Hsp70 in mitochondrial biogenesis

The family of hsp70 (70 kilodalton heat shock protein) molecular chaperones plays an essential and diverse role in cellular physiology, Hsp70 proteins appear to elicit their effects by interacting with polypeptides that present domains which exhibit non-native conformations at distinct stages during their life in the cell. In this paper we review work pertaining to the functions of hsp70 proteins in chaperoning mitochondrial protein biogenesis. Hsp70 proteins function in protein synthesis, protein translocation across mitochondrial membranes, protein folding and finally the delivery of misfolded proteins to proteolytic enzymes in the mitochondrial matrix

Heat Shock Protein 70 Protects the Heart from Ischemia/Reperfusion Injury through Inhibition of p38 MAPK Signaling.

BackgroundHeat shock protein 70 (Hsp70) has been shown to exert cardioprotection. Intracellular calcium ([Ca2+]i) overload induced by p38 mitogen-activated protein kinase (p38 MAPK) activation contributes to cardiac ischemia/reperfusion (I/R) injury. However, whether Hsp70 interacts with p38 MAPK signaling is unclear. Therefore, this study investigated the regulation of p38 MAPK by Hsp70 in I/R-induced cardiac injury.MethodsNeonatal rat cardiomyocytes were subjected to oxygen-glucose deprivation for 6 h followed by 2 h reoxygenation (OGD/R), and rats underwent left anterior artery ligation for 30 min followed by 30 min of reperfusion. The p38 MAPK inhibitor (SB203580), Hsp70 inhibitor (Quercetin), and Hsp70 short hairpin RNA (shRNA) were used prior to OGD/R or I/R. Cell viability, lactate dehydrogenase (LDH) release, serum cardiac troponin I (cTnI), [Ca2+]i levels, cell apoptosis, myocardial infarct size, mRNA level of IL-1β and IL-6, and protein expression of Hsp70, phosphorylated p38 MAPK (p-p38 MAPK), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase2 (SERCA2), phosphorylated signal transducer and activator of transcription3 (p-STAT3), and cleaved caspase3 were assessed.ResultsPretreatment with a p38 MAPK inhibitor, SB203580, significantly attenuated OGD/R-induced cell injury or I/R-induced myocardial injury, as evidenced by improved cell viability and lower LDH release, resulted in lower serum cTnI and myocardial infarct size, alleviation of [Ca2+]i overload and cell apoptosis, inhibition of IL-1β and IL-6, and modulation of protein expressions of p-p38 MAPK, SERCA2, p-STAT3, and cleaved-caspase3. Knockdown of Hsp70 by shRNA exacerbated OGD/R-induced cell injury, which was effectively abolished by SB203580. Moreover, inhibition of Hsp70 by quercetin enhanced I/R-induced myocardial injury, while SB203580 pretreatment reversed the harmful effects caused by quercetin.ConclusionsInhibition of Hsp70 aggravates [Ca2+]i overload, inflammation, and apoptosis through regulating p38 MAPK signaling during cardiac I/R injury, which may help provide novel insight into cardioprotective strategies

Mitochondrial heat shock protein 70, a molecular chaperone for proteins encoded by mitochondrial DNA

Mitochondrial heat shock protein 70 (mt-Hsp70) has been shown to play an important role in facilitating import into, as well as folding and assembly of nuclear-encoded proteins in the mitochondrial matrix. Here, we describe a role for mt-Hsp70 in chaperoning proteins encoded by mitochondrial DNA and synthesized within mitochondria. The availability of mt-Hsp70 function influences the pattern of proteins synthesized in mitochondria of yeast both in vivo and in vitro. In particular, we show that mt-Hsp70 acts in maintaining the var1 protein, the only mitochondrially encoded subunit of mitochondrial ribosomes, in an assembly competent state, especially under heat stress conditions. Furthermore, mt-Hsp70 helps to facilitate assembly of mitochondrially encoded subunits of the ATP synthase complex. By interacting with the ATP-ase 9 oligomer, mt-Hsp70 promotes assembly of ATP-ase 6, and thereby protects the latter protein from proteolytic degradation. Thus mt-Hsp70 by acting as a chaperone for proteins encoded by the mitochondrial DNA, has a critical role in the assembly of supra- molecular complexes

Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC50 values from 30 nM to 1.6 μM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents. © 2009 Elsevier Ltd. All rights reserved

Serum Hsp70 antigen: Early diagnosis marker in perinatal asphyxia

BACKGROUND: Perinatal asphyxia is an important cause of mortality and permanent neurological and developmental deficit. Early and accurate diagnosis would help to establish the likely prognosis and may also help in determining the most appropriate treatment. Studies in experimental animal models suggest that a protein called Hsp70 may be a good and potentially useful marker of cellular stress that may be clinically useful in determining the presence of neonatal asphyxia. OBJECTIVES: Regarding the importance of early and accurate diagnosis of asphyxia, we conducted this study, which is the first investigation of the comparison of the serum Hsp70 antigen level between asphyxiated and healthy infants. PATIENTS AND METHODS: In this observational study, the serum concentrations of Hsp70 antigen were compared between neonates suffering from perinatal asphyxia (n = 50) and normal neonates (n = 51). The inclusion criteria for the cases were neonates who had reached term and had at least two clinical criteria of asphyxia. Exclusion criteria were babies with gestational age < 37 weeks, infants with congenital abnormalities or positive blood culture. Exclusion criteria in this group were the requirement to hospital stay during first week of the life or babies whose mothers had difficulties during pregnancy or delivery. Term neonates without major anomalies who had asphyxia during delivery were enrolled in the first six hours after delivery, and control group consisted of healthy term neonates without problems and normal delivery process in the first week of life. The cord blood was taken during labor to measure Hsp70 antigen level by using an in-house ELISA (The enzyme-linked immunosorbent assay). RESULTS: The median values of serum anti Hsp70 titers were significantly higher in asphyxiated neonates compared with non-asphyxiated neonates (0.36 [0.04 - 1.14] vs 0.24 [0.01 - 0.63]). At cutoff point = 0.3125 ng/mL, sensitivity was 58% and specificity 76% based on ROC curve. CONCLUSIONS: A significant difference between the serum concentrations of Hsp70 of the control and patient group was observed in this study. It is inferred serum concentrations of Hsp70 antigen may be a useful marker for the early diagnosis of that prenatal hypoxia

Additional sex combs interacts with enhancer of zeste and trithorax and modulates levels of trimethylation on histone H3K4 and H3K27 during transcription of hsp70.

