Heat Shock Protein 20 (HSP20) is a novel substrate for Protein Kinase D1 (PKD1)

Heat shock protein 20 (HSP20) has cardioprotective qualities, which are triggered by PKA phosphorylation. PKD1 is also a binding partner for HSP20, and this prompted us to investigate whether the chaperone was a substrate for PKD1. We delineate the PKD1 binding sites on HSP20 and show for the first time HSP20 is a substrate for PKD1. Phosphorylation of HSP20 by PKD1 is diminished by pharmacological or siRNA reduction of PKD1 activity and is enhanced following PKD1 activation. Our results suggest that both PKA and PKD1 can both phosphorylate HSP20 on serine 16 but that PKA is the most dominant

The cellular heat shock response monitored by chemical exchange saturation transfer MRI

CEST-MRI of the rNOE signal has been demonstrated in vitro to be closely linked to the protein conformational state. As the detectability of denaturation and aggregation processes on a physiologically relevant scale in living organisms has yet to be verified, the aim of this study was to perform heat-shock experiments with living cells to monitor the cellular heat-shock response of the rNOE CEST signal. Cancer cells (HepG2) were dynamically investigated after a mild, non-lethal heat-shock of 42 °C for 20 min using an MR-compatible bioreactor system at 9.4 T. Reliable and fast high-resolution CEST imaging was realized by a relaxation-compensated 2-point contrast metric. After the heat-shock, a substantial decrease of the rNOE CEST signal by 8.0 ± 0.4% followed by a steady signal recovery within a time of 99.1 ± 1.3 min was observed in two independent trials. This continuous signal recovery is in coherence with chaperone-induced refolding of heat-shock induced protein aggregates. We demonstrated that protein denaturation processes influence the CEST-MRI signal on a physiologically relevant scale. Thus, the protein folding state is, along with concentration changes, a relevant physiological parameter for the interpretation of CEST signal changes in diseases that are associated with pathological changes in protein expression, like cancer and neurodegenerative diseases

Phosphoproteomic analysis of mammalian infective Trypanosoma brucei subjected to heat shock suggests atypical mechanisms for thermotolerance

The symptoms of African sleeping sickness, caused by the parasite Trypanosoma brucei, can include periods of fever as high as 41 °C which triggers a heat shock response in the parasite. To capture events involved in sensing and responding to heat shock in the mammalian infective form we have conducted a SILAC-based quantitative proteomic and phosphoproteomic analysis of T. brucei cells treated at 41 °C for 1h. Our analysis identified 193 heat shock responsive phosphorylation sites with an average of 5-fold change in abundance, but only 20 heat shock responsive proteins with average of 1.5-fold change. These data indicate that protein abundance does not rapidly respond (≤1 h) to heat shock, and that the changes observed in phosphorylation site abundance are larger and more widespread. The heat shock responsive phosphorylation sites showed enrichment of RNA binding proteins with putative roles in heat shock response included P-body / stress granules and the eukaryotic translation initiation 4F complex. The ZC3H11-MKT1 complex, which stabilises mRNAs of thermotolerance proteins, appears to represent a key signal integration node in the heat shock response

Microwave radiation can alter protein conformation without bulk heating

AbstractExposure to microwave radiation enhances the aggregation of bovine serum albumin in vitro in a time- and temperature-dependent manner. Microwave radiation also promotes amyloid fibril formation by bovine insulin at 60°C. These alterations in protein conformation are not accompanied by measurable temperature changes, consistent with estimates from field modelling of the specific absorbed radiation (15–20 mW kg−1). Limited denaturation of cellular proteins could explain our previous observation that modest heat-shock responses are induced by microwave exposure in Caenorhabditis elegans. We also show that heat-shock responses both to heat and microwaves are suppressed after RNA interference ablating heat-shock factor function

Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Protonophores are the agents that dissipate the proton-motive-force (PMF) across <it>E. coli </it>plasma membrane. As the PMF is known to be an energy source for the translocation of membrane and periplasmic proteins after their initial syntheses in cell cytoplasm, protonophores therefore inhibit the translocation phenomenon. In addition, protonophores also induce heat-shock-like stress response in <it>E. coli </it>cell. In this study, our motivation was to investigate that how the protonophores-mediated phenomena like inhibition of protein translocation and induction of heat-shock proteins in <it>E. coli </it>were correlated.</p> <p>Results</p> <p>Induction of heat-shock-like response in <it>E. coli </it>attained the maximum level after about 20 minutes of cell growth in the presence of a protonophore like carbonyl cyanide m-chloro phenylhydrazone (CCCP) or 2, 4-dinitrophenol (DNP). With induction, cellular level of the heat-shock regulator protein sigma-32 also increased. The increase in sigma-32 level was resulted solely from its stabilization, not from its increased synthesis. On the other hand, the protonophores inhibited the translocation of the periplasmic protein alkaline phosphatase (AP), resulting its accumulation in cell cytosol partly in aggregated and partly in dispersed form. On further cell growth, after withdrawal of the protonophores, the previously accumulated AP could not be translocated out; instead the AP-aggregate had been degraded perhaps by an induced heat-shock protease ClpP. Moreover, the non-translocated AP formed binary complex with the induced heat-shock chaperone DnaK and the excess cellular concentration of DnaK disallowed the induction of heat-shock response by the protonophores.</p> <p>Conclusion</p> <p>Our experimental results suggested that the protonophores-mediated accumulation and aggregation of membrane proteins (like AP) in cell cytosol had signaled the induction of heat-shock proteins in <it>E. coli </it>and the non-translocated protein aggregates were possibly degraded by an induced heat-shock protease ClpP. Moreover, the induction of heat-shock response occurred by the stabilization of sigma-32. As, normally the DnaK-bound sigma-32 was known to be degraded by the heat-shock protease FtsH, our experimental results further suggested that the engagement of DnaK with the non-translocated proteins (like AP) had made the sigma-32 free and stable.</p

Novologues Containing a Benzamide Side Chain Manifest Anti-proliferative Activity Against Two Breast Cancer Cell Lines

The heat shock protein 90 (Hsp90) folding machinery is essential for the maturation of nascent polypeptides into their biologically active three-dimensional-structures and for the rematuration/clearance of misfolded proteins that form under cellular stress.1–3 As a prosurvival chaperone, Hsp90 overexpression is commonly observed in transformed cells, which is required to sustain the hostile tumor micro-environment associated with nutrient deprivation and hypoxia. Pharmacological inhibition of Hsp90 has been shown to induce the degradation of oncogenic proteins associated with all six hallmarks of cancer that rely upon Hsp90.4–8 Consequently, Hsp90 represents a highly sought after target for the treatment of cancer. In fact, 17 small molecules that bind competitively to the N-terminal ATP-binding pocket are under clinical evaluation against various cancers.9,10 However, heat shock factor 1 (HSF-1), the master regulator of the pro-survival heat shock response also binds Hsp90. Ultimately, Hsp90 N-terminal inhibition results in HSF-1 release, and upon phosphorylation, trimerizes and translocates to the nucleus wherein it binds the heat shock elements to activate the pro-survival, heat shock response (HSR). The HSR serves to expand the cellular buffering capacity and to assist in the maturation of mutated and oncogenic substrates.11 This concomitant heat shock response is detrimental to the treatment of cancer and may lead to drug resistance and tumor metastasis.12 Recent studies have demonstrated that allosteric modulation of the Hsp90 C-terminus can separate the pro-survival heat shock response from pro-apoptotic, client protein degradation.13–20 Two classes of small molecules derived from novobiocin 1, (Figure 1) the first identified Hsp90 C-terminal inhibitor, were discovered via the structure-activity relationship studies. KU-32 (2), which lacks a 4-hydroxyl, the 3’-carbamate, and contains an acetamide in lieu of a prenylated benzamide, represents a lead compound that induces the heat shock response at concentrations much lower than that needed for client protein degradation.2,21 Consequently, this class of analogues has been evaluated as neuroprotective agents to refold protein aggregates.22–24 In contrast, KU-174 (3) contains a biarylamide side chain in lieu of the acetamide, and induces Hsp90 client protein degradation without induction of the heat shock response.25–26 Therefore, this class of novobiocin analogues manifests optimal properties for the treatment of cancer, as no HSR is observed with such compounds

Short-term reliability of inflammatory mediators and response to exercise in the heat.

