Seasonal variation in expression pattern of genes under HSP70

Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.     

Identification of cyclophilin-40-interacting proteins reveals potential cellular function of cyclophilin-40

Cyclophilin-40 (CyP40) is part of the immunophilin family and is found in Hsp90-containing protein complexes. We were interested in identifying proteins that interact with CyP40. CyP40-interacting proteins in HeLa cells were identified using the tandem affinity purification approach. Adenovirus expressing human CyP40 protein (Ad–CyP40), fused with streptavidin and calmodulin binding peptides at the N terminus, was generated. Proteins were separated on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel after tandem affinity purification. Here 10 silver-stained protein bands that were enriched in the Ad–CyP40-infected lysate and the corresponding regions in the control lysate were excised, digested by trypsin, and identified by tandem mass spectrometric analysis. Of 11 interacting proteins that were identified, 4 (RACK1, Ku70, RPS3, and NF45) were expressed in rabbit reticulocyte lysate, bacteria, and MCF-7 cells. We confirmed that these proteins interact with CyP40. We observed that RACK1 suppressed the cobalt chloride-induced, hypoxia response element-dependent luciferase activity in MCF-7 cells but not in MCF-7 stable cells expressing approximately 10% of the cellular CyP40 content. In addition, RACK1 reduced the HIF-1α protein accumulation after cobalt chloride treatment, which was not observed when the CyP40 content was down-regulated. Collectively, we conclude that reduction of the HIF-1α protein by RACK1 is CyP40-mediated.     

Increased stability of Bcl-2 in HSP70-mediated protection against apoptosis induced by oxidative stress

We have previously shown that heat shock protein 70 (HSP70) markedly inhibits H₂O₂-induced apoptosis in mouse C2C12 myogenic cells by reducing the release of Smac. However, the molecular mechanism by which HSP70 interferes with Smac release during oxidative stress-induced apoptosis is not understood. In the current study, we showed that HSP70 increased the stability of Bcl-2 during oxidative stress. An antisense phosphorothioate oligonucleotide against Bcl-2 caused selective inhibition of Bcl-2 protein expression induced by HSP70 and significantly attenuated HSP70-mediated cell protection against H₂O₂-induced release of Smac and apoptosis. Taken together, our results indicate that there are important relationships among HSP70, Bcl-2, release of Smac, and induction of apoptosis by oxidative stress.     

BiPPred: Combined sequence‐ and structure‐based prediction of peptide binding to the Hsp70 chaperone BiP

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi‐scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence‐based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence‐based prediction models were fitted using this and other peptide binding data. A structure‐based position‐specific scoring matrix (SB‐PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB‐PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA‐based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi‐scale pipeline can readily be applied to other protein‐peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence‐based prediction models is not available. Proteins 2016; 84:1390–1407. © 2016 Wiley Periodicals, Inc.     

Is hypothermia a stress condition in HepG2 cells? Expression and localization of Hsp70 in human hepatoma cell line

To understand hypothermia as a stress condition we determined the expression and localization of Hsp70 under hyperthermic and hypothermic stress in human hepatoma HepG2 cells. Western blot analysis indicates that there was a statistically significant increase of Hsp70 expression under thermal stresses. Immunohistochemically, the distribution of inducible Hsp70 in stressed cells showed a granular pattern mostly in the cytoplasm. At subcellular level, Hsp70 was localized in the nucleus, vacuoles, cytoskeletal components and dispersed throughout the cytoplasm. Accumulation of Hsp70 in cells under hypothermia could be related to restitution of cell equilibrium modified by this thermal stress condition. The protective effect of hypothermia could be associated with promotion of Hsp expression. We suggest that hypothermia is a stress capable of inducing Hsp70 expression in human HepG2 cells.     

All roads lead to Rome (but some may be harder to travel): SRP-independent translocation into the endoplasmic reticulum

Abstract

Translocation into the endoplasmic reticulum (ER) is the first biogenesis step for hundreds of eukaryotic secretome proteins. Over the past 30 years, groundbreaking biochemical, structural and genetic studies have delineated one conserved pathway that enables ER translocation- the signal recognition particle (SRP) pathway. However, it is clear that this is not the only pathway which can mediate ER targeting and insertion. In fact, over the past decade, several SRP-independent pathways have been uncovered, which recognize proteins that cannot engage the SRP and ensure their subsequent translocation into the ER. These SRP-independent pathways face the same challenges that the SRP pathway overcomes: chaperoning the preinserted protein while in the cytosol, targeting it rapidly to the ER surface and generating vectorial movement that inserts the protein into the ER. This review strives to summarize the various mechanisms and machineries which mediate these stages of SRP-independent translocation, as well as examine why SRP-independent translocation is utilized by the cell. This emerging understanding of the various pathways utilized by secretory proteins to insert into the ER draws light to the complexity of the translocational task, and underlines that insertion into the ER might be more varied and tailored than previously appreciated.     

Antioxidant responses of Chilo suppressalis (Lepidoptera: Pyralidae) larvae exposed to thermal stress

High temperatures cause a variety of physiological stress responses in insects, including increased generation of reactive oxygen species (ROS), which can cause oxidative damage. This study investigated the effects of thermal stress on ROS generation, the expression of heat shock protein 70 (Hsp70) at the mRNA and protein levels, the activity of antioxidant enzymes (SOD, CAT), and apoptosis in hemocytes of Chilo suppressalis larvae. Results indicated that thermal stress significantly elevated the level of ROS and antioxidant enzyme activity in C. suppressalis larvae. Real-time quantitative PCR showed that hsp70 gene expression was induced by heat stress. Flow cytometric results revealed that the expression profile of Hsp70 at the protein level was in agreement with that at the mRNA level. The expression of Hsp70 at both the mRNA and protein levels reached a maximum at 36 °C in larval hemocytes. Exposure to tested temperatures did not cause any significant change in the rate of apoptosis in larval hemocytes. These results suggest that thermal stress leads to oxidative stress and that antioxidant enzymes and the Hsp70 play an important role in reducing oxidative damage in C. suppressalis larvae.     

A conserved histidine in human DNLZ/HEP is required for stimulation of HSPA9 ATPase activity

The DNL-type zinc-finger protein DNLZ regulates the activity and solubility of the human mitochondrial chaperone HSPA9. To identify DNLZ residues that are critical for chaperone regulation, we carried out an alanine mutagenesis scan of charged residues in a W115I mutant of human DNLZ and assessed the effect of each mutation on interactions with HSPA9. All mutants analyzed promote the solubility of HSPA9 upon expression in Escherichia coli. However, binding studies examining the effect of DNLZ mutants on chaperone tryptophan fluorescence identified three mutations (R81A, H107A, and D111A) that decrease DNLZ binding affinity for nucleotide-free chaperone. In addition, ATPase measurements revealed that DNLZ–R81A and DNLZ–D111A both stimulate the catalytic activity HSPA9, whereas DNLZ–H107A does not elicit an increase in activity even when present at a concentration that is 10-fold higher than the level required for half-maximal stimulation by DNLZ. These findings implicate a conserved histidine as critical for DNLZ regulation of mitochondrial HSPA9 catalytic activity.