Recovery from Heat Shock in Heat-Tolerant and Nontolerant Variants of Creeping Bentgrass

Recovery from the heat-shock response was tested in heat-tolerant (selected bentgrass [SB]) and nontolerant (nonselected bentgrass [NSB]) variants of creeping bentgrass (Agrostis palustris Huds.) SB increased incorporation of radioactive amino acids into protein 2 h earlier than NSB when leaf blades were incubated at the recovery temperature following heat shock. Electrophoresis indicated that heat-shock protein (HSP) synthesis decreased and normal protein synthesis increased at 4 h in SB and at 6 to 8 h in NSB. Increased synthesis of normal proteins was not due to increased abundance of normal mRNAs, which were equivalent in SB and NSB at 4 h. But at 4 h, more of the normal mRNA population was associated with polysomes in SB than in NSB. Synthesis of HSP70 and HSP18 decreased earlier in SB than in NSB. The decreased synthesis of these HSPs appeared to be correlated with decreased mRNA abundance. But at 4 h, some of the HSP18 mRNA may have been associated with heat-shock granules in SB. Synthesis of HSP25 continued through the 8-h recovery in both variants. Although the abundance of HSP25 was equivalent in SB and NSB during heat shock and recovery, more HSP25 mRNA was associated with polysomes in SB than in NSB.     

Recruitment of the Host Plant Heat Shock Protein 70 by Tomato Yellow Leaf Curl Virus Coat Protein Is Required for Virus Infection

A functional capsid protein (CP) is essential for host plant infection and insect transmission of Tomato yellow leaf curl virus (TYLCV) and other monopartite begomoviruses. We have previously shown that TYLCV CP specifically interacts with the heat shock protein 70 (HSP70) of the virus insect vector, Bemisia tabaci. Here we demonstrate that during the development of tomato plant infection with TYLCV, a significant amount of HSP70 shifts from a soluble form into insoluble aggregates. CP and HSP70 co-localize in these aggregates, first in the cytoplasm, then in the nucleus of cells associated with the vascular system. CP-HSP70 interaction was demonstrated by co-immunopreciptation in cytoplasmic - but not in nuclear extracts from leaf and stem. Inhibition of HSP70 expression by quercetin caused a decrease in the amount of nuclear CP aggregates and a re-localization of a GFP-CP fusion protein from the nucleus to the cytoplasm. HSP70 inactivation resulted in a decrease of TYLCV DNA levels, demonstrating the role of HSP70 in TYLCV multiplication in planta. The current study reveals for the first time the involvement of plant HSP70 in TYLCV CP intracellular movement. As described earlier, nuclear aggregates contained TYLCV DNA-CP complexes and infectious virions. Showing that HSP70 localizes in these large nuclear aggregates infers that these structures operate as nuclear virus factories.     

Heat shock gene expression in Xenopus laevis A6 cells in response to heat shock and sodium arsenite treatments

Continuous exposure of a Xenopus laevis kidney epithelial cell line, A6, to either heat shock (33 degrees C) or sodium arsenite (50 microM) resulted in transient but markedly different temporal patterns of heat-shock protein (HSP) synthesis and HSP 70 and 30 mRNA accumulation. Heat-shock-induced synthesis of HSPs was detectable within 1 h and reached maximum levels by 2-3 h. While sodium arsenite induced the synthesis of some HSPs within 1 h, maximal HSP synthesis did not occur until 12 h. The pattern of HSP 70 and 30 mRNA accumulation was similar to the response observed at the protein level. During recovery from heat shock, a coordinate decline in HSPs and HSP 70 and 30 mRNA was observed. During recovery from sodium arsenite, a similar phenomenon occurred during the initial stages. However, after 6 h of recovery, HSP 70 mRNA levels persisted in contrast to the declining HSP 30 mRNA levels. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of 5 HSPs in the HSP 70 family, of which two were constitutive, and 16 different stress-inducible proteins in the HSP 30 family. In conclusion, heat shock and sodium arsenite induce a similar set of HSPs but maximum synthesis of the HSP is temporally separated by 12-24 h.     