Prospective application of serum cytokines, lipopolysaccharide (LPS), and heat shock proteins (eHSPs) requires reliable measurement of these biomarkers that can signify exercise-induced heat stress in hot conditions. To accomplish this, both short-term (7 day) reliability (at rest, n = 12) and the acute responsiveness of each biomarker to exercise in the heat (pre and post 60-min cycling, 34.5°C and 70% RH, n = 20) were evaluated. Serum was analysed for the concentration of C-reactive protein (CRP), interleukin-6 (IL-6), heat shock protein 72 (eHSP72), immunoglobulin M (IgM) and LPS. Test–retest reliability was determined as the coefficient of variation (CV). Biomarkers with the least short-term within-participant variation were IL-6 (19%, ±20%; CV, ±95% confidence limits (CL)) and LPS (23%, ±13%). Greater variability was observed for IgM, eHSP72 and CRP (CV range 28–38%). IL-6 exhibited the largest increase in response to acute exercise (95%, ±11%, P = < 0.001) and although CRP had a modest CV (12%, ±7%), it increased substantially post-exercise (P = 0.02, ES; 0.78). In contrast, eHSP72 and LPS exhibited trivial changes post-exercise. It appears variation of common inflammatory markers after exercise in the heat is not always discernible from short-term (weekly) variation

Short-term reliability of inflammatory mediators and response to exercise in the heat

Prospective application of serum cytokines, lipopolysaccharide, and heat shock proteins requires reliable measurement of these biomarkers that can signify exercise-induced heat stress in hot conditions. To accomplish this, both short-term (seven day) reliability (at rest, n=12) and the acute responsiveness of each biomarker to exercise in the heat (pre and post 60 min cycling, 34.5oC and 70% RH, n=20) were evaluated. Serum was analysed for the concentration of C-reactive protein (CRP), interleukin (IL-6), heat shock protein 72 (eHSP72), immunoglobulin M (IgM) and lipopolysaccharide (LPS). Test-retest reliability was determined as the coefficient of variation (CV). Biomarkers with the least short-term within-subject variation were IL-6 (19%, ± 20%; CV, ± 95% confidence limits) and LPS (23%, ± 13%). Greater variability was observed for IgM, eHSP72 and CRP (CV range 28-38%). IL-6 exhibited the largest increase in response to acute exercise (95%, ± 11%, p = <0.001) and although CRP had a modest CV (12%, ± 7%) it increased substantially post-exercise (p = 0.02, ES; 0.78). In contrast, eHSP72 and LPS exhibited trivial changes post-exercise. It appears variation of common inflammatory markers after exercise in the heat is not always discernible from short-term (weekly) variation

Multi-stress proteomics: The global protein response to multiple environmental stressors in the porcelain crab Petrolisthes cinctipes

Global climate change is increasing the number of hot days along the California coast as well as increasing the incidence of off-shore upwelling events that lower the pH of intertidal seawater; thus, intertidal organisms are experiencing an increase in more than one stress simultaneously. This study seeks to characterize the global protein response of the eurythermal porcelain crab Petrolisthes cinctipes to changes in thermal, pH, and tidal regime treatments, either combined or individually. The first experiment examined temperature stress alone and sought to determine the effect of chronic temperature acclimation on the acute heat shock response. We compared the proteomic response of cheliped muscle tissue following a month-long acclimation to either (1) constant 10°C, (2) daily fluctuation from 10-20°C, or (3) daily fluctuation from 10-30°C, all followed by either a 30°C acute heat shock or 10°C control. We found that ATP supply via the phosphagen system, changes in glycolytic enzymes, muscle fiber restructuring, respiratory protein fragmentation, and immunity were primarily affected by acclimation and subsequent heat shock. Acclimation to the “extreme” regimes (10°C and 10-30°C) resulted in the greatest proteomic changes, while acclimation to the moderate regime (10-20°C) resulted in a more mild response to heat shock (i.e., fewer adjustments to relative protein abundance). The second experiment sought to determine the proteomic response of gill tissue following a 17 d acclimation to daily changes in pH (ambient pH 8.1 vs low pH 7.6), tidal regime (constant immersion vs 6 h emersion), and temperature (ambient 11°C vs 22-31°C heat shock during emersion). Low pH alone reduced expression of molecular chaperones of the endoplasmic reticulum, lectins, and serine proteases involved in activating the prophenoloxidase cascade. It also increased the abundance of Na+/K+-ATPase, nitrogen metabolism enzymes, and induced changes in tubulin expression, all suggesting an increase in ammonium excretion. Addition of emersion during low pH reduced the abundance of several metabolic proteins including those involved in the proposed ammonium excretion mechanism, suggesting a decrease in metabolic function in part to prevent toxic accumulation of ammonium in the branchial chambers. Combined pH, emersion, and thermal stress increased the abundance of proteins involved in cuticle binding and crosslinking. These results indicate that the responses to pH, tidal cycle, and temperature are highly dependent on one another and that changes in ER protein maturation, ion transport, immunity, and cuticle structure are the primary biochemical systems impacted by these environmental stressors in crustacean gill