Hamartin-Hsp70 Interaction Is Necessary for Akt-Dependent Tuberin Phosphorylation during Heat Shock

Hamartin and tuberin interact directly to regulate cell growth negatively. In this study, far-western blotting revealed that hamartin binds directly Heat shock protein 70 (Hsp70), even in the absence of tuberin. While the hamartin-tuberin complex acts as a sensor for a variety of types of stress, it is unclear how the complex is regulated under stress conditions. We found that the hamartin-Hsp70 interaction is stabilized during heat shock. On the other hand, tuberin underwent degradation through phosphorylation in an Akt-dependent manner. Furthermore, we found that when Hsp70 expression was inhibited by N-formyl-3,4-methylenedioxy-benzylidene-γ-butyrolactam (KNK437), Akt phosphorylation on site Ser308 diminished and tuberin was not phosphorylated at Thr1462 during heat shock. We conclude that both hamartin and Hsp70 increase in response to heat shock, whereas tuberin is phosphorylated and thereafter degraded via the PI3K/Akt pathway. Through this pathway, hamartin-Hsp70 plays a crucial role as a scaffolding protein that transfers the Akt signal to tuberin.     

Small molecule sensitization to TRAIL is mediated via nuclear localization,phosphorylation and inhibition of chaperone activity of Hsp27

The small chaperone protein Hsp27 confers resistance to apoptosis, and therefore is an attractive anticancer drug target. We report here a novel mechanism underlying the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitizing activity of the small molecule {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, an inactive analog of the phosphoinositide 3-kinase inhibitor inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, in HeLa cells that are refractory to TRAIL-induced apoptosis. On the basis of the fact that {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511 is derived from {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, itself derived from quercetin, and earlier findings indicating that quercetin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 affected Hsp27 expression, we investigated whether {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511 sensitized cancer cells to TRAIL via a conserved inhibitory effect on Hsp27. We provide evidence that upon treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, Hsp27 is progressively sequestered in the nucleus, thus reducing its protective effect in the cytosol during the apoptotic process. {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511-induced nuclear translocation of Hsp27 is linked to its sustained phosphorylation via activation of p38 kinase and MAPKAP kinase 2 and the inhibition of PP2A. Furthermore, Hsp27 phosphorylation leads to the subsequent dissociation of its large oligomers and a decrease in its chaperone activity, thereby further compromising the death inhibitory activity of Hsp27. Furthermore, genetic manipulation of Hsp27 expression significantly affected the TRAIL sensitizing activity of {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, which corroborated the Hsp27 targeting activity of {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511. Taken together, these data indicate a novel mechanism of small molecule sensitization to TRAIL through targeting of Hsp27 functions, rather than its overall expression, leading to decreased cellular protection, which could have therapeutic implications for overcoming chemotherapy resistance in tumor cells.     

Heat stress but not inbreeding affects offensive sperm competitiveness in Callosobruchus maculatus

Environmental and genetic stress have well‐known detrimental effects on ejaculate quality, but their concomitant effect on male fitness remains poorly understood. We used competitive fertilization assays to expose the effects of stress on offensive sperm competitive ability in the beetle Callosobruchus maculatus, a species where ejaculates make up more than 5% of male body mass. To examine the effects of environmental and genetic stress, males derived from outcrosses or sib matings were heat shocked at 50°C for 50 min during the pupal stage, while their siblings were maintained at a standard rearing temperature of 28°C. Heat‐shocked males achieved only half the offensive paternity success of their siblings. While this population exhibited inbreeding depression in body size, sperm competitiveness was unaffected by inbreeding, nor did the effect of heat shock stress on sperm competitiveness depend on inbreeding status. In contrast, pupal emergence success was increased by 34% among heat‐stressed individuals, regardless of their inbreeding status. Heat‐shocked males' ejaculate size was 19% reduced, but they exhibited 25% increased mating duration in single mating trials. Our results highlight both the importance of stress in postcopulatory sexual selection, and the variability among stressors in affecting male fitness.     

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.     

