The N-terminal arm of small heat shock proteins is important for both chaperone activity and substrate specificity

Small heat shock proteins (sHSPs) are a ubiquitous class of molecular chaperones that interacts with substrates to prevent their irreversible insolubilization during denaturation. How sHSPs interact with substrates remains poorly defined. To investigate the role of the conserved C-terminal alpha-crystallin domain versus the variable N-terminal arm in substrate interactions, we compared two closely related dodecameric plant sHSPs, Hsp18.1 and Hsp16.9, and four chimeras of these two sHSPs, in which all or part of the N-terminal arm was switched. The efficiency of substrate protection and formation of sHSP-substrate complexes by these sHSPs with three different model substrates, firefly luciferase, citrate synthase, and malate dehydrogenase (MDH) provide new insights into sHSP/substrate interactions. Results indicate that different substrates have varying affinities for different domains of the sHSP. For luciferase and citrate synthase, the efficiency of substrate protection was determined by the identity of the N-terminal arm in the chimeric proteins. In contrast, for MDH, efficient protection clearly required interactions with the alpha-crystallin domain in addition to the N-terminal arm. Furthermore, we show that sHSP-substrate complexes with varying stability and composition can protect substrate equally, and substrate protection is not correlated with sHSP oligomeric stability for all substrates. Protection of MDH by the dimeric chimera composed of the Hsp16.9 N-terminal arm and Hsp18.1 alpha-crystallin domain supports the model that a dimeric form of the sHSP can bind and protect substrate. In total, results demonstrate that sHSP-substrate interactions are complex, likely involve multiple sites on the sHSP, and vary depending on substrate.     

Insights into small heat shock protein and substrate structure during chaperone action derived from hydrogen/deuterium exchange and mass spectrometry

Small heat shock proteins (sHSPs) and the related alpha-crystallins are ubiquitous chaperones linked to neurodegenerative diseases, myopathies, and cataract. To better define their mechanism of chaperone action, we used hydrogen/deuterium exchange and mass spectrometry (HXMS) to monitor conformational changes during complex formation between the structurally defined sHSPs, pea PsHsp18.1, and wheat TaHsp16.9, and the heat-denatured model substrates malate dehydrogenase (MDH) and firefly luciferase. Remarkably, we found that even when complexed with substrate, the highly dynamic local structure of the sHSPs, especially in the N-terminal arm (>70% exchange in 5 s), remains unchanged. These results, coupled with sHSP-substrate complex stability, indicate that sHSPs do not adopt new secondary structure when binding substrate and suggest sHSPs are tethered to substrate at multiple sites that are locally dynamic, a feature that likely facilitates recognition and refolding of sHSP-bound substrate by the Hsp70/DnaK chaperone system. Both substrates were found to be stabilized in a partially unfolded state that is observed only in the presence of sHSP. Furthermore, peptide-level HXMS showed MDH was substantially protected in two core regions (residues 95-156 and 228-252), which overlap with the MDH structure protected in the GroEL-bound MDH refolding intermediate. Significantly, despite differences in the size and structure of TaHsp16.9-MDH and PsHsp18.1-MDH complexes, peptide-level HXMS patterns for MDH in both complexes are virtually identical, indicating that stabilized MDH thermal unfolding intermediates are not determined by the identity of the sHSP.     

Binding determinants of the small heat shock protein, αB‐crystallin: recognition of the ‘IxI’ motif

Small heat shock proteins (sHSPs) play a central role in protein homeostasis under conditions of stress by binding partly unfolded, aggregate‐prone proteins and keeping them soluble. Like many sHSPs, the widely expressed human sHSP, αB‐crystallin (‘αB’), forms large polydisperse multimeric assemblies. Molecular interactions involved in both sHSP function and oligomer formation remain to be delineated. A growing database of structural information reveals that a central conserved α‐crystallin domain (ACD) forms dimeric building blocks, while flanking N‐ and C‐termini direct the formation of larger sHSP oligomers. The most commonly observed inter‐subunit interaction involves a highly conserved C‐terminal ‘IxI/V’ motif and a groove in the ACD that is also implicated in client binding. To investigate the inherent properties of this interaction, peptides mimicking the IxI/V motif of αB and other human sHSPs were tested for binding to dimeric αB‐ACD. IxI‐mimicking peptides bind the isolated ACD at 22°C in a manner similar to interactions observed in the oligomer at low temperature, confirming these interactions are likely to exist in functional αB oligomers.     

