Altered expression of a heat shock protein in the mammalian nervous system in the presence of agents which affect microtubule stability

In vitro incubation of the isolated rabbit retina at elevated temperature results in the synthesis of a heat shock protein of M.W. 74,000 (hsp74). Recently we have demonstrated that this protein is associated with preparations of purified retinal microtubules and intermediate filaments. In order to examine the possibility that hsp74 synthesis is related to cytoskeletal stability, the effects of agents known to specifically affect microtubules were examined using an in vitro retinal system. Taxol, an antimitotic agent which stabilizes microtubules, was found to reduce the level of hsp74 synthesized in response to elevated temperature. Colchicine, a potent microtubule de-stabilizing agent, did not induce hsp74 synthesis in the absence of elevated temperature, however, under heat shock conditions, hsp74 synthesis was elevated in the presence of colchicine. Kinetics of microtubule assembly were similar in preparations isolated from cerebral hemispheres of control and hyperthermic animals however, microtubules from the latter were altered in appearance and exhibited a higher degree of crosslinking.     

The CNGCb and CNGCd genes from Physcomitrella patens moss encode for thermosensory calcium channels responding to fluidity changes in the plasma membrane

Land plants need precise thermosensors to timely establish molecular defenses in anticipation of upcoming noxious heat waves. The plasma membrane-embedded cyclic nucleotide-gated Ca²⁺ channels (CNGCs) can translate mild variations of membrane fluidity into an effective heat shock response, leading to the accumulation of heat shock proteins (HSP) that prevent heat damages in labile proteins and membranes. Here, we deleted by targeted mutagenesis the CNGCd gene in two Physcomitrella patens transgenic moss lines containing either the heat-inducible HSP-GUS reporter cassette or the constitutive UBI-Aequorin cassette. The stable CNGCd knockout mutation caused a hyper-thermosensitive moss phenotype, in which the heat-induced entry of apoplastic Ca²⁺ and the cytosolic accumulation of GUS were triggered at lower temperatures than in wild type. The combined effects of an artificial membrane fluidizer and elevated temperatures suggested that the gene products of CNGCd and CNGCb are paralogous subunits of Ca²⁺channels acting as a sensitive proteolipid thermocouple. Depending on the rate of temperature increase, the duration and intensity of the heat priming preconditions, terrestrial plants may thus acquire an array of HSP-based thermotolerance mechanisms against upcoming, otherwise lethal, extreme heat waves.     

Heat shock augments angiotensin II-induced vascular contraction through increased production of reactive oxygen species

A temporal increase in temperature triggers a series of stress responses and alters vascular smooth muscle (VSM) contraction induced by agonist stimulation. Here we examined the role of reactive oxygen species (ROS) in heat shock-dependent augmentation of angiotensin II (AngII)-induced VSM contraction. Endothelium-denuded rat aortic rings were treated with heat shock for 45 min at 42 °C and then subjected to assays for the production of force, ROS, and the expression of ROS-related enzymes. AngII-induced contraction was enhanced in heat shock-treated aorta. AngII-induced production of hydrogen peroxide and superoxide were elevated in response to the heat shock treatment. Pre-treatment with superoxide dismutases (SOD) mimetic and inhibitors for glutathione peroxidase and NADPH oxidase but not for xanthine oxidase eliminated an increase in the AngII-induced contraction in the heat shock-treated aorta. Heat shock increased the expression of p47phox, a cytosolic subunit of NADPH oxidase, but not Cu-Zn-SOD and Mn-SOD. In addition, heat shock increased contraction that was evoked by hydrogen peroxide and pyrogallol. These results suggest that heat shock causes an elevation of ROS as well as a sensitization of ROS signal resulting in an augmentation of VSM contraction in response to agonist.     