Changes in synthesis and localization of members of the 70-kDa class of heat-shock proteins accompany the induction of embryogenesis inBrassica napus L. microspores

Elevation of the culture temperature to 32°C for approximately 8 h can irreversibly change the developmental fate of isolatedBrassica napus microspores from pollen development to embryogenesis. This stress treatment was accompanied by de-novo synthesis of a number of heat-shock proteins (HSPs) of the 70-kDa class: HSP68 and HSP70. A detailed biochemical and cytological analysis was performed of the HSP68 and HSP70 isoforms. Eight HSP68 isoforms, one of which was induced three fold by the stress treatment, were detected on two-dimensional immunoblots. Immunocytochemistry revealed a co-distribution of HSP68 with DNA-containing organelles, presumably mitochondria. Six HSP70 isoforms were detected, one of which was induced six fold under embryogenic culture conditions. During normal pollen development, HSP70 was localized in the nucleoplasm during the S phase of the cell cycle, and predominantly in the cytoplasm during the remainder. Induction of embryogenic development in late unicellular microspores was accompanied by an intense anti-HSP70 labeling of the nucleoplasm during an elongated S phase. In early bicellular pollen the nucleus of the vegetative cell, which normally does not divide and never expresses HSP70, showed intense labeling of the nucleoplasm with anti-HSP70 after 8 h of culture under embryogenic conditions. These results demonstrate a strong correlation between the phase of the cell cycle, the nuclear localization of HSP70 and the induction of embryogenesis. As temperature stress alone is responsible for the induction of embryogenic development, and causes an altered pattern of cell division, there might be a direct involvement of HSP70 in this process.Abbreviations HSP heat-shock protein - 2-D two-dimensional - DAPI 4,6-diamidino-2-phenylindole. 1-D = one-dimensional - pI isoelectric point     

人骨保护素-热休克蛋白70融合蛋白在昆虫细胞中的表达和活性分析

目的:利用BaculoDirect杆状病毒表达系统融合表达人OPG功能片段p22-194和分枝杆菌HSP70 p111-125基因,并鉴定重组蛋白及其生物学活性。方法:将编码人OPG功能片段和分枝杆菌HSP70功能片段基因克隆至杆状病毒转座载体,将重组转座载体与BaculoDirectTM Linear DNA进行LR重组连接反应,构建出重组杆状病毒DNA,转染Sf9昆虫细胞,获得重组病毒。在Sf9细胞中进行表达,并对表达产物进行SDS-PAGE电泳、Western blotting分析,用Ni柱纯化。采用破骨细胞生成抑制试验和抑炎试验鉴定表达产物的生物学活性。结果:重组病毒在感染昆虫细胞后48h开始出现一相对分子质量为28 kDa大小的特异条带,感染后72～96 h蛋白量达到高峰。破骨细胞生成抑制实验及抑炎试验结果显示,重组蛋白能明显抑制破骨细胞的生长和分化,同时亦具有抑制炎症反应的作用。结论:利用杆状病毒表达系统在昆虫细胞中成功表达OPG-HSP70融合蛋白,该融合蛋白具有抑制破骨细胞生成和抑制炎症反应生物学活性。     

Heat shock-induced redistribution of a 160-kDa nuclear matrix protein.