棉铃虫Hsp70的多克隆抗体制备及鉴定

HSPs(热休克蛋白)是机体在不利环境条件刺激下合成的一类蛋白质,在进化上高度保守,普遍存在于生物体,其中Hsp70是最为保守的成员。研究发现,昆虫滞育过程中普遍存在Hsp70表达上调的现象。然而,现有的研究均是在基因水平的检测结果。为了能从蛋白水平检测棉铃虫中Hsp70的表达,本研究制备了棉铃虫Hsp70的多克隆抗体。构建了hsp70的原核表达载体并在大肠杆菌BL21(DE3)中成功表达,重组蛋白经镍柱纯化后免疫兔子,制备了棉铃虫Hsp70的多克隆抗体。抗体的效价较高,达到了1∶256 000,此外Western结果表明,制备的抗体能检测出热诱导的Hsp70蛋白。本研究制备了高效价且较为特异的Hsp70对克隆抗体,该抗体为后续从蛋白水平研究棉铃虫滞育过程中Hsp70的表达及其分子机制奠定了基础。     

Dynamic Aha1 co‐chaperone binding to human Hsp90

Hsp90 is an essential chaperone that requires large allosteric changes to determine its ATPase activity and client binding. The co‐chaperone Aha1, which is the major ATPase stimulator in eukaryotes, is important for regulation of Hsp90's allosteric timing. Little is known, however, about the structure of the Hsp90/Aha1 complex. Here, we characterize the solution structure of unmodified human Hsp90/Aha1 complex using NMR spectroscopy. We show that the 214‐kDa complex forms by a two‐step binding mechanism and adopts multiple conformations in the absence of nucleotide. Aha1 induces structural changes near Hsp90's nucleotide‐binding site, providing a basis for its ATPase‐enhancing activity. Our data reveal important aspects of this pivotal chaperone/co‐chaperone interaction and emphasize the relevance of characterizing dynamic chaperone structures in solution.     

Heat shock proteins: A dual carrier-adjuvant for an anti-drug vaccine against heroin

Heroin is a highly abused opioid that has reached epidemic status within the United States. Yet, existing therapies to treat addiction are inadequate and frequently result into rates of high recidivism. Vaccination against heroin offers a promising alternative therapeutic option but requires further development to enhance the vaccine’s performance. Hsp70 is a conserved protein with known immunomodulatory properties and is considered an excellent immunodominant antigen. Within an antidrug vaccine context, we envisioned Hsp70 as a potential dual carrier-adjuvant, wherein immunogenicity would be increased by co-localization of adjuvant and antigenic drug hapten. Recombinant Mycobacterium tuberculosis Hsp70 was appended with heroin haptens and the resulting immunoconjugate granted anti-heroin antibody production and blunted heroin-induced antinociception. Moreover, Hsp70 as a carrier protein surpassed our benchmark Her-KLH cocktail through antibody-mediated blockade of 6-acetylmorphine, the main mediator of heroin’s psychoactivity. The work presents a new avenue for exploration in the use of hapten-Hsp70 conjugates to elicit anti-drug immune responses.     

Induction of biochemical stress markers and apoptosis in transgenic Drosophila melanogaster against complex chemical mixtures: role of reactive oxygen species

The study was aimed to investigate the effect of leachates of solid waste from a flashlight battery factory and a pigment plant on 70 kDa heat shock protein (Hsp70) expression, generation of reactive oxygen species (ROS), antioxidant enzymes activities and apoptosis in Drosophila. Third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) were fed on diet mixed with leachates of solid wastes (0.05-2.0%, v/v) released from two industrial plants at three different pHs (7.00, 4.93 and 2.88) for 2-48 h. A concentration- and time-dependent significant change in Hsp70 expression, ROS generation, antioxidant enzymes activities and MDA content was observed in the exposed larvae preceding the antioxidant enzymes activities. Mitochondria-mediated, caspase-dependent apoptotic cell death in the larvae exposed to 1.0 and 2.0% leachates of flashlight battery factory was concurrent with a significant regression in Hsp70 expression and a higher ROS generation. A positive correlation drawn between ROS generation and apoptotic markers and a negative correlation between apoptotic markers and Hsp70 expression in these groups indicated the important role of ROS in the leachate-induced cellular damage. Hsp70 along with antioxidant enzymes offered protection to the organisms exposed to all the tested concentrations of the leachates of pigment plant waste and 0.5% leachate of flashlight battery factory in a cooperative manner when ROS generation was less induced. Conversely, higher levels of ROS generation in the organisms treated with 1.0 and 2.0% leachate of flashlight battery factory after 24 and 48 h resulted in regression of Hsp70 expression in them leading to cell death. The study suggests that (1) leachates of flashlight battery factory waste more adversely affected the organisms in comparison to the leachates of pigment plant waste. (2) Hsp70 may be used as a biomarker of cellular damage in organisms exposed to leachates. (3) Cell based assays using D. melanogaster as an in vivo model may provide important mechanistic information about the adverse effect of xenobiotics.