Chaperone function and mechanism of small heat-shock proteins

Small heat-shock proteins （sHSPs） are ubiquitous ATP-independent molecular chaperones that play crucial roles in protein quality control in cells. They are able to prevent the aggregation and/or inactivation of various non-native sub- strate proteins and assist the refolding of these substrates independently or under the help of other ATP-dependent chaperones. Substrate recognition and binding by sHSPs are essential for their chaperone functions. This review focuses on what natural substrate proteins an sHSP pro- tects and how it binds the substrates in cells under fluctuat- ing conditions. It appears that sHSPs of prokaryotes, although being able to bind a wide range of cellular pro- teins, preferentially protect certain classes of functional proteins, such as translation-related proteins and metabolic enzymes, which may well explain why they could increase the resistance of host cells against various stresses. Mechanistically, the sHSPs of prokaryotes appear to possess numerous multi-type substrate-binding residues and are able to hierarchically activate these residues in a temperature-dependent manner, and thus act as tempera- ture-regulated chaperones. The mechanism of hierarchical activation of substrate-binding residues is also discussed regarding its implication for eukaryotic sHSPs.     

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.     

Chaperone activity of tobacco HSP18, a small heat-shock protein, is inhibited by ATP

NtHSP18P (HSP18), a cytosolic class I small heat-shock protein from tobacco pollen grains, was expressed in Escherichia coli. The viability of these cells was improved by 50% at 50 degrees C, demonstrating its functionality in vivo. Purified recombinant protein formed 240 kDa HSP18 oligomers, irrespective of temperature. These oligomers interacted with the model substrate citrate synthase (CS) to form large complexes in a temperature-dependent manner. Furthermore, HSP18 prevented thermally induced aggregation of CS at 45 degrees C. The fluorescence probe bis-ANS revealed the exposure of HSP18 hydrophobic surfaces at this temperature. Reactivation of chemically denatured CS was also significantly enhanced by HSP18. Surprisingly, HSP18 function was inhibited (in contrast to the related chaperone alphabeta-crystallin and plant sHSPs studied so far) by the presence of ATP in a concentration-dependent manner. The conformational changes of HSP18 imposed by ATP binding were indicated by the difference in the quenching of intrinsic tryptophan fluorescence, and implied more compact structure with ATP. Fluorescence measurements with bis-ANS showed that the conformational shift of HSP18 is suppressed in the presence of ATP. Decreased chaperone activity of HSP18 in the presence of ATP is caused by the lower affinity of conformationally blocked HSP18 for the substrate, as demonstrated by a higher susceptibility of model substrate, malate dehydrogenase, to proteolytic cleavage. Our results suggest that the chaperone activity of some plant sHSPs could be regulated by the availability of ATP in the cytoplasm, which would provide a mechanism to monitor the cell environment, control biological activity of sHSPs, and coordinate it with other ATP-dependent chaperones such as HSP70.     

Tsp36, a tapeworm small heat-shock protein with a duplicated alpha-crystallin domain, forms dimers and tetramers with good chaperone-like activity

Small heat shock proteins (sHSPs), which range in monomer size between 12 and 42 kDa, are characterized by a conserved C-terminal alpha-crystallin domain of 80-100 residues. They generally form large homo- or heteromeric complexes, and typically have in vitro chaperone-like activity, keeping unfolding proteins in solution. A special type of sHSP, with a duplicated alpha-crystallin domain, is present in parasitic flatworms (Platyhelminthes). Considering that an alpha-crystallin domain is essential for the oligomerization and chaperone-like properties of sHSPs, we characterized Tsp36 from the tapeworm Taenia saginata. Both wild-type Tsp36 and a mutant (Tsp36C-->R) in which the single cysteine has been replaced by arginine were expressed and purified. Far-UV CD measurements of Tsp36 were in agreement with secondary structure predictions, which indicated alpha-helical structure in the N-terminal region and the expected beta-sandwich structure for the two alpha-crystallin domains. Gel permeation chromatography and nano-ESI-MS showed that wild type Tsp36 forms dimers in a reducing environment, and tetramers in a non-reducing environment. The tetramers are stabilized by disulfide bridges involving a large proportion of the Tsp36 monomers. Tsp36C-->R exclusively occurs as dimers according to gel permeation chromatography, while the nondisulfide bonded fraction of wild type Tsp36 dissociates from tetramers into dimers under nonreducing conditions at increased temperature (43 degrees C). The tetrameric form of Tsp36 has a greater chaperone-like activity than the dimeric form.     

Enhanced molecular chaperone activity of the small heat-shock protein alphaB-cystallin following covalent immobilization onto a solid-phase support

The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface; alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.     

