Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Replacement of Arg in the conserved N-terminal RLFDQxFG motif affects physico-chemical properties and chaperone-like activity of human small heat shock protein HspB8 (Hsp22).

ABSTRACT: The small heat shock protein (sHsp) called HspB8 (formerly, Hsp22) is one of the least typical sHsp members, whose oligomerization status remains debatable. Here we analyze the effect of mutations in a highly conservative sequence located in the N-terminal domain of human HspB8 on its physico-chemical properties and chaperone-like activity. According to size-exclusion chromatography coupled to multi-angle light scattering, the wild type (WT) HspB8 is present as dominating monomeric species (~24 kDa) and a small fraction of oligomers (~60 kDa). The R29A amino acid substitution leads to the predominant formation of 60-kDa oligomers, leaving only a small fraction of monomers. Deletion of the 28-32 pentapeptide (Δ mutant) results in the formation of minor quantities of dimers (~49 kDa) and large quantities of the 24-kDa monomers. Both the WT protein and its Δ mutant efficiently bind a hydrophobic probe bis-ANS and are relatively rapidly hydrolyzed by chymotrypsin, whereas the R29A mutant weakly binds bis-ANS and resists chymotrypsinolysis. In contrast to HspB8 WT and its Δ mutant, which are well phosphorylated by cAMP-dependent and ERK1 protein kinases, the R29A mutant is poorly phosphorylated. R29A mutation affects the chaperone-like activity of HspB8 measured in vitro. It is concluded that the irreplaceable Arg residue located in the only highly conservative motif in the N-terminal domain of all sHsp proteins affects the oligomeric structure and key properties of HspB8.

SUBMITTER: Shatov VM

PROVIDER: S-EPMC8213154 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity.

Project description:A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic alpha-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.

| S-EPMC1133843 | biostudies-literature

The Mitochondrial Small Heat Shock Protein HSP22 from Pea is a Thermosoluble Chaperone Prone to Co-Precipitate with Unfolding Client Proteins.

Project description:The small heat shock proteins (sHSPs) are molecular chaperones that share an alpha-crystallin domain but display a high diversity of sequence, expression, and localization. They are especially prominent in plants, populating most cellular compartments. In pea, mitochondrial HSP22 is induced by heat or oxidative stress in leaves but also strongly accumulates during seed development. The molecular function of HSP22 was addressed by studying the effect of temperature on its structural properties and chaperone effects using a recombinant or native protein. Overexpression of HSP22 significantly increased bacterial thermotolerance. The secondary structure of the recombinant protein was not affected by temperature in contrast with its quaternary structure. The purified protein formed large polydisperse oligomers that dissociated upon heating (42 °C) into smaller species (mainly monomers). The recombinant protein appeared thermosoluble but precipitated with thermosensitive proteins upon heat stress in assays either with single protein clients or within complex extracts. As shown by in vitro protection assays, HSP22 at high molar ratio could partly prevent the heat aggregation of rhodanese but not of malate dehydrogenase. HSP22 appears as a holdase that could possibly prevent the aggregation of some proteins while co-precipitating with others to facilitate their subsequent refolding by disaggregases or clearance by proteases.

| S-EPMC6981728 | biostudies-literature

Heat shock proteins HSPB8 and DNAJC5B have HCV antiviral activity.

Project description:Hepatitis C is a disease caused by the hepatitis C virus (HCV), and an estimated 3% of the world population is infected with the virus. During replication, HCV interacts with several cellular proteins. Studies have shown that several heat shock proteins (HSPs) have an altered expression profile in the presence of the virus, and some HSPs interact directly with HCV proteins. In the present study, we evaluated the expression levels of heat shock proteins in vitro in the presence and absence of HCV. The differential expression of 84 HSPs and chaperones was observed using a qPCR array, comparing HCV uninfected and infected Huh7.5 cells. To validate qPCR array, the differentially expressed genes were tested by real-time PCR in three different HCV models: subgenomic HCV replicon cells (SGR-JFH-1), JFH-1 infected cells (both genotype 2a) and subgenomic S52 cells (genotype 3). The HSPB8 gene showed increased expression in all three viral models. We silenced HSPB8 expression and observed an increase in viral replication. In contrast, when we increased the expression of HSPB8, a decrease in the HCV replication rate was observed. The same procedure was adopted for DNAJC5B, and HCV showed a similar replication pattern as that observed for HSPB8. These results suggest that HSPB8 may act as an intracellular factor against hepatitis C virus replication and that DNAJC5B has the same function, with more relevant results for genotype 3. We also evaluated the direct interactions between HCV and HSP proteins, and the IP experiments showed that the HCV NS4B protein interacts with HSPB8. These results contribute to a better understanding of the mechanisms involved in HCV replication.

| S-EPMC5705118 | biostudies-literature

Oligomeric structure and chaperone-like activity of Drosophila melanogaster mitochondrial small heat shock protein Hsp22 and arginine mutants in the alpha-crystallin domain.