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

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

Interaction of human HSP22 (HSPB8) with other small heat shock proteins

Mammalian small heat shock proteins (sHSP) are abundant in muscles and are implicated in both muscle function and myopathies. Recently a new sHSP, HSP22 (HSPB8, H11), was identified in the human heart by its interaction with HSP27 (HSPB1). Using phylogenetic analysis we show that HSP22 is a true member of the sHSP superfamily. sHSPs interact with each other and form homo- and hetero-oligomeric complexes. The function of these complexes is poorly understood. Using gel filtration HPLC, the yeast two-hybrid method, immunoprecipitation, cross-linking, and fluorescence resonance energy transfer microscopy, we report that (i). HSP22 forms high molecular mass complexes in the heart, (ii). HSP22 interacts with itself, cvHSP (HSPB7), MKBP (HSPB2) and HSP27, and (iii). HSP22 has two binding domains (N- and C-terminal) that are specific for different binding partners. HSP22 homo-dimers are formed through N-N and N-C interactions, and HSP22-cvHSP hetero-dimers through C-C interaction. HSP22-MKBP and HSP22-HSP27 hetero-dimers involve the N and C termini of HSP22 and HSP27, respectively, but appear to require full-length protein as a binding partner.     

The diversification of plant cytosolic small heat shock proteins preceded the divergence of mosses.

A cDNA library was constructed with mRNA isolated from heat-stressed cell cultures of Funaria hygrometrica (Bryophyta, Musci, Funariaceae). cDNA clones encoding six cytosolic small heat shock proteins (sHSPs) were identified using differential screening. Phylogenetic analysis of these sHSP sequences with other known sHSPs identified them as members of the previously described higher plant cytosolic class I and II families. Four of the F. hygrometrica sHSPs are members of the cytosolic class I family, and the other two are members of the cytosolic class II family. The presence of members of the cytosolic I and II sHSP families in a bryophyte indicates that these gene families are ancient, and evolved at least 450 MYA. This result also indicates that the plant sHSP gene families duplicated much earlier than did the well-studied phytochrome gene family. Members of the cytosolic I and II sHSP families are developmentally regulated in seeds and flowers in higher plants. Our findings show that the two cytosolic sHSP families evolved before the appearance of these specialized structures. Previous analysis of angiosperm sHSPs had identified class- or family-specific amino acid consensus regions and determined that rate heterogeneity exists among the different sHSP families. The analysis of the F. hygrometrica sHSP sequences reveals patterns and rates of evolution distinct from those seen among angiosperm sHSPs. Some, but not all, of the amino acid consensus regions identified in seed plants are conserved in the F. hygrometrica sHSPs. Taken together, the results of this study illuminate the evolution of the sHSP gene families and illustrate the importance of including representatives of basal land plant lineages in plant molecular evolutionary studies.     

Muscle develops a specific form of small heat shock protein complex composed of MKBP/HSPB2 and HSPB3 during myogenic differentiation

