The cytosolic/nuclear HSC70 and HSP90 molecular chaperones are important for stomatal closure and modulate abscisic acid-dependent physiological responses in Arabidopsis

Cytosolic/nuclear molecular chaperones of the heat shock protein families HSP90 and HSC70 are conserved and essential proteins in eukaryotes. These proteins have essentially been implicated in the innate immunity and abiotic stress tolerance in higher plants. Here, we demonstrate that both chaperones are recruited in Arabidopsis (Arabidopsis thaliana) for stomatal closure induced by several environmental signals. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are compromised in the dark-, CO(2)-, flagellin 22 peptide-, and abscisic acid (ABA)-induced stomatal closure. HSC70-1 and HSP90 proteins are needed to establish basal expression levels of several ABA-responsive genes, suggesting that these chaperones might also be involved in ABA signaling events. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are hypersensitive to ABA in seed germination assays, suggesting that several chaperone complexes with distinct substrates might tune tissue-specific responses to ABA and the other biotic and abiotic stimuli studied. This study demonstrates that the HSC70/HSP90 machinery is important for stomatal closure and serves essential functions in plants to integrate signals from their biotic and abiotic environments.     

HSP70 inhibition by the small-molecule 2-phenylethynesulfonamide impairs protein clearance pathways in tumor cells

The evolutionarily conserved stress-inducible HSP70 molecular chaperone plays a central role in maintaining protein quality control in response to various forms of stress. Constitutively elevated HSP70 expression is a characteristic of many tumor cells and contributes to their survival. We recently identified the small-molecule 2-phenylethyenesulfonamide (PES) as a novel HSP70 inhibitor. Here, we present evidence that PES-mediated inhibition of HSP70 family proteins in tumor cells results in an impairment of the two major protein degradation systems, namely, the autophagy-lysosome system and the proteasome pathway. HSP70 family proteins work closely with the HSP90 molecular chaperone to maintain the stability and activities of their many client proteins, and PES causes a disruption in the HSP70/HSP90 chaperone system. As a consequence, many cellular proteins, including known HSP70/HSP90 substrates, accumulate in detergent-insoluble cell fractions, indicative of aggregation and functional inactivation. Overall, PES simultaneously disrupts several cancer critical survival pathways, supporting the idea of targeting HSP70 as a potential approach for cancer therapeutics.     

The HSP90 chaperone complex, an emerging force in plant development and phenotypic plasticity

The essential cellular functions of the molecular chaperone HSP90 have been intensively investigated in fungal and mammalian model systems. Several recent publications have highlighted the importance of this chaperone complex in plant development and responsiveness to external stimuli. In particular, HSP90 is crucial for R-protein-mediated defense against pathogens. Other facets of HSP90 function in plants include its involvement in phenotypic plasticity, developmental stability, and buffering of genetic variation. Plants have emerged as powerful tools that complement other model systems in attempts to extend our knowledge of the myriad impacts of protein folding and chaperone function.     

热激蛋白90在植物发育和疾病抗性中的作用

相对分子质量90000的热激蛋白（heatshock protein,HSP90）是真核细胞必需的分子伴侣。拟南芥中HSP90有7个成员,其中AtHSP90-1、AtHSP90-2、AtHSP90-3和AtHSP90-4组成细胞质亚族;AtHSP90-5、AtHSP90-6、AtHSP90-7分别位于叶绿体、线粒体和内质网。HSP90分子伴侣复合物在植物发育和对外部刺激应答中非常重要,尤其是在抗性（resistance R）蛋白介导的抵抗病毒侵入的过程中起重要作用。     

A rhizosphere fungus enhances Arabidopsis thermotolerance through production of an HSP90 inhibitor

The molecular chaperone HEAT SHOCK PROTEIN90 (HSP90) is essential for the maturation of key regulatory proteins in eukaryotes and for the response to temperature stress. Earlier, we have reported that fungi living in association with plants of the Sonoran desert produce small molecule inhibitors of mammalian HSP90. Here, we address whether elaboration of the HSP90 inhibitor monocillin I (MON) by the rhizosphere fungus Paraphaeosphaeria quadriseptata affects plant HSP90 and plant environmental responsiveness. We demonstrate that MON binds Arabidopsis (Arabidopsis thaliana) HSP90 and can inhibit the function of HSP90 in lysates of wheat (Triticum aestivum) germ. MON treatment of Arabidopsis seedlings induced HSP101 and HSP70, conserved components of the stress response. Application of MON, or growth in the presence of MON, allowed Arabidopsis wild type but not AtHSP101 knockout mutant seedlings to survive otherwise lethal temperature stress. Finally, cocultivation of P. quadriseptata with Arabidopsis enhanced plant heat stress tolerance. These data demonstrate that HSP90-inhibitory compounds produced by fungi can influence plant growth and responses to the environment.     