In this paper we describe a 160-kDa protein (p160) which is present in the nuclear matrix of rat, mouse, and human cells. Biochemical and ultrastructural analysis shows that p160 is associated with the internal matrix and is not present in the lamina-pore complex. Immunoelectron microscopy shows that the protein is part of the extranucleolar, fibrogranular network of the nuclear matrix. During an in vivo 42 degrees C heat treatment of HeLa cells, A431 human epidermoid cells, and T24 human bladder carcinoma cells, p160 transiently formed large clusters inside the nucleus. These p160 clusters are associated with the nuclear matrix network, as judged by immunolabeling on isolated nuclear matrices. The percentage of cells showing p160 clusters increased proportionally with longer heat treatments, reaching a maximum after a period of 3 h. At this time 70 +/- 5% of the cells displayed these clusters. Clustering decreased after longer heat treatments and the anti-p160 staining pattern became diffuse granular again. Other nuclear components, such as the A1 antigen of hnRNP (ribonucleoprotein), the Sm antigen of snRNPs, and lamins A and C, did not cluster during the 42 degrees C treatment, indicating that this reallocation is characteristic for the p160 matrix protein. These results demonstrate that p160 is an internal nuclear matrix element with a dynamic spatial distribution.     

Withaferin A Induces Proteasome Inhibition,Endoplasmic Reticulum Stress,the Heat Shock Response and Acquisition of Thermotolerance

In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge.     

Region-specific regulation of glucocorticoid receptor/HSP90 expression and interaction in brain

The hippocampal glucocorticoid receptor (GR) is involved in negative feedback regulation of the hypothalamo-pituitary-adrenal axis and is believed to transduce the deleterious effects of glucocorticoids in depression and age-related memory loss. Regulation and intracellular trafficking of the GR are critical determinants of GR action in both health and disease. Here, we show dynamic regulation of GR and its interaction with its principal intracellular chaperone, heat-shock protein (HSP) 90, across the circadian cycle. Our initial experiments indicate that cytosolic hippocampal GR protein is elevated in the evening (PM), whereas nuclear GR and cytosolic HSP90, HSP70 and heat-shock cognate 70 (HSC70), are unchanged. In contrast, there are no changes in examined proteins in the hypothalamus. Immunoprecipitation experiments reveal increased GR-HSP90 associations in the hippocampus in the PM, whereas binding in the hypothalamus is decreased in the PM. Given that GR requires HSP90 for ligand binding, the data suggest that circadian GR signaling capacity is regulated in a region-specific pattern.     

热激蛋白70的研究进展

热激蛋白(HSP70)作为分子伴侣参与细胞内许多重要反应,从而对生物体起着重要的作用。随着研究的深入,其生物学功能不断被发现和利用的同时,HSP70的应用前景也变得越来越广泛。已有研究者对HSP70的生物学功能做了详细介绍,我们主要对近年来HSP70在医学及环境监测等方面的应用进行综述。     

PP2A Mediated AMPK Inhibition Promotes HSP70 Expression in Heat Shock Response

Background

Under stress, AMP-activated protein kinase (AMPK) plays a central role in energy balance, and the heat shock response is a protective mechanism for cell survival. The relationship between AMPK activity and heat shock protein (HSP) expression under stress is unclear.

Methodology/Principal Findings

We found that heat stress induced dephosphorylation of AMPKα subunit (AMPKα) in various cell types from human and rodent. In HepG2 cells, the dephosphorylation of AMPKα under heat stress in turn caused dephosphorylation of acetyl-CoA carboxylase and upregulation of phosphoenolpyruvate carboxykinase, two downstream targets of AMPK, confirming the inhibition of AMPK activity by heat stress. Treatment of HepG2 cells with phosphatase 2A (PP2A) inhibitor okadaic acid or inhibition of PP2A expression by RNA interference efficiently reversed heat stress-induced AMPKα dephosphorylation, suggesting that heat stress inhibited AMPK through activation of PP2A. Heat stress- and other HSP inducer (CdCl₂, celastrol, MG132)-induced HSP70 expression could be inhibited by AICAR, an AMPK specific activator. Inhibition of AMPKα expression by RNA interference reversed the inhibitory effect of AICAR on HSP70 expression under heat stress. These results indicate that AMPK inhibition under stress contribute to HSP70 expression. Mechanistic studies showed that activation of AMPK by AICAR had no effect on heat stress-induced HSF1 nuclear translocation, phosphorylation and binding with heat response element in the promoter region of HSP70 gene, but significantly decreased HSP70 mRNA stability.

Conclusions/Significance

These results demonstrate that during heat shock response, PP2A mediated AMPK inhibition upregulates HSP70 expression at least partially through stabilizing its mRNA, which suggests a novel mechanism for HSP induction under stress.     

Gene Silencing of Selenoprotein K Induces Inflammatory Response and Activates Heat Shock Proteins Expression in Chicken Myoblasts

In the present study, specific small interfering RNA (siRNA) for selenoprotein K (Selk) gene was designed and transfected into chicken myoblasts. Then, the expressions of inflammatory factors (including induced nitric oxide synthase [iNOS], nuclear factor-kappa B [NF-κB], heme-oxygenase-1 [HO-1], cyclooxygenase-2 [COX-2], and prostaglandin E synthase [PTGEs]), inflammation-related cytokines (including interleukin [IL]-1β, IL-6, IL-7, IL-8, IL-17, and interferon [IFN]-γ), and heat shock proteins (HSPs) (including HSP27, HSP40, HSP60, HSP70, and HSP90) were examined at 24 and 72 h after transfection. The results showed that messenger RNA (mRNA) expressions of iNOS, NF-κB, HO-1, COX-2, IL-6, IL-7, IL-8, HSP 27, HSP 40, HSP 60, HSP 70, and HSP 90 were significantly increased (p < 0.05) at 24 and 72 h after siRNA transfection, and the mRNA expressions of PTGEs, IL-1β, IL-17, and IFN-γ were significantly increased and decreased (p < 0.05) at 24 and 72 h after siRNA transfection. The results also showed that the protein expressions of iNOS, NF-κB, HO-1, COX-2, HSP60, HSP70, and HSP90 were significantly increased (p < 0.05) at 24 and 72 h after siRNA transfection. The correlation analysis and principal component analysis (PCA) showed that PTGEs, IL-1β, IL-17, IFN-γ, HSP40, and HSP90 might play special roles in response to Selk silencing in chicken myoblasts. These results indicated that Selk silencing induced inflammation response by affecting the expression levels of inflammatory factors and inflammation-related cytokines, and the heat shock proteins might play protective roles in this response in chicken myoblasts.     

The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein

Background

Since the identification of poly-alanine expanded poly(A) binding protein nuclear 1 (PABPN1) as the genetic cause of oculopharyngeal muscular dystrophy (OPMD), considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear.

Results

In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90). Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs). Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP). The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells.

Conclusions

Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD.     

HeLa细胞热休克蛋白的代谢动力学

 HeLa细胞经45℃,15min应激后,可诱导一组分子量为100,85,73,70,54kD的热休克蛋白,其中分子量为73/70kD的HSP产量最高。在产生HSP的同时,正常蛋白质合成受到抑制,并在随后数小时内恢复。HSP73/70在应激后能迅速诱导产生,应激后4—6小时为其合成高峰,10小时后明显减少,24小时恢复正常。其分解遵循指数规律,半衰期为49.9小时。     

Osmotic induction of stress-responsive gene expression in the lobster Homarus americanus

The American lobster, Homarus americanus, encounters osmotic stress throughout its life cycle. To understand the molecular basis of osmotic stress responses in vivo, we used homologous cDNA probes to characterize the mRNA patterns of lobster HSP70 (=70-kDa heat-shock protein), HSP90 (=90-kDa heat-shock protein), and polyubiquitin during hypo- and hyper-osmotic stress in abdominal muscle and hepatopancreas (a digestive tissue) at 30, 60, and 120 min of osmotic stress. Hypo- and hyper-osmotic stress significantly increased the levels of the mRNAs encoding HSP70 and HSP90 in abdominal muscle. Hyper-osmotic stress increased HSP90 mRNA levels in hepatopancreas, but hypo-osmotic stress did not. Both abdominal muscle and hepatopancreas exhibited significant changes in polyubiquitin gene expression during osmotic stress. In abdominal muscle, polyubiquitin mRNA levels increased during both hypo- and hyper-osmotic stress. Hepatopancreas, however, showed a significant elevation in polyubiquitin mRNA only during hypo-osmotic stress.     