Previously, we identified a new mammalian sHSP, MKBP, as a myotonic dystrophy protein kinase-binding protein, and suggested its important role in muscle maintenance (Suzuki, A., Sugiyama, Y., Hayashi, Y., Nyu-i, N., Yoshida, M., Nonaka, I., Ishiura, S., Arahata, K., and Ohno, S. (1998) J. Cell Biol. 140, 1113-1124). In this paper, we develop the former work by performing extensive characterization of five of the six sHSPs so far identified, that is, HSP27, alphaB-crystallin, p20, MKBP/HSPB2, and HSPB3, omitting lens-specific alphaA-crystallin. Tissue distribution analysis revealed that although each sHSP shows differential constitutive expression in restricted tissues, tissues that express all five sHSPs are only muscle-related tissues. Especially, the expressions of HSPB3, identified for the first time as a 17-kDa protein in this paper, and MKBP/HSPB2 are distinctly specific to muscles. Moreover, these sHSPs form an oligomeric complex with an apparent molecular mass of 150 kDa that is completely independent of the oligomers formed by HSP27, alphaB-crystallin, and p20. The expressions of MKBP/HSPB2 and HSPB3 are induced during muscle differentiation under the control of MyoD, suggesting that the sHSP oligomer comprising MKBP/HSPB2 and HSPB3 represents an additional system closely related to muscle function. The functional divergence among sHSPs in different oligomers is also demonstrated in several ways: 1) an interaction with myotonic dystrophy protein kinase, which has been suggested to be important for the maintenance of myofibril integrity, was observed only for MKBP/HSPB2; 2) a myotube-specific association with actin bundles was observed for HSP27 and alphaB-crystallin, but not for MKBP/HSPB2; and 3) sHSPs whose mRNAs are induced by heat shock are alphaB-crystallin and HSP27. Taken together, the results suggest that muscle cells develop two kinds of stress response systems composed of diverged sHSP members, and that these systems work independently in muscle maintenance and differentiation.     

Comparison of the small heat shock proteins alphaB-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 (alphaB-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 alphaB-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes alphaB-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of alphaB-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.     

Small heat-shock proteins function in the insoluble protein complex

Small heat-shock proteins (sHSPs) represent an abundant and ubiquitous family of molecular chaperones. The current model proposes that sHSPs function to prevent irreversible aggregation of non-native proteins by forming soluble complex. The chaperone activity of sHSPs is usually determined by the capacity to suppress thermally or chemically induced protein aggregation. However, sHSPs were frequently found in the insoluble complex particularly in vivo. In this report, it is clearly revealed that the insoluble sHSP/substrate complex is formed when sHSP is overloaded with non-native substrates, which is the very case under in vivo conditions. The proposal that sHSPs function to prevent the protein aggregation seems misleading. sHSPs appear to promote the elimination of protein aggregates by incorporating into the insoluble protein complex.     

Structural Properties of Silkworm Small Heat-Shock Proteins: sHSP19.9 and sHSP20.8

sHSP20.8 and sHSP19.9 are silkworm small-heat shock proteins (sHSPs) comprising a number of polypeptides of molecular sizes of several tens of kilodaltons as subunits. The structural properties of sHSPs were investigated. sHSP19.9 was found to be aggregated by itself during incubation at 60 °C. Aggregation was suppressed in the presence of dithiothreitol and at high ionic strength. In contrast, sHSP20.8 was not aggregated. Aggregation of sHSP19.9 was partially suppressed by sHSP20.8 and in the presence of catalase as a target protein. Based on changes in small-angle X-ray scattering, it is possible that the molecular size of sHSP19.9 is larger than that of sHSP20.8, and that their molecular sizes increase with increasing temperature in a reversible, biphasic manner. sHSPs did not protect catalase from thermal inactivation, but protected it from precipitation by forming a soluble complex. sHSP20.8 and sHSP19.9 with dithiothreitol were stable against lyophilization, autoclaving at 120 °C, and boiling.     

Interactions of HSP22 (HSPB8) with HSP20, alphaB-crystallin, and HSPB3

Seven of the 10 mammalian small heat shock proteins (sHSP) are expressed in muscle where they constitute 3% or more of total protein. sHSPs interact with one another, and these interactions are believed to be important for their functions. In cell types expressing multiple sHSPs, it is of interest to know which sHSPs interact with one another. We have previously shown that HSP22 interacts with itself as well as with HSP27, MKBP, and cvHSP. Using yeast two-hybrid assays and F?rster resonance energy transfer microscopy, we now show that HSP22 also can interact with two additional members of the sHSP family, alphaB-crystallin and HSP20. We also show that HSP22 is found in HPLC fractions of primate cardiac muscle containing high molecular weight complexes that include alphaB-crystallin and HSP20. Our results suggest that a variety of oligomers composed of different proportions of different sHSPs may form in cell types expressing multiple sHSPs.     

