Interaction of the Neurospora crassa heat shock factor with the heat shock element during heat shock and different developmental stages

The interaction of the heat shock factor (HSF) with the heat shock element (HSE) was determined by a non-radioactive electrophoretic mobility shift assay, in order to analyze HSF regulation in Neurospora crassa. HSF binds to HSE under normal, non-stress conditions and is thus constitutively trimerized. Upon heat shock, the HSF-HSE complex shows a retarded mobility. This was also observed in Saccharomyces cerevisiae, where this mobility shift was shown to be due to HSF phosphorylation [Sorger and Pelham (1988) Cell 54, 855-864]. In N. crassa, HSE-dependent electrophoretic mobility shift is temperature- and time-dependent. Under normal growth conditions, the HSF is located in the cytoplasm as well as in the nucleus. In germinating conidia the HSF shows a retarded mobility typical for heat shock even at normal growth temperatures. No HSF-dependent mobility shift was detectable in aerial hyphae.     

Reversibility of heat shock in Chlamydia trachomatis

The heat shock effect on chlamydia development was studied. We report here that the reversibility of the heat shock response did not depend on the stage of chlamydial morphogenesis at which transfer to high temperature occurred, and the infectivity of the particles produced was not affected significantly, so long as the heat shock exposure was not prolonged. Exposure to heat shock for more than 9 h resulted in stagnation of the growth cycle, appearance of aberrant reticulate body particles and loss of infectivity. SDS-PAGE analysis of proteins synthesized under prolonged heat shock showed increased relative abundance of heat shock proteins in common with other procaryotic organisms.     

人口腔螺旋体热休克蛋白的初步研究

为分析牙周病的发病与人口爱螺旋体的热休克蛋白的关系。通过ＳＤＳ－ＰＡＧＥ电泳将菌体蛋白分离,转移电泳（Ｗｅｓｔｅｒｎ ｂｌｏｔ）检查螺旋体的热休克蛋白抗原,牙周病患者血清与螺旋体的热休克蛋白进行免疫印迹试验检查灯克蛋白抗体。结果为实验所用的螺旋体有４种可诱导产生质变休克蛋白,患者血清中有多种对于口腔螺旋体蛋白能起作用的本,其中有两名患者的血清对Ｔ．ｓｏｃｒａｎｓｋｉｉ３５５３５菌株的６０ｋＤ或６５     

Dobutamine mediates cytoprotection by induction of heat shock protein 70 in vitro

Aims

Dobutamine is cytoprotective when applied before a subsequent stress. However, the underlying molecular mechanism is unknown. Dobutamine also inhibits nuclear factor (NF)-κB in human T lymphocytes. Other inhibitors of NF-κB induce a so-called heat shock response. We hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response.

Main methods

Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed by electrophoretic mobility shift assay, mRNA-expression of heat shock protein (hsp)70 and hsp90 by Northern Blot, activity of caspase-3 by fluorogenic caspase activity assay and cleavage of pro-caspase-3 by Western Blot. Apoptosis was assessed by flow cytometry after annexin V-fluorescein isothiocyanate staining. Hsp70 and hsp90 were inhibited using N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam and 17-allylamino-17-demethoxygeldana-mycin, respectively. All data are given as median and 25/75% percentile.

Key findings

Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20–32] % vs. 12 [9–15] % for dobutamine 0.1 mM and 7 [5–12] % for dobutamine 0.5 mM, p < 0.001), cleavage of pro-caspase-3 as well as caspase-3-like activity (0.46 [0.40–0.48] vs. 0.32 [0.27–0.39] for Dobutamine 0.1 mM and 0.20 [0.19–0.23] for Dobutamine 0.5 mM, p < 0.01). Dobutamine induced DNA-binding of HSF-1 and mRNA-expression of hsp70 and hsp90. While inhibition of Hsp90 had no effect, inhibition of Hsp70 increased the number of annexin V-positive cells (33 [32–36] % vs. 18 [16–24] %) and caspase-3-like activity (0.21 [0.19–0.23] vs. 0.16 [0.13–0.17], p < 0.05).

Significance

Dobutamine protects from apoptotic cell death via the induction of Hsp70.     

Heat shock protein 70 and albuminuria in patients with type 2 diabetes: a matched case control study

Here, we aimed to study serum heat shock protein (HSP) 70 levels in diabetic patients with and without albuminuria. We performed a 1:1 matched case control study on 40 diabetic patients with albuminuria as cases and 40 age, sex, body mass index matched diabetic patients without albuminuria (normoalbuminuria) as controls. Normoalbuminuria was defined as urinary albumin excretion rate <15 mg/12 h, and albuminuria was defined as urinary albumin excretion rate between 100–400 mg/12 h. Patients with albuminuria had a higher HSP70 than controls (0.83 ± 0.50 vs. 0.63 ± 0.06; p = 0.02), while they did not differ in any other studied variables. In ten of the studied pairs, the controls had higher HSP70 levels than cases (reverse relationship). Patients in the “direct relationship group” had higher HbA1c values than the patients in the “reverse relationship group” (8.9 ± 0.3 vs. 7.3 ± 0.6, p = 0.04). Cases in the reverse pairs had a lower low density lipoprotein cholesterol levels than their controls. The odds ratio of HSP70 in the prediction of albuminuria was (28.69 (3.2–250.1), p = 0.002). In conclusion, we have shown an increased HSP70 levels in diabetic patients with albuminuria.     