真菌Hsp90研究进展

近年来,随着广谱抗生素、化疗以及器官移植技术的广泛应用,真菌感染日益严重,从分子水平揭示病原真菌的致病机制对真菌感染的防控、治疗至关重要。微生物适应宿主微环境压力的能力在其共生与感染过程中发挥着关键作用,heat shock protein 90（Hsp90）是真核生物参与压力应答响应的分子伴侣,它不仅参与胞内蛋白质的折叠,还与许多底物蛋白相互作用共同调节病原真菌的形态发育、生物被膜形成、有性生殖、毒力以及耐药性。本文从真菌Hsp90的活性调节、底物蛋白,以及Hsp90与病原真菌形态发生、有性生殖、耐药性调控等方面综述了近年来真菌Hsp90信号通路的研究进展。     

Changes in albumin precursor and heat shock protein 70 expression and their potential role in response to corneal epithelial wound repair

Many proteins displayed differential expression (either up- or down-regulation) when proteome of migrating and non-migrating epithelium was assessed using 2-DE and ESI-Q-TOF MS/MS. From the up-regulated set, we have identified for the first time a 69-kDa albumin precursor protein with four peptides sequences and 70-kDa heat shock protein (hsp70) with one peptide in the active phase of cell migration (48 h) during the healing process. Western blot analysis was used to further characterize these proteins at different phases (24, 48 and 72 h) of healing. An increase in the mRNA expression (measured using RT-PCR) in the active migration phase (48 h) for albumin precursor and hsp70 was also observed. Furthermore, co-immunoprecipitation studies with anti-albumin precursor and anti-hsp70 antibodies, followed by immunoblotting with anti-fibronectin antibody demonstrated a novel and biologically relevant interaction between albumin precursor protein and fibronectin in corneal epithelial wound healing but not with hsp70. The increased gene and protein expression of albumin and hsp70 during the active phase of cell migration (48 h) in the corneal epithelium suggests their possible role in corneal wound healing. These findings may have broader implications for developing therapeutic strategies for treating wound healing disorders.     

Dynamic processes that reflect anti-apoptotic strategies set up by HspB1 (Hsp27)

Human HspB1 (also denoted Hsp27) is an oligomeric anti-apoptotic protein that has tumorigenic and metastatic roles. To approach the structural organizations of HspB1 that are active in response to apoptosis inducers acting through different pathways, we have analyzed the relative protective efficiency induced by this protein as well its localization, oligomerization and phosphorylation. HeLa cells, that constitutively express high levels of HspB1 were treated with either etoposide, Fas agonist antibody, staurosporine or cytochalasin D. Variability in HspB1 efficiency to interfere with the different apoptotic transduction pathways induced by these agents were detected. Moreover, inducer-specific dynamic changes in HspB1 localization, native size and phosphorylation were observed, that differed from those observed after heat shock. Etoposide and Fas treatments gradually shifted HspB1 towards large but differently phosphorylated oligomeric structures. In contrast, staurosporine and cytochalasin D induced the rapid but transient formation of small oligomers before large structures were formed. These events correlated with inducer-specific phosphorylations of HspB1. Of interest, the formation of small oligomers in response to staurosporine and cytochalasin D was time correlated with the rapid disruption of F-actin. The subsequent, or gradual in the case of etoposide and Fas, formation of large oligomeric structures was a later event concomitant with the early phase of caspase activation. These observations support the hypothesis that HspB1 has the ability, through specific changes in its structural organization, to adapt and interfere at several levels with challenges triggered by different signal transduction pathways upstream of the execution phase of apoptosis.     

The role of alkylhydroxybenzenes in the adaptation of <Emphasis Type="Italic">Micrococcus luteus</Emphasis> to heat shock

The response of the gram-positive bacterium micrococcus luteus to heat shock (4°C, 15 min) and the adaptogenic activity of alkylhydroxybenzenes (AHBs), which are extracellular growth-regulating substances of these bacteria, were studied. The perception of stress and the postshock behavior of M. luteus cells proved to depend on the growth phase and medium. The magnitude of the stress response was more pronounced in cultures grown on synthetic medium than in cultures grown on rich medium (nutrient broth). During exponential or linear growth, the cells were more sensitive to the temperature effect than during decelerated growth. In linearly growing m. luteus cultures, the amount of total intra- and extracellular alkylhydroxybenzenes, the anabiosis inducers, increased in response to heat shock. AHB redistribution between cells and culture liquid occurred in the course of stress and after stress. In micrococci exposed to heat shock, an increase in the AHB concentration both in cells and in culture liquid is likely a defense reaction of stress resistance. This conclusion was confirmed in experiments with the addition 30 min before the heat shock of a chemical analogue of the anabiosis inducer, C₇-AHB (12 mM), which protected M. luteus cells so that their intense growth was observed after shock without any lag. The protective effect of AHBs is a result of their ability to form complexes with enzyme macromolecules and stabilize them. The data obtained extend the knowledge of the stress-protective functions of low-molecular-weight autoregulators and of the role of intercellular communications in the stress response of bacterial cultures.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 26–33.Original Russian Text Copyright © 2005 by Stepanenko, Mulyukin, Kozlova, Nikolaev, El-Registan.     