Immunomodulation of function of small heat shock proteins prevents their assembly into heat stress granules and results in cell death at sublethal temperatures

The conformational dynamism and aggregate state of small heat shock proteins (sHSPs) may be crucial for their functions in thermoprotection of plant cells from the detrimental effects of heat stress. Ectopic expression of single chain fragment variable (scFv) antibodies against cytosolic sHSPs was used as new tool to generate sHSP loss-of-function mutants by antibody-mediated prevention of the sHSP assembly in vivo . Anti-sHSP scFv antibodies transiently expressed in heat-stressed tobacco protoplasts were not only able to recognize the endogenous sHSPs but also prevented their assembly into heat stress granula (HSGs). Constitutive expression of the same scFv antibodies in transgenic plants did not alter their phenotype at normal growth temperatures, but their leaves turned yellow and died after prolonged stress at sublethal temperatures. Structural analysis revealed a regular cytosolic distribution of stress-induced sHSPs in mesophyll cells of stress-treated transgenic plants, whereas extensive formation of HSGs was observed in control cells. After prolonged stress at sublethal temperatures, mesophyll cells of transgenic plants suffered destruction of all cellular membranes and finally underwent cell death. In contrast, mesophyll cells of the stressed controls showed HSG disintegration accompanied by appearance of polysomes, dictyosomes and rough endoplasmic reticulum indicating normalization of cell functions. Apparently, the ability of sHSPs to assemble into HSGs as well as the HSG disintegration is a prerequisite for survival of plant cells under continuous stress conditions at sublethal temperatures.     

Detection and Architecture of Small Heat Shock Protein Monomers

Background

Small Heat Shock Proteins (sHSPs) are chaperone-like proteins involved in the prevention of the irreversible aggregation of misfolded proteins. Although many studies have already been conducted on sHSPs, the molecular mechanisms and structural properties of these proteins remain unclear. Here, we propose a better understanding of the architecture, organization and properties of the sHSP family through structural and functional annotations. We focused on the Alpha Crystallin Domain (ACD), a sandwich fold that is the hallmark of the sHSP family.

Methodology/Principal Findings

We developed a new approach for detecting sHSPs and delineating ACDs based on an iterative Hidden Markov Model algorithm using a multiple alignment profile generated from structural data on ACD. Using this procedure on the UniProt databank, we found 4478 sequences identified as sHSPs, showing a very good coverage with the corresponding PROSITE and Pfam profiles. ACD was then delimited and structurally annotated. We showed that taxonomic-based groups of sHSPs (animals, plants, bacteria) have unique features regarding the length of their ACD and, more specifically, the length of a large loop within ACD. We detailed highly conserved residues and patterns specific to the whole family or to some groups of sHSPs. For 96% of studied sHSPs, we identified in the C-terminal region a conserved I/V/L-X-I/V/L motif that acts as an anchor in the oligomerization process. The fragment defined from the end of ACD to the end of this motif has a mean length of 14 residues and was named the C-terminal Anchoring Module (CAM).

Conclusions/Significance

This work annotates structural components of ACD and quantifies properties of several thousand sHSPs. It gives a more accurate overview of the architecture of sHSP monomers.     