Molecular mechanisms of canalization: Hsp90 and beyond

The Hsp90 chaperone machine facilitates the maturation of a diverse set of ‘client’ proteins. Many of these Hsp90 clients are essential nodes in signal transduction pathways and regulatory circuits, accounting for the important role Hsp90 plays in organismal development and responses to the environment. Recent findings suggest a broader impact of the chaperone on phenotype: fully functional Hsp90 canalizes wild-type phenotypes by suppressing underlying genetic and epigenetic variation. This variation can be expressed upon challenging the Hsp90 machinery by environmental stress, genetic or pharmaceutical targeting of Hsp90. The existence of Hsp90-buffered genetic and epigenetic variation together with plausible release mechanisms has wide-ranging implication for phenotype and possibly evolutionary processes. Here, we discuss the role of Hsp90 in canalization and organismal plasticity, and highlight important questions for future experimental inquiry.     

Structural and functional analysis of SGT1 reveals that its interaction with HSP90 is required for the accumulation of Rx, an R protein involved in plant immunity

SGT1 (for suppressor of G2 allele of skp1) and RAR1 (for required for Mla12 resistance) are highly conserved eukaryotic proteins that interact with the molecular chaperone HSP90 (for heat shock protein90). In plants, SGT1, RAR1, and HSP90 are essential for disease resistance triggered by a number of resistance (R) proteins. Here, we present structural and functional characterization of plant SGT1 proteins. Random mutagenesis of Arabidopsis thaliana SGT1b revealed that its CS (for CHORD-SGT1) and SGS (for SGT1 specific) domains are essential for disease resistance. NMR-based interaction surface mapping and mutational analyses of the CS domain showed that the CHORD II domain of RAR1 and the N-terminal domain of HSP90 interact with opposite sides of the CS domain. Functional analysis of the CS mutations indicated that the interaction between SGT1 and HSP90 is required for the accumulation of Rx, a potato (Solanum tuberosum) R protein. Biochemical reconstitution experiments suggest that RAR1 may function to enhance the SGT1-HSP90 interaction by promoting ternary complex formation.     

Death by chaperone: HSP90, HSP70 or both?

HSP70 family members are highly conserved proteins that function as molecular chaperones. Their principle role is to aid protein folding and promote the correct cellular localisations of their respective substrates. The function of HSP70 isoforms can be exhibited independently or with the HSP90 chaperone system in which HSP70 is important for substrate recruitment. In addition to their chaperone role, HSP70 isoforms promote cell survival by inhibiting apoptosis at multiple points within both the intrinsic and extrinsic cell death pathways. Consistent with this cytoprotective function, increased expression of HSP70 isoforms is commonly associated with the malignant phenotype. We recently reported that dual silencing of the major constitutive (HSC70) and inducible (HSP72) isoforms of HSP70 in cancer cells could phenocopy the effects of a pharmacologic HSP90 inhibitor to induce proteasome-dependent degradation of HSP90 client proteins CRAF, CDK4 and ERBB2. This was accompanied by a G1 cell cycle arrest and extensive apoptosis which was not seen in non-tumorigenic human cell lines. Here we discuss the possible implications of our research for the development of HSP70 family modulators which offer not only the possibility of inhibiting HSP70 activity but also the simultaneous inhibition of HSP90, resulting in extensive tumour-specific apoptosis.     

Inhibitors of the heat shock protein 90 activity: a novel class of tumor radiosensitizers

The 90 kDa heat shock protein (HSP90) is one of major chaperones of eukaryotes which catalyzes maturation and activation of its client proteins. Among the identified client proteins there are oncogene products, hormone or growth factor receptors and key components of signaling pathways responsible for the malignant growth of tumors or their resistance to chemotherapy and radiotherapy. In the case of inhibition of the HSP90 chaperone function, such proteins are inactivated and degraded soon that leads to simultaneous blocking several pathways essential for proliferation and survival of malignant cells; therefore, pharmacological inhibitors of the HSP90 chaperone activity could be used in anticancer therapy. At present, several HSP90 inhibitors are in preclinical testing or I-III Phase clinical trials as mono-agents or in combinations with other anticancer drugs or radiation. In the present review, all the data are summarized which characterize HSP90 inhibitors as effective radiosensitizers of tumor cells. Molecular mechanisms and selectivity of the radiosensitizing action of HSP90 inhibitors are here discussed as well as a possibility of their application to improve the outcome of radiotherapy.     

Hsp90 recognizes a common surface on client kinases

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.     

The role of HSP90 molecular chaperones in hepatocellular carcinoma

Misfolded proteins have enhanced formation of toxic oligomers and nonfunctional protein copies lead to recruiting wild-type protein types. Heat shock protein 90 (HSP90) is a molecular chaperone generated by cells that are involved in many cellular functions through regulation of folding and/or localization of large multi-protein complexes as well as client proteins. HSP90 can regulate a number of different cellular processes including cell proliferation, motility, angiogenesis, signal transduction, and adaptation to stress. HSP90 makes the mutated oncoproteins able to avoid misfolding and degradation and permits the malignant transformation. As a result, HSP90 is an important factor in several signaling pathways associated with tumorigenicity, therapy resistance, and inhibiting apoptosis. Clinically, the upregulation of HSP90 expression in hepatocellular carcinoma (HCC) is linked with advanced stages and inappropriate survival in cases suffering from this kind of cancer. The present review comprehensively assesses HSP90 functions and its possible usefulness as a potential diagnostic biomarker and therapeutic option for HCC.     