Structure and properties of small heat shock proteins (sHsp) and their interaction with cytoskeleton proteins

The modern classification of small heat shock proteins (sHsp) is presented and peculiarities of their primary structure and the mechanism of formation of oligomeric complexes are described. Data on phosphorylation of sHsp by different protein kinases are presented and the effect of phosphorylation on oligomeric state and chaperone activity of sHsp is discussed. Intracellular location of sHsp under normal and stress conditions is described and it is emphasized that under certain condition sHsp interact with different elements of cytoskeleton. The literature concerning the effect of sHsp on polymerization of actin in vitro is analyzed. An attempt is made to compare effects of sHsp on polymerization of actin in vitro with the results obtained on living cells under normal conditions and after heat shock or hormone action. The literature concerning possible effects of sHsp on cell motility is also analyzed.     

Proteomic analysis of the expression of proteins related to rice quality during caryopsis development and the effect of high temperature on expression

Proteins are essential to rice caryopsis development and quality formation. High temperature is an important environmental factor, which may decrease grain quality. In the present study rice caryopsis proteins were profiled by two-dimensional polyacrylamide gel electrophoresis, and differentially expressed proteins were analyzed by liquid chromatography/tandem mass spectrometry. Expressions of more than 400 polypeptide spots during caryopsis development, in response to temperature treatments or between varieties were monitored. Among them, more than 70 differentially expressed polypeptides were analyzed by liquid chromatography/tandem mass spectrometry. We identified 54 proteins with known functions. Of these, 21 were involved with carbohydrate metabolism, 14 with protein synthesis and sorting, and 9 with stress responses. Waxy (Wx) proteins and glutelins were the most significant spots, which increased significantly during development. Allergen-like proteins, PPDK and NADH-SDH, also were expressed during development, implying their physiological roles in caryopsis. Expression of large isoforms of Wx proteins was correlated with the amylose content of rice caryopses. One protein with high GC content in its DNA sequence was correlated with the chalky trait of kernels. High temperature (35/30 degrees C) decreased the expression of Wx proteins, allergen-like proteins, and elongation factor 1beta, but increased the expression of small heat shock proteins (sHSP), glyceraldehyde-3-phosphate dehydrogenase, and prolamin. sHSP was positively correlated with the appearance of chalky kernels. During development, glutelins were phosphorylated and glycosylated, indicating that these molecules were post-translationally modified. Possible functions of the expression of candidate proteins on the grain quality are discussed.     

Interactome analysis reveals versatile functions of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 in RNA processing within the nucleus and cytoplasm

Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3) shares an RNA chaperone function with E. coli cold shock proteins and regulates freezing tolerance during cold acclimation. Here, we screened for AtCSP3-interacting proteins using a yeast two-hybrid system and 38 candidate interactors were identified. Sixteen of these were further confirmed in planta interaction between AtCSP3 by a bi-molecular fluorescence complementation assay. We found that AtCSP3 interacts with CONSTANS-LIKE protein 15 and nuclear poly(A)-binding proteins in nuclear speckles. Three 60S ribosomal proteins (RPL26A, RPL40A/UBQ2, and RPL36aB) and the Gar1 RNA-binding protein interacted with AtCSP3 in the nucleolus and nucleoplasm, suggesting that AtCSP3 functions in ribosome biogenesis. Interactions with LOS2/enolase and glycine-rich RNA-binding protein 7 that are cold inducible, and an mRNA decapping protein 5 (DCP5) were observed in the cytoplasm. These data suggest that AtCSP3 participates in multiple complexes that reside in nuclear and cytoplasmic compartments and possibly regulates RNA processing and functioning.