Project description:The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.

| S-EPMC5465034 | biostudies-literature

The Role of the Heat Shock Protein B8 (HSPB8) in Motoneuron Diseases.

Project description:Amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA) are two motoneuron diseases (MNDs) characterized by aberrant protein behavior in affected cells. In familial ALS (fALS) and in SBMA specific gene mutations lead to the production of neurotoxic proteins or peptides prone to misfold, which then accumulate in form of aggregates. Notably, some of these proteins accumulate into aggregates also in sporadic ALS (sALS) even if not mutated. To prevent proteotoxic stresses detrimental to cells, misfolded and/or aggregated proteins must be rapidly removed by the protein quality control (PQC) system. The small heat shock protein B8 (HSPB8) is a chaperone induced by harmful events, like proteasome inhibition. HSPB8 is expressed both in motoneuron and muscle cells, which are both targets of misfolded protein toxicity in MNDs. In ALS mice models, in presence of the mutant proteins, HSPB8 is upregulated both in spinal cord and muscle. HSPB8 interacts with the HSP70 co-chaperone BAG3 and enhances the degradation of misfolded proteins linked to sALS, or causative of fALS and of SBMA. HSPB8 acts by facilitating autophagy, thereby preventing misfolded protein accumulation in affected cells. BAG3 and BAG1 compete for HSP70-bound clients and target them for disposal to the autophagy or proteasome, respectively. Enhancing the selective targeting of misfolded proteins by HSPB8-BAG3-HSP70 to autophagy may also decrease their delivery to the proteasome by the BAG1-HSP70 complex, thereby limiting possible proteasome overwhelming. Thus, approaches aimed at potentiating HSPB8-BAG3 may contribute to the maintenance of proteostasis and may delay MNDs progression.

| S-EPMC5478700 | biostudies-literature

The Role of the Arginine in the Conserved N-Terminal Domain RLFDQxFG Motif of Human Small Heat Shock Proteins HspB1, HspB4, HspB5, HspB6, and HspB8.

Project description:Although the N-terminal domain of vertebrate small heat shock proteins (sHsp) is poorly conserved, it contains a core motif preserved in many members of the sHsp family. The role of this RLFDQxFG motif remains elusive. We analyzed the specific role of the first arginine residue of this conserved octet sequence in five human sHsps (HspB1, HspB4, HspB5, HspB6, and HspB8). Substitution of this arginine with an alanine induced changes in thermal stability and/or intrinsic fluorescence of the related HspB1 and HspB8, but yielded only modest changes in the same biophysical properties of HspB4, HspB5, and HspB6 which together belong to another clade of vertebrate sHsps. Removal of the positively charged Arg side chain resulted in destabilization of the large oligomers of HspB1 and formation of smaller size oligomers of HspB5. The mutation induced only minor changes in the structure of HspB4 and HspB6. In contrast, the mutation in HspB8 was accompanied by shifting the equilibrium from dimers towards the formation of larger oligomers. We conclude that the RLFDQxFG motif plays distinct roles in the structure of several sHsp orthologs. This role correlates with the evolutionary relationship of the respective sHsps, but ultimately, it reflects the sequence context of this motif.

| S-EPMC6073470 | biostudies-literature

The small heat shock protein B8 (HSPB8) modulates proliferation and migration of breast cancer cells.