Toxoplasma gondii Hsp20 is a stripe-arranged chaperone-like protein associated with the outer leaflet of the inner membrane complex

Background information. Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. T. gondii has five sHsps [small Hsps (heat‐shock proteins)] located in different subcellular compartments. Among them, Hsp20 showed to be localized at the periphery of the parasite body. sHsps are widespread, constituting the most poorly conserved family of molecular chaperones. The presence of sHsps in membrane structures is unusual. Results. The localization of Hsp20 was further analysed using high‐resolution fluorescent light microscopy as well as electron microscopy, which revealed that Hsp20 is associated with the outer surface of the IMC (inner membrane complex), in a set of discontinuous stripes following the same spiralling trajectories as the subpellicular microtubules. The detergent extraction profile of Hsp20 was similar to that of GAP45 [45 kDa GAP (gliding‐associated protein)], a glideosome protein associated with the IMC, but was different from that of IMC1 protein. Although we were unable to detect interacting protein partners of Hsp20 either in normal or stressed tachyzoites, an interaction of Hsp20 with phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate phospholipids could be observed. Conclusions. Hsp20 was shown to be associated with a specialized membranous structure of the parasite, the IMC. This discontinuous striped‐arrangement is unique in T. gondii, indicating that the topology of the outer leaflet of the IMC is not homogeneous.     

Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase

The molecular mechanism whereby the small heat-shock protein (sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase (CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,3'-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross-linker captured Hsp21 in dodecameric form, even at increased temperature (47 degrees C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21-Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21-CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21.     

Genealogy of the α-crystallin—small heat-shock protein superfamily

Sequences of 40 very diverse representatives of the α-crystallin–small heat-shock protein (α-Hsp) superfamily are compared. Their characteristic C-terminal ‘α-crystallin domain' of 80–100 residues contains short consensus sequences that are highly conserved from prokaryotes to eukaryotes. There are, in addition, some positions that clearly distinguish animal from non-animal α-Hsps. The α-crystallin domain is predicted to consist of two hydrophobic β-sheet motifs, separated by a hydrophilic region which is variable in length. Combination of a conserved α-crystallin domain with a variable N-terminal domain and C-terminal extension probably modulates the properties of the various α-Hsps as stress-protective and structural oligomeric proteins. Phylogeny reconstruction indicates that multiple α-Hsps were already present in the last common ancestor of pro- and eukaryotes. It is suggested that during eukaryote evolution, animal and non-animal α-Hsps originated from different ancestral gene copies. Repeated gene duplications gave rise to the multiple α-Hsps present in most organisms.     

AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

The nucleotide sequence of a cDNA clone fromArabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)⁺ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity.In vitro, recombinant AtJ1 stimulated the ATPase activity of bothE. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized inE. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.Abbreviations MBP maltose binding protein - PCR polymerase chain reaction - Stress70c the cytosolic member of the 70 kDA family of stress-related proteins     

Expression of heat-shock protein 70 kDa in Tetrahymena pyriformis during cell adaptation to salinity changes in the medium

The alteration of the content of heat-shock protein 70 kDa (Hsp70) was studied in cells of the freshwater ciliate Tetrahymena pyriformis after the salinity of the medium had been changed. It was shown that ciliates acclimated to fresh (0%) or salt (2 and 10%) water have similar levels of constitutive Hsp70. Neither pronounced induction nor a decrease in the Hsp70 level were revealed in ciliates after salinity stress. These data differ from the results we obtained previously with more euryhaline ciliates, Paramecium nephridiatum and P. jenningsi. In those species, we observed both the induced synthesis of Hsp70 after salinity stress and changes (decrease or increase) in the constitutive Hsp70 level after the acclimation of ciliates to the altered medium salinity. We presume that the differences in the chaperone system reaction of these ciliates species may be connected with their different salinity resistances, least of all in P. jenningsi, intermediate in T. pyriformis, and most pronounced in P. nephridiatum.     

Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process

The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H₂O₂, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins.     

Basic peroxidases in isolated vacuoles of nicotiana tabacum L.

Vacuoles of tobacco mesophyll and of suspension-cultured cells were isolated in order to study the localization of peroxidase isoenzymes. Only basic peroxidases were detectable by electrophoretic separation of the vacuolar sap. Some of the basic peroxidases have formerly been described as an ionically bound cell-wall fraction. This fraction, however, was found to be an artifact produced by incomplete cell breakage. Reinvestigation of isolated cell walls confirmed that mainly acidic peroxidases are localized in the cell walls where they move freely or are bound. As a consequence of former and present results we think it probable that all of the peroxidase isoenzymes are secretory proteins because they have to be transported from the sites of synthesis in the cytoplasm to the sites of function, the extracytoplasmic spaces, cell wall (acidic peroxidases), and vacuole (basic peroxidases).Abbreviation ER endoplasmic reticulum - PAGE polyacrylamide gel electrophoresis     