A small HSP gene of bloody clam (Tegillarca granosa) involved in the immune response against Vibrio parahaemolyticus and lipopolysaccharide

Small heat shock proteins (sHSPs) associate with nuclei, cytoskeleton and membranes, and as molecular chaperones they bind partially denatured proteins, thereby preventing irreversible protein aggregation during stress. In the present study, the small heat shock proteins of Tegillarca granosa (Tg-sHSP) were identified from hemocytes by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consisted of 1005 bp with a 594 bp open reading frame encoding 197 amino acids. Sequence comparison showed that Tg-sHSP had low degree of homology to sHSP of other organisms, such as 47.8% similarity with sHSP from Zhikong scallop Chlamys farreri (AAR11780), 34.8% similarity with silkworm Bombyx mori (NP_001036941). A sHSP feature domain Alpha-crystallin domain (ACD) and V/IXI/V motif in the C-terminal extension were identified in Tg-sHSP, indicating that Tg-sHSP should be a new member of sHSP family. Quantitative RT-PCR assay was developed to detect the mRNA expression of Tg-sHSP in five different tissues. Higher-level mRNA expression of Tg-sHSP was detected in the tissues of hemocytes and mantle. The up-regulation of Tg-sHSP after bacteria Vibrio parahaemolyticus and lipopolysaccharide (LPS) challenge showed that sHSPs play a pivotal role in anti-bacterial immunity. These results together indicated that Tg-sHSP would provide candidate promising therapeutic or prophylactic agents in health management and diseases control of clam aquaculture.     

Interactions between small heat shock protein subunits and substrate in small heat shock protein-substrate complexes

Small heat shock proteins (sHSPs) are dynamic oligomeric proteins that bind unfolding proteins and protect them from irreversible aggregation. This binding results in the formation of sHSP-substrate complexes from which substrate can later be refolded. Interactions between sHSP and substrate in sHSP-substrate complexes and the mechanism by which substrate is transferred to ATP-dependent chaperones for refolding are poorly defined. We have established C-terminal affinity-tagged sHSPs from a eukaryote (pea HSP18.1) and a prokaryote (Synechocystis HSP16.6) as tools to investigate these issues. We demonstrate that sHSP subunit exchange for HSP18.1 and HSP16.6 is temperature-dependent and rapid at the optimal growth temperature for the organism of origin. Increasing the ratio of sHSP to substrate during substrate denaturation decreased sHSP-substrate complex size, and accordingly, addition of substrate to pre-formed sHSP-substrate complexes increased complex size. However, the size of pre-formed sHSP-substrate complexes could not be reduced by addition of more sHSP, and substrate could not be observed to transfer to added sHSP, although added sHSP subunits continued to exchange with subunits in sHSP-substrate complexes. Thus, although some number of sHSP subunits within complexes remain dynamic and may be important for complex structure/solubility, association of substrate with the sHSP does not appear to be similarly dynamic. These observations are consistent with a model in which ATP-dependent chaperones associate directly with sHSP-bound substrate to initiate refolding.     

Developmental control of small heat shock protein expression during pea seed maturation

Plants synthesize four classes of small heat shock proteins (sHSPs); two classes are targeted to the plastid and endoplasmic reticulum, respectively, and two are found in the cytoplasm. In this paper, we describe a new role for the two classes of cytoplasmic HSPs in maturing embryos of developing seeds. The expression of each class of sHSPs was examined in pea seeds grown under non-stress conditions using Western and Northern analysis. Class I and class II cytoplasmic sHSPs are coordinately expressed in the embryo and accumulate to levels seen in moderately heat-stressed leaves. Their induction in cotyledons coincides with the mid-maturation phase of seed development, and induction in axes roughly coincides with abscission of the seed from the ovary wall. Both classes of sHSPs persisted in cotyledons for 4 days after the onset of imbibition, but disappeared from axes shortly after germination. Neither class of cytoplasmic sHSP is expressed in non-embryonic organs associated with the seed. The timing and organ specificity of sHSP expression is paralleled by the expression of the corresponding mRNAs. Neither the plastid nor the endoplasmic reticulum sHSPs were consistently expressed during seed development, but both could be induced by heat-stressing the developing seed. Developmental regulation of the cytoplasmic sHSPs is evidence that these proteins function not only in responding to heat-stress but also during seed development and/or germination.