization of cytoskeletal structures and apoptosis. Mutations in the αB-crystallin gene can have detrimental effects, leading to pathologies such as cataract and cardiomyopathy. This review describes the biological roles of αB-crystallin, with a special focus on its function in the eye lens, heart muscle and brain. In addition its therapeutic potential is discussed.     

Inhibition of Cu2+-mediated generation of reactive oxygen species by the small heat shock protein αB-crystallin: the relative contributions of the N- and C-terminal domains

Oxidative stress, Cu²⁺ homeostasis, and small heat shock proteins (sHsp's) have important implications in several neurodegenerative diseases. The ubiquitous sHsp αB-crystallin is an oligomeric protein that binds Cu²⁺. We have investigated the relative contributions of the N- and C-terminal (C-TDαB-crystallin) domains of αB-crystallin to its Cu²⁺-binding and redox-attenuation properties and mapped the Cu²⁺-binding regions. C-TDαB-crystallin binds Cu²⁺ with slightly less affinity and inhibits Cu²⁺-catalyzed, ascorbate-mediated generation of ROS to a lesser extent than αB-crystallin. [Cu²⁺]/[subunit] stoichiometries for redox attenuation by αB-crystallin and C-TDαB-crystallin are 5 and 2, respectively. Both αB-crystallin and C-TDαB-crystallin also inhibit the Fenton reaction of hydroxyl radical formation. Trypsinization of αB-crystallin bound to a Cu²⁺-NTA column and MALDI-TOF analysis of column-bound peptides yielded three peptides located in the N-terminal domain, and in-solution trypsinization of αB-crystallin followed by Cu²⁺-NTA column chromatography identified four additional Cu²⁺-binding peptides located in the C-terminal domain. Thus, Cu²⁺-binding regions are distributed in the N- and C-terminal domains. Small-angle X-ray scattering and sedimentation-velocity measurements indicate quaternary structural changes in αB-crystallin upon Cu²⁺ binding. Our study indicates that an oligomer of αB-crystallin can sequester a large number (~ 150) of Cu²⁺ ions. It acts like a “Cu²⁺ sponge,” exhibits redox attenuation of Cu²⁺, and has potential roles in Cu²⁺ homeostasis and in preventing oxidative stress.     

The identification and characterization of IbpA,a novel α-crystallin-type heat shock protein from mycoplasma

α-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present in Acholeplasma laidlawii. Importantly, similar to the endogenously expressed sHsp proteins, the recombinant IbpA protein was able to spontaneously generate oligomers in vitro and to rescue chemically denatured bovine insulin from irreversible denaturation and aggregation. Collectively, these data suggest that IbpA is a bona fide member of the sHsps family. The immune-electron microscopy data using specific antibodies against IbpA have revealed different intracellular localization of this protein in A. laidlawii cells upon heat shock, which suggests that IbpA not only may participate in the stabilization of individual polypeptides, but may also play a protective role in the maintenance of various cellular structures upon temperature stress.