BACKGROUND: Maintenance of cell fate determination requires the Polycomb group for repression; the trithorax group for gene activation; and the enhancer of trithorax and Polycomb (ETP) group for both repression and activation. Additional sex combs (Asx) is a genetically identified ETP for the Hox loci, but the molecular basis of its dual function is unclear. RESULTS: We show that in vitro, Asx binds directly to the SET domains of the histone methyltransferases (HMT) enhancer of zeste [E(z)] (H3K27me3) and Trx (H3K4me3) through a bipartite interaction site separated by 846 amino acid residues. In Drosophila S2 cell nuclei, Asx interacts with E(z) and Trx in vivo. Drosophila Asx is required for repression of heat-shock gene hsp70 and is recruited downstream of the hsp70 promoter. Changes in the levels of H3K4me3 and H3K27me3 downstream of the hsp70 promoter in Asx mutants relative to wild type show that Asx regulates H3K4 and H3K27 trimethylation. CONCLUSIONS: We propose that during transcription Asx modulates the ratio of H3K4me3 to H3K27me3 by selectively recruiting the antagonistic HMTs, E(z) and Trx or other nucleosome-modifying enzymes to hsp70

Heat-shock pretreatment inhibits sorbitol-induced apoptosis in K562, U937 and HeLa cells.

The aim of this study was to determine whether heat-shock pretreatment exerted a protective effect against sorbitol-induced apoptotic cell death in K562, U937 and HeLa cell lines and whether such protection was associated with a decreased cytochrome c release from mithocondria and a decreased activation of caspase-9 and -3. Following heat-shock pretreatment (42 6 0.3C for 1 hr), these cell lines were exposed to sorbitol for 1 hr. Apoptosis was evaluated by DNA fragmentation, whereas caspase-9,-3 activation, cytochrome c release and heat-shock protein70 (HSP70) were assayed by Western Blot. Sorbitol exposure-induced apoptosis in these different cell lines with a marked activation of caspase-9 and caspase- 3, whereas heat-shock pretreatment before sorbitol exposure, induced expression of HSP70 and inhibited sorbitol-mediated cytochrome c release and subsequent activation of caspase-9 and caspase- 3. Similarly, overexpression of HSP70 in the three cell lines studied prevented caspase-9 cleavage and activation as well as cell death. Furthermore, we showed that the mRNA expression of iNOS decreased during both the heat-shock treatment and heat-shock pretreatment before sorbitol exposure. By contrast, the expression of Cu-Zn superoxide dismutase (SOD) and Mn-SOD proteins increased during heat-shock pretreatment before sorbitol exposure. We conclude that, heat-shock pretreatment protects different cell lines against sorbitol-induced apoptosis through a mechanism that is likely to involve SOD family members

Bacterial Infection Elicits Heat Shock Protein 72 Release from Pleural Mesothelial Cells

Heat shock protein 70 (HSP70) has been implicated in infection-related processes and has been found in body fluids during infection. This study aimed to determine whether pleural mesothelial cells release HSP70 in response to bacterial infection in vitro and in mouse models of serosal infection. In addition, the in vitro cytokine effects of the HSP70 isoform, Hsp72, on mesothelial cells were examined. Further, Hsp72 was measured in human pleural effusions and levels compared between non-infectious and infectious patients to determine the diagnostic accuracy of pleural fluid Hsp72 compared to traditional pleural fluid parameters. We showed that mesothelial release of Hsp72 was significantly raised when cells were treated with live and heat-killed Streptococcus pneumoniae. In mice, intraperitoneal injection of S. pneumoniae stimulated a 2-fold increase in Hsp72 levels in peritoneal lavage (p<0.01). Extracellular Hsp72 did not induce or inhibit mediator release from cultured mesothelial cells. Hsp72 levels were significantly higher in effusions of infectious origin compared to non-infectious effusions (p<0.05). The data establish that pleural mesothelial cells can release Hsp72 in response to bacterial infection and levels are raised in infectious pleural effusions. The biological role of HSP70 in pleural infection warrants exploration

Lack of an HSP70 heat shock response in two Antarctic marine invertebrates

Members of the HSP70 gene family comprising the inducible (HSP70) genes and GRP78 (glucose-regulated protein 78 kDa) were identified in an Antarctic sea star (Odontaster validus) and an Antarctic gammarid (Paraceradocus gibber). These genes were surveyed for expression levels via Q-PCR after an acute 2-hour heat shock experiment in both animals and a time course assay in O. validus. No significant up-regulation was detected for any of the genes in either of the animals during the acute heat shock. The time course experiment in O. validus produced slightly different results with an initial down regulation in these genes at 2°C, but no significant up-regulation of the genes either at 2 or 6°C. Therefore, the classical heat shock response is absent in both species. The data is discussed in the context of the organisms’ thermal tolerance and the applicability of HSP70 to monitor thermal stress in Antarctic marine organisms