Project description:Breast cancer (BC) is one of the major causes of cancer death in women and is closely related to hormonal dysregulation. Estrogen receptor (ER)-positive BCs are generally treated with anti hormone therapy using antiestrogens or aromatase inhibitors. However, BC cells may become resistant to endocrine therapy, a process facilitated by autophagy, which may either promote or suppress tumor expansion. The autophagy facilitator HSPB8 has been found overexpressed in some BC. Here we found that HSPB8 is highly expressed and differentially modulated by natural or synthetic selective ER modulators (SERMs), in the triple-positive hormone-sensitive BC (MCF-7) cells, but not in triple-negative MDA-MB-231 BC cells. Specific SERMs induced MCF-7 cells proliferation in a HSPB8 dependent manner whereas, did not modify MDA-MB-231 cell growth. ER expression was unaffected in HSPB8-depleted MCF-7 cells. HSPB8 over-expression did not alter the distribution of MCF-7 cells in the various phases of the cell cycle. Conversely and intriguingly, HSPB8 downregulation resulted in an increased number of cells resting in the G0/G1 phase, thus possibly reducing the ability of the cells to pass through the restriction point. In addition, HSPB8 downregulation reduced the migratory ability of MCF-7 cells. None of these modifications were observed, when another small HSP (HSPB1), also expressed in MCF-7 cells, was downregulated. In conclusion, our data suggest that HSPB8 is involved in the mechanisms that regulate cell cycle and cell migration in MCF-7 cells.

| S-EPMC5354667 | biostudies-literature

Two distinct classes of cochaperones compete for the EEVD motif in heat shock protein 70 to tune its chaperone activities.

Project description:Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions with many cochaperones. One "hotspot" for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70's EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70-CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70-DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein-protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded.

| S-EPMC8913300 | biostudies-literature

Abnormal interaction of motor neuropathy-associated mutant HspB8 (Hsp22) forms with the RNA helicase Ddx20 (gemin3).

Project description:A number of missense mutations in the two related small heat shock proteins HspB8 (Hsp22) and HspB1 (Hsp27) have been associated with the inherited motor neuron diseases (MND) distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 and HspB1 interact with each other, suggesting that these two etiologic factors may act through a common biochemical mechanism. However, their role in neuron biology and in MND is not understood. In a yeast two-hybrid screen, we identified the DEAD box protein Ddx20 (gemin3, DP103) as interacting partner of HspB8. Using co-immunoprecipitation, chemical cross-linking, and in vivo quantitative fluorescence resonance energy transfer, we confirmed this interaction. We also show that the two disease-associated mutant HspB8 forms have abnormally increased binding to Ddx20. Ddx20 itself binds to the survival-of-motor-neurons protein (SMN protein), and mutations in the SMN1 gene cause spinal muscular atrophy, another MND and one of the most prevalent genetic causes of infant mortality. Thus, these protein interaction data have linked the three etiologic factors HspB8, HspB1, and SMN protein, and mutations in any of their genes cause the various forms of MND. Ddx20 and SMN protein are involved in spliceosome assembly and pre-mRNA processing. RNase treatment affected the interaction of the mutant HspB8 with Ddx20 suggesting RNA involvement in this interaction and a potential role of HspB8 in ribonucleoprotein processing.

| S-EPMC3006614 | biostudies-literature

Induction of Hsp22 (HspB8) by estrogen and the metalloestrogen cadmium in estrogen receptor-positive breast cancer cells.

Project description:Estrogen (E2) plays a critical role in the etiology and progression of human breast cancer. The estrogenic response is complex and not completely understood, including in terms of the involved responsive genes. Here we show that Hsp22 (synonyms: HspB8, E2lG1, H11), a member of the small heat shock protein (sHSP) superfamily, was induced by E2 in estrogen receptor-positive MCF-7 breast cancer cells, resulting in an elevated Hsp22 protein level, whereas it was not induced in estrogen receptor-negative MDA-MB-231 cells. This induction was prevented by the pure anti-estrogen ICI182780 (faslodex, fulvestrant), whereas tamoxifen, a substance with mixed estrogenic and antiestrogenic properties, had no major inhibitory effect on this induction, nor did it induce Hsp22 on its own. Cadmium (Cd) is an environmental pollutant with estrogenic properties (metalloestrogen) that has been implicated in breast cancer. Treatment of MCF-7 cells with Cd also resulted in induction of Hsp22, and this induction was also inhibited by ICI182780. In live MCF-7 cells, Hsp22 interacted at the level of dimers with Hsp27, a related sHSP, as was shown by quantitative fluorescence resonance energy transfer measurements. In cytosolic extracts of MCF-7 cells, most of the E2- and Cd-induced Hsp22 was incorporated into high-molecular mass complexes. In part, Hsp22 and Hsp27 were components of distinct populations of these complexes. Finally, candidate elements in the Hsp22 promoter were identified by sequence analysis that could account for the induction of Hsp22 by E2 and Cd. Taken together, Hsp22 induction represents a new aspect of the estrogenic response with potential significance for the biology of estrogen receptor-positive breast cancer cells.

| S-EPMC2134793 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data