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The molecular chaperone Hsp90 is an essential and highly abundant central node in the interactome of eukaryotic cells. Many of its large number of client proteins are relevant to cancer. A hallmark of Hsp90-dependent proteins is that their accumulation is compromised by Hsp90 inhibitors. Combined with the anecdotal observation that cancer cells may be more sensitive to Hsp90 inhibitors, this has led to clinical trials aiming to develop Hsp90 inhibitors as anti-cancer agents. However, the sensitivity to Hsp90 inhibitors has not been studied in rigorously matched normal versus cancer cells, and despite the discovery of important regulators of Hsp90 activity and inhibitor sensitivity, it has remained unclear, why cancer cells might be more sensitive. To revisit this issue more systematically, we have generated an isogenic pair of normal and oncogenically transformed NIH-3T3 cell lines. Our proteomic analysis of the impact of three chemically different Hsp90 inhibitors shows that these affect a substantial portion of the oncogenic program and that indeed, transformed cells are hypersensitive. Targeting the oncogenic signaling pathway reverses the hypersensitivity, and so do inhibitors of DNA replication, cell growth, translation and energy metabolism. Conversely, stimulating normal cells with growth factors or challenging their proteostasis by overexpressing an aggregation-prone sensitizes them to Hsp90 inhibitors. Thus, the differential sensitivity to Hsp90 inhibitors may not stem from any particular intrinsic difference between normal and cancer cells, but rather from a shift in the balance between cellular quiescence and activity. [ABSTRACT FROM AUTHOR]

The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters. This requirement has been very conveniently met with the popular dual luciferase assay. Its disadvantages are the requirement for cell lysis, the inability to analyze the same cells repeatedly, and the cost, at least in its most commonly used commercial format. Here we describe a novel dual reporter assay with the naturally secreted luciferase from Gaussia princeps as the main reporter protein and a secreted version of the red fluorescent protein mCherry as internal standard. After first measuring mCherry fluorescence in the medium, an enzyme buffer with coelenterazine as substrate is added to the same sample to trigger a glow-type luminescence of the luciferase. The simple and cheap assay can easily be adapted to a variety of experimental situations. As a case in point, we have developed a panel of Gaussia luciferase reporter genes for transcriptional activation assays with estrogen and glucocorticoid response elements, and with response elements for fusion proteins with the Gal4 DNA binding domain for use in mammalian cells. Our secreted dual reporter assay should be an attractive alternative to the currently available commercial kits. [ABSTRACT FROM AUTHOR]

Understanding the functions of proteins requires information about their protein-protein interactions (PPI). The collective effort of the scientific community generates far more data on any given protein than individual experimental approaches. The latter are often too limited to reveal an interactome comprehensively. We developed a workflow for parallel mining of all major PPI databases, containing data from several model organisms, and to integrate data from the literature for a protein of interest. We applied this novel approach to build the PPI network of the human Hsp90 molecular chaperone machine (Hsp90Int) for which previous efforts have yielded limited and poorly overlapping sets of interactors. We demonstrate the power of the Hsp90Int database as a discovery tool by validating the prediction that the Hsp90 cochaperone Aha1 is involved in nucleocytoplasmic transport. Thus, we both describe how to build a custom database and introduce a powerful new resource for the scientific community. [ABSTRACT FROM AUTHOR]

Background: To make sense out of gene expression profiles, such analyses must be pushed beyond the mere listing of affected genes. For example, if a group of genes persistently display similar changes in expression levels under particular experimental conditions, and the proteins encoded by these genes interact and function in the same cellular compartments, this could be taken as very strong indicators for coregulated protein complexes. One of the key requirements is having appropriate tools to detect such regulatory patterns. Results: We have analyzed the global adaptations in gene expression patterns in the budding yeast when the Hsp90 molecular chaperone complex is perturbed either pharmacologically or genetically. We integrated these results with publicly accessible expression, protein-protein interaction and intracellular localization data. But most importantly, all experimental conditions were simultaneously and dynamically visualized with an animation. This critically facilitated the detection of patterns of gene expression changes that suggested underlying regulatory networks that a standard analysis by pairwise comparison and clustering could not have revealed. Conclusions: The results of the animation-assisted detection of changes in gene regulatory patterns make predictions about the potential roles of Hsp90 and its cochaperone p23 in regulating whole sets of genes. The simultaneous dynamic visualization of microarray experiments, represented in networks built by integrating one's own experimental with publicly accessible data, represents a powerful discovery tool that allows the generation of new interpretations and hypotheses. [ABSTRACT FROM AUTHOR]

Abstract: The abundant molecular chaperone Hsp90 functions in association with co-chaperones including p23 to promote the folding and maturation of a subset of cytosolic proteins. “Fluorescence recovery after photobleaching” (FRAP) experiments showed that the dynamics of p23 in live cells is dictated by Hsp90. Since Hsp90 is present in large excess over p23, the mobility of Hsp90 could conceivably be quite different. To facilitate the analysis and to allow a direct comparison with p23, we developed a 2-color FRAP technique. Two test proteins are expressed as fusion proteins with the two spectrally separable fluorescent proteins mCherry and enhanced green fluorescent protein (EGFP). The 2-color FRAP technique is powerful for the concomitant recording of two proteins located in the same area of a cell, two components of the same protein complex, or mutant and wild-type versions of the same protein under identical experimental conditions. 2-color FRAP of Hsp90 and p23 is virtually indistinguishable, consistent with the notion that they are both engaged in a multitude of large protein complexes. However, when Hsp90–p23 complexes are disrupted by the Hsp90 inhibitor geldanamycin, p23 moves by free diffusion while Hsp90 maintains its low mobility because it remains bound in remodeled multicomponent complexes. [Copyright &y& Elsevier]