Golgi fragmentation induced by heat shock or inhibition of heat shock proteins is mediated by non-muscle myosin IIA via its interaction with glycosyltransferases

The Golgi apparatus is a highly dynamic organelle which frequently undergoes morphological changes in certain normal physiological processes or in response to stress. The mechanisms are largely not known. We have found that heat shock of Panc1 cells expressing core 2 N-acetylglucosaminyltransferase-M (Panc1-C2GnT-M) induces Golgi disorganization by increasing non-muscle myosin IIA (NMIIA)–C2GnT-M complexes and polyubiquitination and proteasomal degradation of C2GnT-M. These effects are prevented by inhibition or knockdown of NMIIA. Also, the speed of Golgi fragmentation induced by heat shock is found to be positively correlated with the levels of C2GnT-M in the Golgi. The results are reproduced in LNCaP cells expressing high levels of two endogenous glycosyltransferases—core 2 N-acetylglucosaminyltransferase-L:1 and β-galactoside:α2-3 sialyltransferase 1. Further, during recovery after heat shock, Golgi reassembly as monitored by a Golgi matrix protein giantin precedes the return of C2GnT-M to the Golgi. The results are consistent with the roles of giantin as a building block of the Golgi architecture and a docking site for transport vesicles carrying glycosyltransferases. In addition, inhibition/depletion of HSP70 or HSP90 in Panc1-C2GnT-M cells also causes an increase of NMIIA–C2GnT-M complexes and NMIIA-mediated Golgi fragmentation but results in accumulation or degradation of C2GnT-M, respectively. These results can be explained by the known functions of these two HSP: participation of HSP90 in protein folding and HSP70 in protein folding and degradation. We conclude that NMIIA is the master regulator of Golgi fragmentation induced by heat shock or inhibition/depletion of HSP70/90.     

The heat shock proteins: Their roles as multi-component machines for protein folding

The roles played by heat shock proteins in fungi have been subjected to intense scrutiny. Presuming they only played roles in tolerance to stress gave way to the realization that many of them were essential for maintenance of cell physiology under all conditions. Recent progress has revealed their action as multi-component machines, playing roles in signalling and expansion of phenotypic plasticity, as well as their well-established function as molecular chaperones.     

A heat shock protein 90 inhibitor that modulates the immunophilins and regulates hormone receptors without inducing the heat shock response

When a cell encounters external stressors, such as lack of nutrients, elevated temperatures, changes in pH or other stressful environments, a key set of evolutionarily conserved proteins, the heat shock proteins (hsps), become overexpressed. Hsps are classified into six major families with the hsp90 family being the best understood; an increase in cell stress leads to increased levels of hsp90, which leads to cellular protection. A hallmark of hsp90 inhibitors is that they induce a cell rescue mechanism, the heat shock response. We define the unique molecular profile of a compound (SM145) that regulates hormone receptor protein levels through hsp90 inhibition without inducing the heat shock response. Modulation of the binding event between heat shock protein 90 and the immunophilins/homologs using SM145, leads to a decrease in hormone receptor protein levels. Unlike N-terminal hsp90 inhibitors, this hsp90 inhibitor does not induce a heat shock response. This work is proof of principle that controlling hormone receptor expression can occur by inhibiting hsp90 without inducing pro-survival protein heat shock protein 70 (hsp70) or other proteins associated with the heat shock response. Innovatively, we show that blocking the heat shock response, in addition to hsp90, is key to regulating hsp90-associated pathways.     

Sublethal heat shock induces premature senescence rather than apoptosis in human mesenchymal stem cells

Stem cells in adult organism are responsible for cell turnover and tissue regeneration. The study of stem cell stress response contributes to our knowledge on the mechanisms of damaged tissue repair. Previously, we demonstrated that sublethal heat shock (HS) induced apoptosis in human embryonic stem cells. This study aimed to investigate HS response of human adult stem cells. Human mesenchymal stem cells (MSCs) cultivated in vitro were challenged with sublethal HS. It was found that sublethal HS did not affect the cell viability assessed by annexin V/propidium staining. However, MSCs subjected to severe HS exhibited features of stress-induced premature senescence (SIPS): irreversible cell cycle arrest, altered morphology, increased expression of senescence-associated β-galactosidase (SA-β-gal) activity, and induction of cyclin-dependent kinase inhibitor p21 protein. High level of Hsp70 accumulation induced by sublethal HS did not return to the basal level, at least, after 72 h of the cell recovery when most cells exhibited SIPS hallmarks. MSCs survived sublethal HS, and resumed proliferation sustained the properties of parental MSCs: diploid karyotype, replicative senescence, expression of the cell surface markers, and capacity for multilineage differentiation. Our results showed for the first time that in human MSCs, sublethal HS induced premature senescence rather than apoptosis or necrosis. MSC progeny that survived sublethal HS manifested stem cell properties of the parental cells: limited replicative life span and multilineage capacity.     

Cell signaling and heat shock protein expression

Exposure of cells and organs to heat shock is associated with numerous changes in various cellular metabolic parameters and overexpression of proteins collectively known as heat shock proteins (HSP). In this communication we review the cell-signaling events that are altered in response to heat shock as they relate to the subsequent induction of HSP 70 kd (HSP-70) expression. We also review the mechanisms by which HSP-70 is involved in conferring cytoprotective effects. The possibility of altering HSP expression through manipulations of the cell-signal process has clinical importance.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or Department of Defense.     

A mutant in a major heat shock protein of Escherichia coli continues to show inducible thermotolerance

Summary Escherichia coli cells carrying the dnaK756 mutation, were inactivated at 52°C faster than control cells. This suggests that the intact dnaK gene product plays a role in protecting the cell from lethal damage at 52°C. The effect of the dnaK mutation on induced thermotolerance was examined. Prior heat shock at 42°C greatly lowered the subsequent inactivation rate in both mutant and control cells. This result suggests that, although produced in large amounts in response to thermal stress, mutation in the DnaK protein has little or no effect on induced thermotolerance.