The role of Hsp90 in protein complex assembly

Hsp90 is a ubiquitous and essential molecular chaperone that plays central roles in many signaling and other cellular pathways. The in vivo and in vitro activity of Hsp90 depends on its association with a wide variety of cochaperones and cofactors, which form large multi-protein complexes involved in folding client proteins. Based on our proteomic work mapping the molecular chaperone interaction networks in yeast, especially that of Hsp90, as well as, on experiments and results presented in the published literature, one major role of Hsp90 appears to be the promotion and maintenance of proper assembly of protein complexes. To highlight this role of Hsp90, the effect of the chaperone on the assembly of the following seven complexes is discussed in this review: snoRNP, RNA polymerase II, phosphatidylinositol-3 kinase-related protein kinase (PIKK), telomere complex, kinetochore, RNA induced silencing complexes (RISC), and 26S proteasome. For some complexes, it is observed that Hsp90 mediates complex assembly by stabilizing an unstable protein subunit and facilitating its incorporation into the complex; for other complexes, Hsp90 promotes change in the composition of that complex. In all cases, Hsp90 does not appear to be part of the final assembled complex. This article is part of a Special Issue entitled:Heat Shock Protein 90 (HSP90).     

The HSP90-SGT1-RAR1 molecular chaperone complex: A core modulator in plant immunity

The HSP90 (heat shock protein 90), SGT1 (suppressor of G-two allele ofSkp1), and RAR1 (required forMla12 resistance) proteins in plants form a molecular chaperone complex which is involved in diverse biological signaling including development and disease resistance. The three components of this complex interact via specific protein binding motifs and recruit client proteins to initiate a specific signaling cascade in response to cellular or environmental cues. Although the functions of this chaperone complex during development/growth have not been well characterized, the HSP90 chaperone and SGT1 and RAR1 co-chaperones have been demonstrated to be essential signaling components of plant immune responses. These three proteins also play important roles in activation of the mammalian Nod genes, which possess a structurally conserved plant resistance (R) protein motif, NB-LRR (nucleotide binding site-leucine rich repeat). In this review, we summarize the structures and functions of these molecular chaperones, and discuss their putative modes of action in plant immune responses.     

Stress,genomes, and evolution

Evolutionary change, whether in populations of organisms or malignant tumor cells, is contingent on the availability of inherited variation for natural selection to act upon. It is becoming clear that the Hsp90 chaperone, which normally functions to buffer client proteins against the effects of genetic variation, plays a central role in this process. Severe environmental stress can overwhelm the chaperone's buffering capacity, causing previously cryptic genetic variation to be expressed. Recent studies now indicate that in addition to exposing existing variation, Hsp90 can induce novel epigenetic and genetic changes. We discuss key findings that suggest a rich set of pathways by which Hsp90 can mediate the influences of the environment on the genome.     

Generation of a novel anti-geldanamycin antibody

Geldanamycin (GA) and herbimycin A are benzoquinone ansamycins (BAs) that inhibit the molecular chaperone HSP90. The central role of HSP90 in maintaining the conformation, stability, and function of key oncogenic proteins involved in signal transduction pathways renders BAs attractive candidates for clinical development. Two GA derivatives, 17-allylamino-17-demethoxygeldanamycin and 17-demethoxy-17-N,N-dimethylaminoethylamino-geldanamycin are currently evaluated in clinical trials. The present study demonstrates generation of a polyclonal antibody elicited against GA that was conjugated to keyhole limpet hemocyanin via its 17 position. The anti-GA antibody recognizes GA as well as other BAs, suggesting its possible application for monitoring plasma levels of GA derivatives. The specificity of the antibody towards BAs is demonstrated by its inability to recognize radicicol, an HSP90 inhibitor not related to BAs. This antibody thus presents a novel research tool as well as a possible alternative approach for monitoring drug levels in patients.     

Beyond Arabidopsis. Translational biology meets evolutionary developmental biology

Developmental processes shape plant morphologies, which constitute important adaptive traits selected for during evolution. Identifying the genes that act in developmental pathways and determining how they are modified during evolution is the focus of the field of evolutionary developmental biology, or evo-devo. Knowledge of genetic pathways in the plant model Arabidopsis serves as the starting point for investigating how the toolkit of developmental pathways has been used and reused to form different plant body plans. One productive approach is to identify genes in other species that are orthologous to genes known to control developmental pathways in Arabidopsis and then determine what changes have occurred in the protein coding sequence or in the gene's expression to produce an altered morphology. A second approach relies on natural variation among wild populations or crop plants. Natural variation can be exploited to identify quantitative trait loci that underlie important developmental traits and, thus, define those genes that are responsible for adaptive changes. The possibility of applying comparative genomics approaches to Arabidopsis and related species promises profound new insights into the interplay of evolution and development.