Structure,function, and biogenesis of SecY,an integral membrane protein involved in protein export

TheE. coli secY (prlA) gene, located in the operator-distal part of thespec ribosomal protein operon, codes for an integral membrane protein, SecY. The phenotypes of temperature-sensitive and cold-sensitive mutations insecY suggest that the SecY protein plays an essential rolein vivo to facilitate protein translocation, whereas theprlA mutations in this gene suggest that SecY may interact with the signal sequence of translocating polypeptides. SecY contains most probably six cytoplasmic and five periplasmic domains, as well as 10 transmembrane segments. Such membrane-embedded structure may confer the SecY protein a translocator function, in which it provides a proteinaceous pathway for passage of secreted as well as membrane proteins. Results obtained byin vitro analyses of the translocation reactions, as well as some new phenotypes of thesecY mutants, are consistent with this notion. Possible interaction of SecY with other secretion and chaperone-like factors is also discussed.     

Functional complementation of a yeast vesicular transport mutation ypt1-1 by a Brassica napus cDNA clone encoding a small GTP-binding protein

A cDNA clone (bra) encoding a small GTP-binding protein was isolated from Brassica napus by screening a root cDNA library with a degenerate oligonucleotide probe that corresponds to a highly conserved GTP-binding domain of the Ras superfamily. Sequence analysis shows that the clone contains an open reading frame of 219 amino acid residues with the estimated molecular mass of 24379 Da and this coding region contains all the conserved motifs of the Ras superfamily. The deduced amino acid sequence of the bra gene is most closely related to the Ypt/Rab family that functions in the vesicular transport (46% and 47% amino acid identity to the yeast Ypt1 and to the human Rab1, respectively) and is more distantly related to the other Ras-related families. The protein encoded by the bra gene, when expressed in Escherichia coli, shows the ability to bind GTP. Furthermore, when the bra gene is introduced into Saccharomyces cerevisiae under the regulation of the yeast GAL1 promoter, the gene can complement the temperature-sensitive yeast mutation ypt1-1 that has defects in vesicular transport function. The amino acid sequence similarity and the functional complementation of the yeast mutation suggest that this gene is likely to be involved in the vesicular transport in plants. Genomic Southern analysis shows that this gene is a member of a small gene family in Brassica napus.     

Correlation between thylakoid protein phosphorylation and molecular organization of the photosynthetic apparatus in a dynamic system

In-vitro thylakoid protein phosphorylation has been studied in synchronized cells of Scenedesmus obliquus at the 8- and 16-h of the life cycle, stages which are characterized by the maximum and minimum photosynthetic activities, respectively. The stage of maximum photosynthetic activity (8-h) is characterized by the highest protein phosphorylation in vitro and in vivo, by the largest proportion of the heavy subfraction of thylakoids, and by maximum oligomerization of the light-harvesting chlorophyll a/b-protein complex, altogether creating the highest energy charge of the thylakoid membranes. Protein phosphorylation in vitro decreases the amount of the heavy subfraction and increases the amount of oligomerization of the antenna of photosystem I (PSI) (increase of chlorophyll b in the light fraction). Concomittantly, PSII units become smaller (longer time for the rise in fluorescence induction) and photosynthetic efficiency increases (decrease of fluorescence yield). In-vivo protein phosphorylation is controlled mainly endogenously during the 8-h of the life cycle but is exogenously modulated by light to optimize the photosynthetic activity by redistribution of pigment-protein complexes. In-vitro protein phosphorylation seems to restore partially the conditions prevalent in vivo and lost during the preparation of membranes. The effect is greater in 16-h cells which have less-stable membranes. The regulatory mechanism between membrane stabilization and oligomerization on the one hand and redistribution of the light-harvesting chlorophyll a/b-protein complex from PSII to PSI on the other hand remains unexplained. We have confirmed that the mechanism of protein phosphorylation is regulated via plastohydroquinone, but experiments with the plastohydroquinone analogue 2,3,5,6-tetramethyl-p-benzoquinone demonstrated that plastohydroquinone is not solely responsible for the differences in protein phosphorylation of 8- and 16-h thylakoids. The inhibitory effect of ADP and the distinct rates of kinase reaction indicate that the adenylate energy charge and changes in the organization of the photosynthetic apparatus also contribute to the observed differences in protein phosphorylation. Phosphorylation in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea indicated that the 32-kDa phosphoprotein and the herbicide-binding Q_B protein may be the same. These experiments also indicated that 3-(3,4-dichlorophenyl)-1,1-dimethylurea-binding reduces kinase activity directly and not only by inhibiting electron transport.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSI, II photosystem I, II - TMQ 2,3,5,6-tetramethyl-p-benzoquinone Dedicated to Professor Dr. W. Nultsch on the occasion of his 60th brithday     

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode.