Showing papers on "Heat shock protein published in 2008"

Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging

Abstract: The long-term health of the cell is inextricably linked to protein quality control. Under optimal conditions this is accomplished by protein homeostasis, a highly complex network of molecular interactions that balances protein biosynthesis, folding, translocation, assembly/disassembly, and clearance. This review will examine the consequences of an imbalance in homeostasis on the flux of misfolded proteins that, if unattended, can result in severe molecular damage to the cell. Adaptation and survival requires the ability to sense damaged proteins and to coordinate the activities of protective stress response pathways and chaperone networks. Yet, despite the abundance and apparent capacity of chaperones and other components of homeostasis to restore folding equilibrium, the cell appears poorly adapted for chronic proteotoxic stress when conformationally challenged aggregation-prone proteins are expressed in cancer, metabolic disease, and neurodegenerative disease. The decline in biosynthetic and repair activities that compromises the integrity of the proteome is influenced strongly by genes that control aging, thus linking stress and protein homeostasis with the health and life span of the organism.

Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice

Abstract: Plants respond to adverse environment by initiating a series of signaling processes including activation of transcription factors that can regulate expression of arrays of genes for stress response and adaptation. NAC (NAM, ATAF, and CUC) is a plant specific transcription factor family with diverse roles in development and stress regulation. In this report, a stress-responsive NAC gene (SNAC2) isolated from upland rice IRA109 (Oryza sativa L. ssp japonica) was characterized for its role in stress tolerance. SNAC2 was proven to have transactivation and DNA-binding activities in yeast and the SNAC2-GFP fusion protein was localized in the rice nuclei. Northern blot and SNAC2 promoter activity analyses suggest that SNAC2 gene was induced by drought, salinity, cold, wounding, and abscisic acid (ABA) treatment. The SNAC2 gene was over-expressed in japonica rice Zhonghua 11 to test the effect on improving stress tolerance. More than 50% of the transgenic plants remained vigorous when all WT plants died after severe cold stress (4–8°C for 5 days). The transgenic plants had higher cell membrane stability than wild type during the cold stress. The transgenic rice had significantly higher germination and growth rate than WT under high salinity conditions. Over-expression of SNAC2 can also improve the tolerance to PEG treatment. In addition, the SNAC2-overexpressing plants showed significantly increased sensitivity to ABA. DNA chip profiling analysis of transgenic plants revealed many up-regulated genes related to stress response and adaptation such as peroxidase, ornithine aminotransferase, heavy metal-associated protein, sodium/hydrogen exchanger, heat shock protein, GDSL-like lipase, and phenylalanine ammonia lyase. Interestingly, none of the up-regulated genes in the SNAC2-overexpressing plants matched the genes up-regulated in the transgenic plants over-expressing other stress responsive NAC genes reported previously. These data suggest SNAC2 is a novel stress responsive NAC transcription factor that possesses potential utility in improving stress tolerance of rice.

Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone

Abstract: There are currently no approved antifibrotic therapies for liver cirrhosis. We used vitamin A‐coupled liposomes to deliver small interfering RNA (siRNA) against gp46, the rat homolog of human heat shock protein 47, to hepatic stellate cells. Our approach exploits the key roles of these cells in both fibrogenesis as well as uptake and storage of vitamin A. Five treatments with the siRNA-bearing vitamin A‐coupled liposomes almost completely resolved liver fibrosis and prolonged survival in rats with otherwise lethal dimethylnitrosamine-induced liver cirrhosis in a dose- and duration-dependent manner. Rescue was not related to off-target effects or associated with recruitment of innate immunity. Receptor-specific siRNA delivery was similarly effective in suppressing collagen secretion and treating fibrosis induced by CCl4 or bile duct ligation. The efficacy of the approach using both acute and chronic models of liver fibrosis suggests its therapeutic potential for reversing human liver cirrhosis. Liver cirrhosis, or fibrosis, the ultimate pathological feature of all forms of chronic hepatic damage, is responsible for much morbidity and mortality worldwide. The principal cell type responsible for liver fibrosis is the hepatic stellate (HS) cell, a resident perisinusoidal cell that takes up vitamin A from circulation and stores it. When stimulated by reactive oxygen intermediates or cytokines, HS cells become activated and are transformed to proliferative, fibrogenic and contractile myofibroblasts 1 , which synthesize and secrete procollagen, which accumulates as insoluble collagen after its terminal domains are cleaved by procollagen peptides, causing fibrosis. The collagen-specific chaperone, heat shock protein 47 (HSP47), facilitates collagen secretion by ensuring proper triple-helix formation of procollagen in the endoplasmic reticulum and has also been implicated in translational regulation of procollagen synthesis 2,3 .

HSP72 protects against obesity-induced insulin resistance.

Abstract: Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of κB kinase, and tumor necrosis factor-α, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

Heat shock proteins: essential proteins for apoptosis regulation

Abstract: Many different external and intrinsic apoptotic stimuli induce the accumulation in the cells of a set of proteins known as stress or heat shock proteins (HSPs). HSPs are conserved proteins present in both prokaryotes and eukaryotes. These proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and by preventing their aggregation. HSPs have a protective function, that is they allow the cells to survive to otherwise lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. Several of these proteins have demonstrated to directly interact with components of the cell signalling pathways, for example those of the tightly regulated caspase-dependent programmed cell death machinery, upstream, downstream and at the mitochondrial level. HSPs can also affect caspase-independent apoptosis-like process by interacting with apoptogenic factors such as apoptosis-inducing factor (AIF) or by acting at the lysosome level. This review will describe the different key apoptotic proteins interacting with HSPs and the consequences of these interactions in cell survival, proliferation and apoptotic processes. Our purpose will be illustrated by emerging strategies in targeting these protective proteins to treat haematological malignancies.

Increase in Endoplasmic Reticulum Stress–Related Proteins and Genes in Adipose Tissue of Obese, Insulin-Resistant Individuals

Abstract: OBJECTIVE—To examine fat biopsy samples from lean insulin-sensitive and obese insulin-resistant nondiabetic individuals for evidence of endoplasmic reticulum (ER) stress. RESEARCH DESIGN AND METHODS—Subcutaneous fat biopsies were obtained from the upper thighs of six lean and six obese nondiabetic subjects. Fat homogenates were used for proteomic (two-dimensional gel and MALDI-TOF/TOF), Western blot, and RT-PCR analysis. RESULTS—Proteomic analysis revealed 19 differentially upregulated proteins in fat of obese subjects. Three of these proteins were the ER stress–related unfolded protein response (UPR) proteins calreticulin, protein disulfide-isomerase A3, and glutathione-S-transferase P. Western blotting revealed upregulation of several other UPR stress–related proteins, including calnexin, a membrane-bound chaperone, and phospho c-jun NH2-terminal kinase (JNK)-1, a downstream effector protein of ER stress. RT-PCR analysis revealed upregulation of the spliced form of X-box binding protein-1s, a potent transcription factor and part of the proximal ER stress sensor inositol-requiring enzyme-1 pathway. CONCLUSIONS—These findings represent the first demonstration of UPR activation in subcutaneous adipose tissue of obese human subjects. As JNK can inhibit insulin action and activate proinflammatory pathways, ER stress activation of JNK may be a link between obesity, insulin resistance, and inflammation.

Hsp70 Translocates into the Plasma Membrane after Stress and Is Released into the Extracellular Environment in a Membrane-Associated Form that Activates Macrophages

Abstract: Heat shock proteins (hsps) are intracellular chaperones that play a key role in the recovery from stress. Hsp70, the major stress-induced hsp, has been found in the extracellular medium and is capable of activating immune cells. The mechanism involved in Hsp70 release is controversial because this protein does not present a consensual secretory signal. In this study, we have shown that Hsp70 integrates into artificial lipid bilayer openings of ion conductance pathways. In addition, this protein was found inserted into the plasma membrane of cells after stress. Hsp70 was released into the extracellular environment in a membrane-associated form, sharing the characteristics of this protein in the plasma membrane. Extracellular membranes containing Hsp70 were at least 260-fold more effective than free recombinant protein in inducing TNF-alpha production as an indicator of macrophage activation. These observations suggest that Hsp70 translocates into the plasma membrane after stress and is released within membranous structures from intact cells, which could act as a danger signal to activate the immune system.

Chaperones in control of protein disaggregation

Abstract: The chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. Although the native structure of a protein is principally encoded in its amino-acid sequence, the process of folding in vivo very often requires the assistance of molecular chaperones. Chaperones also play a role in a post-translational quality control system and thus are required to maintain the proper conformation of proteins under changing environmental conditions. Many factors leading to unfolding and misfolding of proteins eventually result in protein aggregation. Stress imposed by high temperature was one of the first aggregation-inducing factors studied and remains one of the main models in this field. With massive protein aggregation occurring in response to heat exposure, the cell needs chaperones to control and counteract the aggregation process. Elimination of aggregates can be achieved by solubilization of aggregates and either refolding of the liberated polypeptides or their proteolysis. Here, we focus on the molecular mechanisms by which heat-shock protein 70 (Hsp70), Hsp100 and small Hsp chaperones liberate and refold polypeptides trapped in protein aggregates.

Structural and functional diversities between members of the human HSPB, HSPH, HSPA, and DNAJ chaperone families

Abstract: Heat shock proteins (HSPs) were originally identified as stress-responsive proteins required to deal with proteotoxic stresses. Besides being stress-protective and possible targets for delaying progression of protein folding diseases, mutations in chaperones also have been shown to cause disease (chaperonopathies). The mechanism of action of the "classical", stress-inducible HSPs in serving as molecular chaperones preventing the irreversible aggregation of stress-unfolded or disease-related misfolded proteins is beginning to emerge. However, the human genome encodes several members for each of the various HSP families that are not stress-related but contain conserved domains. Here, we have reviewed the existing literature on the various members of the human HSPB (HSP27), HSPH (HSP110), HSPA (HSP70), and DNAJ (HSP40) families. Apart from structural and functional homologies, several diversities between members and families can be found that not only point to differences in client specificity but also seem to serve differential client handling and processing. How substrate specificity and client processing is determined is far from being understood.

Regulation of the cellular heat shock response in Caenorhabditis elegans by thermosensory neurons.

Abstract: Temperature pervasively affects all cellular processes. In response to a rapid increase in temperature, all cells undergo a heat shock response, an ancient and highly conserved program of stress-inducible gene expression, to reestablish cellular homeostasis. In isolated cells, the heat shock response is initiated by the presence of misfolded proteins and therefore thought to be cell-autonomous. In contrast, we show that within the metazoan Caenorhabditis elegans, the heat shock response of somatic cells is not cell-autonomous but rather depends on the thermosensory neuron, AFD, which senses ambient temperature and regulates temperature-dependent behavior. We propose a model whereby this loss of cell autonomy serves to integrate behavioral, metabolic, and stress-related responses to establish an organismal response to environmental change.

Heat stress-responsive transcriptome analysis in heat susceptible and tolerant wheat ( Triticum aestivum L.) by using Wheat Genome Array

Abstract: Wheat is a major crop in the world, and the high temperature stress can reduce the yield of wheat by as much as 15%. The molecular changes in response to heat stress are poorly understood. Using GeneChip® Wheat Genome Array, we analyzed genome-wide gene expression profiles in the leaves of two wheat genotypes, namely, heat susceptible 'Chinese Spring' (CS) and heat tolerant 'TAM107' (TAM). A total of 6560 (~10.7%) probe sets displayed 2-fold or more changes in expression in at least one heat treatment (f alse d iscovery r ate, FDR, α = 0.001). Except for heat shock protein (HSP) and heat shock factor (HSF) genes, these putative heat responsive genes encode transcription factors and proteins involved in phytohormone biosynthesis/signaling, calcium and sugar signal pathways, RNA metabolism, ribosomal proteins, primary and secondary metabolisms, as well as proteins related to other stresses. A total of 313 probe sets were differentially expressed between the two genotypes, which could be responsible for the difference in heat tolerance of the two genotypes. Moreover, 1314 were differentially expressed between the heat treatments with and without pre-acclimation, and 4533 were differentially expressed between short and prolonged heat treatments. The differences in heat tolerance in different wheat genotypes may be associated with multiple processes and mechanisms involving HSPs, transcription factors, and other stress related genes. Heat acclimation has little effects on gene expression under prolonged treatments but affects gene expression in wheat under short-term heat stress. The heat stress responsive genes identified in this study will facilitate our understanding of molecular basis for heat tolerance in different wheat genotypes and future improvement of heat tolerance in wheat and other cereals.

Cellular stress response: a novel target for chemoprevention and nutritional neuroprotection in aging, neurodegenerative disorders and longevity

Abstract: The predominant molecular symptom of aging is the accumulation of altered gene products. Moreover, several conditions including protein, lipid or glucose oxidation disrupt redox homeostasis and lead to accumulation of unfolded or misfolded proteins in the aging brain. Alzheimer’s and Parkinson’s diseases or Friedreich ataxia are neurological diseases sharing, as a common denominator, production of abnormal proteins, mitochondrial dysfunction and oxidative stress, which contribute to the pathogenesis of these so called “protein conformational diseases”. The central nervous system has evolved the conserved mechanism of unfolded protein response to cope with the accumulation of misfolded proteins. As one of the main intracellular redox systems involved in neuroprotection, the vitagene system is emerging as a neurohormetic potential target for novel cytoprotective interventions. Vitagenes encode for cytoprotective heat shock proteins (Hsp) Hsp70 and heme oxygenase-1, as well as thioredoxin reductase and sirtuins. Nutritional studies show that ageing in animals can be significantly influenced by dietary restriction. Thus, the impact of dietary factors on health and longevity is an increasingly appreciated area of research. Reducing energy intake by controlled caloric restriction or intermittent fasting increases lifespan and protects various tissues against disease. Genetics has revealed that ageing may be controlled by changes in intracellular NAD/NADH ratio regulating sirtuin, a group of proteins linked to aging, metabolism and stress tolerance in several organisms. Recent findings suggest that several phytochemicals exhibit biphasic dose responses on cells with low doses activating signaling pathways that result in increased expression of vitagenes encoding survival proteins, as in the case of the Keap1/Nrf2/ARE pathway activated by curcumin and NAD/NADH-sirtuin-1 activated by resveratrol. Consistently, the neuroprotective roles of dietary antioxidants including curcumin, acetyl-l-carnitine and carnosine have been demonstrated through the activation of these redox-sensitive intracellular pathways. Although the notion that stress proteins are neuroprotective is broadly accepted, still much work needs to be done in order to associate neuroprotection with specific pattern of stress responses. In this review the importance of vitagenes in the cellular stress response and the potential use of dietary antioxidants in the prevention and treatment of neurodegenerative disorders is discussed.

A Vicious Cycle Involving Release of Heat Shock Protein 60 from Injured Cells and Activation of Toll-Like Receptor 4 Mediates Neurodegeneration in the CNS

Abstract: Infection, ischemia, trauma, and neoplasia elicit a similar inflammatory response in the CNS characterized by activation of microglia, the resident CNS monocyte. The molecular events leading from CNS injury to the activation of innate immunity is not well understood. We show here that the intracellular chaperone heat shock protein 60 (HSP60) serves as a signal of CNS injury by activating microglia through a toll-like receptor 4 (TLR4)-dependent and myeloid differentiation factor 88 (MyD88)-dependent pathway. HSP60 is released from CNS cells undergoing necrotic or apoptotic cell death and specifically binds to microglia. HSP60-induced synthesis of neurotoxic nitric oxide by microglia is dependent on TLR4. HSP60 induces extensive axonal loss and neuronal death in CNS cultures from wild-type but not TLR4 or MyD88 loss-of-function mutant mice. This is the first evidence of an endogenous molecular pathway common to many forms of neuronal injury that bidirectionally links CNS inflammation with neurodegeneration.

A membrane-tethered transcription factor defines a branch of the heat stress response in Arabidopsis thaliana

Abstract: In plants, heat stress responses are controlled by heat stress transcription factors that are conserved among all eukaryotes and can be constitutively expressed or induced by heat. Heat-inducible transcription factors that are distinct from the "classical" heat stress transcription factors have also been reported to contribute to heat tolerance. Here, we show that bZIP28, a gene encoding a putative membrane-tethered transcription factor, is up-regulated in response to heat and that a bZIP28 null mutant has a striking heat-sensitive phenotype. The heat-inducible expression of genes that encode BiP2, an endoplasmic reticulum (ER) chaperone, and HSP26.5-P, a small heat shock protein, is attenuated in the bZIP28 null mutant. An estradiol-inducible bZIP28 transgene induces a variety of heat and ER stress-inducible genes. Moreover, heat stress appears to induce the proteolytic release of the predicted transcription factor domain of bZIP28 from the ER membrane, thereby causing its redistribution to the nucleus. These findings indicate that bZIP28 is an essential component of a membrane-tethered transcription factor-based signaling pathway that contributes to heat tolerance.

Arabidopsis stromal 70-kD heat shock proteins are essential for plant development and important for thermotolerance of germinating seeds.

Abstract: The 70-kD heat shock proteins (Hsp70s) have been shown to be important for protein folding, protein translocation, and stress responses in almost all organisms and in almost all subcellular compartments. However, the function of plastid stromal Hsp70s in higher plants is still uncertain. Genomic surveys have revealed that there are two putative stromal Hsp70s in Arabidopsis thaliana, denoted cpHsc70-1 (At4g24280) and cpHsc70-2 (At5g49910). In this study, we show that cpHsc70-1 and cpHsc70-2 could indeed be imported into the chloroplast stroma. Their corresponding T-DNA insertion knockout mutants were isolated and designated as Δcphsc70-1 and Δcphsc70-2. No visible phenotype was observed in the Δcphsc70-2 mutant under normal growth conditions. In contrast, Δcphsc70-1 mutant plants exhibited variegated cotyledons, malformed leaves, growth retardation, and impaired root growth, even though the protein level of cpHsc70-2 was up-regulated in the Δcphsc70-1 mutant. After heat shock treatment of germinating seeds, root growth from Δcphsc70-1 seeds was further impaired, indicating that cpHsc70-1 is important for thermotolerance of germinating seeds. No Δcphsc70-1 Δcphsc70-2 double mutant could be obtained, suggesting that the Δcphsc70 double knockout was lethal. Genotype analyses of F1 seedlings from various crosses indicated that double-knockout mutation was lethal to the female gametes and reduced the transmission efficiency of the male gametes. These results indicate that cpHsc70s are essential for plant development and the two cpHsc70s most likely have redundant but also distinct functions.

Convergence of Molecular, Modeling, and Systems Approaches for an Understanding of the Escherichia coli Heat Shock Response

Abstract: Summary: The heat shock response (HSR) is a homeostatic response that maintains the proper protein-folding environment in the cell. This response is universal, and many of its components are well conserved from bacteria to humans. In this review, we focus on the regulation of one of the most well-characterized HSRs, that of Escherichia coli. We show that even for this simple model organism, we still do not fully understand the central component of heat shock regulation, a chaperone-mediated negative feedback loop. In addition, we review other components that contribute to the regulation of the HSR in E. coli and discuss how these additional components contribute to regulation. Finally, we discuss recent genomic experiments that reveal additional functional aspects of the HSR.

Proteinuria and Hyperglycemia Induce Endoplasmic Reticulum Stress

Abstract: The endoplasmic reticulum (ER) is an important site for protein folding and becomes “stressed” when its capacity to fold proteins is overwhelmed. In response, “unfolded protein response” (UPR) genes are induced, increasing the capacity to fold proteins; if the response is insufficient, then apoptosis ensues. For investigation of whether proteinuria and hyperglycemia induce ER stress in renal epithelial cells, microarray data from biopsies of established diabetic nephropathy (DN) were analyzed. Expression of UPR genes was significantly different in these biopsies than in control kidneys or biopsies of patients with mild DN, suggesting an association between the degree of DN and UPR gene expression. Expression of the transcription factor XBP1 and the ER chaperones HSPA5 and HYOU1 were increased, but the proapoptotic gene DDIT3 was unchanged. These findings were replicated in an independent cohort of patients with established DN by real-time reverse transcriptase–PCR. Immunofluorescence of renal biopsies from patients with DN confirmed the upregulation for HSPA5 and HYOU1 proteins in tubular epithelia. In biopsies of minimal-change disease, the mRNA levels of some ER stress molecules were also induced, but protein expression of HSPA5 and HYOU1 remained significantly lower than that observed in DN. Exposure of renal tubular epithelial cells to albumin and high glucose in vitro enhanced expression of genes involved in ER stress. These observations suggest that in proteinuric diseases, tubular epithelial cells undergo ER stress, which induces an adaptive, protective UPR. Although this may protect the cells from ER stress, persistence of hyperglycemia and proteinuria may eventually lead to apoptosis.

Role of Acetylation and Extracellular Location of Heat Shock Protein 90α in Tumor Cell Invasion

Abstract: Heat shock protein (hsp) 90 is an ATP-dependent molecular chaperone, which maintains the active conformation of client oncoproteins in cancer cells. An isoform, hsp90α, promotes extra-cellular maturation of matrix metalloproteinase (MMP)-2 involved in tumor invasion and metastasis. Knockdown of histone deacetylase (HDAC) 6, which deacetylates lysine residues in hsp90, induces reversible hyper-acetylation and attenuates ATP binding and chaperone function of hsp90. Here, utilizing mass spectrometry, we identified seven lysine residues in hsp90α that are hyper-acetylated, following treatment of eukaryotic cells with a pan-HDAC inhibitor that also inhibits HDAC6. Depending on the specific lysine residue in the middle domain involved, while acetylation affects ATP, co-chaperone and client protein binding to hsp90α, acetylation of all seven lysines increased the binding of hsp90α to 17-allyl-amino-demethoxy geldanamycin (17-AAG). Notably, following treatment with the pan-HDAC inhibitor panobinostat (LBH589), the extra-cellular hsp90α was hyper-acetylated and it bound to MMP-2, which was associated with increased in vitro tumor cell invasiveness. Treatment with anti-acetylated hsp90α antibody inhibited in vitro invasion by tumor cells. Thus, reversible hyper-acetylation modulates the intra- and extra-cellular chaperone function of hsp90, and targeting extra-cellular hyper-acetylated hsp90α may undermine tumor invasion and metastasis.

Anti-inflammatory effects of the 70 kDa heat shock protein in experimental stroke

Abstract: The 70-kDa heat shock protein (Hsp70) is involved in protecting the brain from a variety of insults including stroke. Although the mechanism has been largely considered to be because of its chaperone functions, recent work indicates that Hsp70 also modulates inflammatory responses. To explore how and whether Hsp70 regulate immune responses in brain ischemia, mice overexpressing Hsp70 (Hsp Tg) were subjected to 2 h middle cerebral artery occlusion, followed by 24 h reperfusion. Parallel experiments were performed using a brain inflammation model. Hsp Tg microglia cocultured with astrocytes were used to evaluate the direct effects of Hsp70 on cytotoxicity of mcrigolia. Compared with wild-type (Wt) littermates, Hsp Tg mice showed decreased infarct size and improved neurological deficits. The number of activated microglia/macrophages were also reduced in ischemic brains of Hsp Tg mice. Similar observations were made in a model of brain inflammation that does not result in brain cell death. Overexpression of Hsp70 in microglia completely prevented microglia-induced cytotoxicity to astrocytes. Activation of the inflammatory transcription factor, nuclear factor-j B( NF-jB) was inhibited significantly in Hsp Tg mice and microglia. This was associated with decreased phosphorylation of NF-jB inhibitor protein, IjBa, and decreased expression of several NFjB-regulated genes. Co-immunoprecipitation studies revealed an interaction of Hsp70 with NF-jB and IjBa, but not with IjB kinase, IKKc, suggesting that Hsp70 binds to the NF-jB:IjB complex preventing IjB phosphorylation by IKK. The findings of the present work establish an anti-inflammatory role for Hsp70 in the context of brain ischemia as a novel mechanism of protection.

The calmodulin-binding protein kinase 3 is part of heat-shock signal transduction in Arabidopsis thaliana

Abstract: SUMMARY: Based on our previous findings, we proposed a pathway for the participation of Ca(2+)/calmodulin (CaM) in heat-shock (HS) signal transduction. The specific mechanism by which CaM regulates activation of heat-shock transcription factors (HSFs) is not known. CaM-binding protein kinases (CBK) are the most poorly understood of the CaM target proteins in plants. In this study, using a yeast two-hybrid assay, we found that AtCBK3 interacts with AtHSFA1a. Fluorescence resonance energy transfer was used to confirm the interaction between AtCBK3-YFP and AtHSFA1a-CFP. Furthermore, we demonstrate that purified recombinant AtCBK3 phosphorylated recombinant AtHSFA1a in vitro. We also describe the results of both downregulation of AtCBK3 expression and ectopic overexpression in Arabidopsis thaliana. The T-DNA insertion AtCBK3 knockout lines had impaired basal thermotolerance, which could be complemented by transformation of plants with the native gene. Overexpression of AtCBK3 resulted in plants with increased basal thermotolerance. Results from real-time quantitative PCR and protein gel-blot analyses suggest that AtCBK3 regulates transcription of heat-shock protein (HSP) genes and synthesis of HSPs. The binding activity of HSF to the heat-shock element (HSE), the mRNA level of HSP genes and synthesis of HSPs were upregulated in AtCBK3-overexpressing lines after HS, but downregulated in AtCBK3 null lines. These results indicate that AtCBK3 controls the binding activity of HSFs to HSEs by phosphorylation of AtHSFA1a, and is an important component of the HS signal transduction pathway.

Interaction between SGT1 and Cytosolic/Nuclear HSC70 Chaperones Regulates Arabidopsis Immune Responses

Abstract: The conserved eukaryotic protein SGT1 (for Suppressor of G2 allele of skp1) has characteristics of an HSP90 (for heat shock protein 90 kD) cochaperone and in plants regulates hormone responses and Resistance gene–triggered immunity. We affinity-purified SGT1-interacting proteins from Arabidopsis thaliana leaf extracts and identified by mass spectrometry cytosolic heat shock cognate 70 (HSC70) chaperones as the major stable SGT1 interactors. Arabidopsis SGT1a and SGT1b proteins associate with HSC70 in vivo and distribute with HSC70 in the cytosol and nucleus. An intact C-terminal SGT1-specific (SGS) domain that is required for all known SGT1b functions in immunity and development is needed for HSC70 interaction and for the nuclear accumulation of SGT1b. Interaction assays of transiently expressed proteins or their domains in Nicotiana benthamiana point to a role of SGT1 as a HSC70 cofactor. Expression of two HSC70 isoforms is upregulated by pathogen challenge, and while loss of function of individual cytosolic HSC70 genes has no defense phenotype, HSC70-1 overexpression disables resistance to virulent and avirulent pathogens. Moreover, mutations in SGT1b lead to a similar degree of heat shock tolerance as deregulation of HSC70-1. We conclude that an HSC70-SGT1 chaperone complex is important for multiple plant environmental responses and that the evolutionarily conserved SGS domain of SGT1 is a key determinant of the HSC70–SGT1 association.