Abstract: p23 is a component of the Hsp90 molecular chaperone machine. It binds and stabilizes the ATP-bound dimeric form of Hsp90. Since Hsp90 binds protein substrates in the ATP conformation, p23 has been proposed to stabilize Hsp90–substrate complexes. In addition, p23 can also function as a molecular chaperone by itself and even possesses an unrelated enzymatic activity. Whether it fulfills the latter functions in cells while bound to Hsp90 remains unknown and is difficult to extrapolate from cell-free biochemical experiments. Using the “fluorescence recovery after photobleaching” (FRAP) technology, I have examined the dynamics of human p23, expressed as a fusion protein with the green fluorescent protein (GFP), in living human HeLa cells. GFP-p23 is distributed throughout the cell, and its mobility is identical in the cytoplasm and in the nucleus. When the Hsp90 interaction is disrupted either with the Hsp90 inhibitor geldanamycin or by introduction of point mutations into p23, the mobility of p23 is greatly accelerated. Under these conditions, its intracellular movement may be diffusion-controlled. In contrast, when wild-type p23 is able to bind Hsp90, a more complex FRAP behavior is observed, suggesting that it is quantitatively bound in Hsp90 complexes undergoing a multitude of other interactions. [Copyright &y& Elsevier]

Cell death and survival play a key role in the immune system as well as during development. The control mechanisms that balance cell survival against cell death are not well understood. Here we report a novel strategy used by a single protein to regulate chronologically cell survival and death. The interferon-induced protein kinase PKR acts as a molecular clock by using catalysis-dependent and -independent activities to temporally induce cell survival prior to cell death. We show that the proapoptotic protein PKR surprisingly activates a survival pathway, which is mediated by NF-κB to delay apoptosis. Cell death is then induced by PKR through the phosphorylation of eIF-2α. This unique temporal control might serve as a paradigm for other kinases whose catalytic activity is not required for all of their functions. [ABSTRACT FROM AUTHOR]

The functions of molecular chaperones have been extensively investigated biochemically in vitro and genetically in bacteria and yeast. We have embarked on a functional genomic analysis of the Hsp90 chaperone machine in the mouse by disrupting the p23 gene using a gene trap approach. p23 is an Hsp90 cochaperone that is thought to stabilize Hsp90-substrate complexes and, independently, to act as the cytosolic prostaglandin E2 synthase. Gene deletions in budding and fission yeasts and knock-down experiments with the worm have not revealed any clear in vivo requirements for p23. We find that p23 is not essential for overall prenatal development and morphogenesis of the mouse, which parallels the observation that it is dispensable for proliferation in yeast. In contrast, p23 is absolutely necessary for perinatal survival. Apart from an incompletely formed skin barrier, the lungs of p23 null embryos display underdeveloped airspaces and substantially reduced expression of surfactant genes. Correlating with the known function of glucocorticoids in promoting lung maturation and the role of p23 in the assembly of a hormone-responsive glucocorticoid receptor-Hsp90 complex, p23 null fibroblast cells have a defective glucocorticoid response. Thus, p23 contributes a nonredundant, temporally restricted, and tissue-specific function during mouse development. [ABSTRACT FROM AUTHOR]

Abstract: Developing novel drugs against the unicellular parasite Plasmodium is complicated by the paucity of simple screening systems. Heat-shock proteins are an essential class of proteins for the parasite''s cyclical life style between different cellular milieus and temperatures. The molecular chaperone Hsp90 assists a large variety of proteins, but its supporting functions for many proteins that are important for cancer have made it into a well-studied drug target. With a better understanding of the differences between Hsp90 of the malarial parasite and Hsp90 of its human host, new therapeutic options might become available. We have generated a set of isogenic strains of the budding yeast Saccharomyces cerevisiae where the essential yeast Hsp90 proteins have been replaced with either of the two human cytosolic isoforms Hsp90α or Hsp90β, or with Hsp90 from Plasmodium falciparum (Pf). All strains express large amounts of the Flag-tagged Hsp90 proteins and are viable. Even though the strain with Pf Hsp90 grows more poorly, it provides a tool to reconstitute additional aspects of the parasite Hsp90 complex and its interactions with substrates in yeast as a living test tube. Upon exposure of the set of Hsp90 test strains to the two Hsp90 inhibitors radicicol (Rd) and geldanamycin (GA), we found that the strain with Pf Hsp90 is relatively more sensitive to GA than to Rd compared to the strains with human Hsp90''s. This indicates that this set of yeast strains could be used to screen for new Pf Hsp90 inhibitors with a wider therapeutic window. [Copyright &y& Elsevier]