Expression of Heat Shock Protein 70 Gene and Its Correlation with Inflammatory Markers in Essential Hypertension

Objectives

Hypertension is characterized by systemic high blood pressure and is the most common and important risk factor for the development of cardiovascular diseases. Studies have shown that the circulating levels of certain inflammatory markers such as tumor necrosis factor-alpha (TNF-alpha), interlukin-6 (IL-6), c-reactive protein (CRP), and tumor suppressor protein-53 (p53) are upregulated and are independently associated with essential hypertension. However, mechanism of increase in the levels of HSP70 protein is not clear. No such studies are reported in the blood circulation of patients with essential hypertension. In the present study, we investigated the expression of circulating HSP70 at mRNA and protein levels and its relationship with other inflammatory markers in patients with essential hypertension.

Materials and Methods

We recruited 132 patients with essential hypertension and 132 normal controls from similar socio-economic-geographical background. The expression of HSP70 at mRNA levels was determined by Real Time PCR and at protein levels by indirect Elisa and Western Blot techniques.

Results

We found a significantly higher expression of HSP70 gene expression (approximately 6.45 fold, P < 0.0001) in hypertensive patients as compared to healthy controls. A significant difference (P < 0.0001) in the protein expression of HSP70 was also observed in plasma of patients as compared to that of controls.

Conclusion

Higher expression of HSP70 is positively correlated with inflammatory markers in patients with essential hypertension and this correlation could play an important role in essential hypertension.     

Defined nuclear changes accompany the reprogramming of the microspore to embryogenesis

The switch of the gametophytic developmental program toward pollen embryogenesis to form a haploid plant represents an important alternative for plant breeding. In the present study, the switch of the gametophytic developmental program toward a sporophytic pathway, "embryogenesis," has been studied in three different plant species, Brassica, tobacco, and pepper. The switch has been induced by stress (heat shock) at the very responsive stage of the microspore, which is the vacuolate period. As a result, the cell nucleus undergoes striking structural changes with regard to late gametophytic development, including alterations of biosynthetic activities and proliferative activity. An enrichment in HSP70 heat-shock protein and in the presence of Ntf6-MAP kinase was observed after inductive treatment in the nuclei during early embryogenesis. This apparently reflected the possible roles of these proteins, specifically the protective role of HSP70 for the nuclear machinery, and signal transduction of Ntf6-MAPK for the entry of cells into proliferation. Importantly, the observed nuclear changes were similar in the three species investigated and represented convenient markers for early monitoring of embryogenesis and selection purposes for obtaining double-haploid plants in plant breeding.     

Role of Heat Shock Proteins in the Effect of NMDA and KCl on Cerebellar Granule Cells Survival

Cerebellar granule cells (CGC) die apoptotically after five days in culture (DIV) at physiological concentrations of potassium (5 mM; K5). When CGC are depolarized (K25) or treated with NMDA (150 M) cell survival is increased. CGC changed from K25 to K5 die after 24–48 h. It is known that heat shock protein (HSP) may protect from cell death. Here, we found that cells in K5 showed an increase in HSP-70 levels after 3 DIV. Similarly, in cells changed from K25 to K5, HSP-70 levels were increased after 6 h. Neither NMDA nor K25 treatment affected HSP-70 levels from 2–7 DIV. Ethanol or thermal stress induced HSP-70, but cell survival was not affected in K5 medium. These results suggest that HSP, particularly HSP-70, are not involved in the mechanisms by which NMDA and KCl promote cell survival.