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Fine tuning of the protein folding environment in subcellular organelles, such as mitochondria, is important for adaptive homeostasis and may participate in human diseases, but the regulators of this process are still largely elusive. Here, we have shown that selective targeting of heat shock protein-90 (Hsp90) chaperones in mitochondria of human tumor cells triggered compensatory autophagy and an organelle unfolded protein response (UPR) centered on upregulation of CCAAT enhancer binding protein (C/EBP) transcription factors. In turn, this transcriptional UPR repressed NF-κB-dependent gene expression, enhanced tumor cell apoptosis initiated by death receptor ligation, and inhibited intracranial glioblastoma growth in mice without detectable toxicity. These data reveal what we believe to be a novel role of Hsp90 chaperones in the regulation of the protein-folding environment in mitochondria of tumor cells. Disabling this general adaptive pathway could potentially be used in treatment of genetically heterogeneous human tumors., Introduction Cells use sensor pathways compartmentalized in subcellular organelles to recognize stress conditions (1), including aberrant protein folding (2), and in response activate gene expression programs aimed at maintaining cell [...]

Activating transcription factor-5 (ATF5) is highly expressed in malignant glioma and has a key role in promoting cell survival. Here we perform a genome-wide RNAi screen to identify transcriptional regulators of ATF5. Our results reveal an essential survival pathway in malignant glioma, whereby activation of a RAS-mitogen-activated protein kinase or phosphoinositide- 3-kinase signaling cascade leads to induction of the transcription factor cAMP response element-binding protein-3-like-2 (CREB3L2), which directly activates ATF5 expression. ATF5, in turn, promotes survival by stimulating transcription of myeloid cell leukemia sequence-1 (MCL1), an antiapoptotic B cell leukemia-2 family member. Analysis of human malignant glioma samples indicates that ATF5 expression inversely correlates with disease prognosis. The RAF kinase inhibitor sorafenib suppresses ATF5 expression in glioma stem cells and inhibits malignant glioma growth in cell culture and mouse models. Our results demonstrate that ATF5 is essential in malignant glioma genesis and reveal that the ATF5-mediated survival pathway described here provides potential therapeutic targets for treatment of malignant glioma., A hallmark of cancer is uncontrolled cellular proliferation, which can arise as a result of overexpression of genes promoting cell survival or downregulation of genes inducing cell death (1). Genes [...]

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5][P.sub.3]). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)[P.sub.3] in higher eukaryotes. Indeed, PI(3,4,5)[P.sub.3] has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, Pl(3,4,5)[P.sub.3] became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)[P.sub.3] levels. By examining various mutants, we show that synthesis of PI(3,4,5)[P.sub.3] in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)[P.sub.3] synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.

Research has been conducted on nitric oxide and its S-nitrosylation effects on cysteine residues. The hypothesis that nitric oxide controls p53 via S-nitrosylation and activation of p53 negative inhibitor Hdm2 has been tested and the results demonstrate that cysteine residue mutation from a cysteine to an alanine eliminates Hdn2 sensitivity to nitric oxide inactivation.

The nerve growth factor (NGF) receptor is a complex of two proteins, gp75 and the tyrosine kinase TrkA. Using fluorescence recovery after photobleaching, we have studied the diffusion properties of the TrkA receptor. For PC 12 cells that express both gp75 and TrkA, TrkA was relatively immobile with only 28 [+ or -] 1% of receptor molecules free to diffuse with D = (3.64 [+ or -] 0.23) x [10.sup.-9] [cm.sup.2]/s. Addition of NGF decreased the mobile fraction to 21 [+ or -] 1% with D = (4.11 [+ or -] 0.18) x [10.sup.-9] [cm.sup.2]/s. Using the Sf9 baculovirus expression system, we were able to study TrkA in the absence and presence of gp75. On Sf9 cells, TrkA showed a mobile fraction of 46 [+ or -] 2% with D = (2.64 [+ or -] 0.21) x [10.sup.-9] [cm.sup.2]/s in the absence of gp75 and 43 [+ or -] 2% with D = (2.31 [+ or -] 0.25) x [10.sup.-9] [cm.sup.2]/s in its presence. Thus, gp75 did not alter TrkA mobility. Addition of NGF to the medium approximately halved the mobile fraction for TrkA in both the absence and presence of gp75. However, using a kinase-deficient mutant of TrkA, we found that ligand-induced immobilization requires an active kinase in the absence of gp75 but not in its presence. In addition, using point mutations at specific TrkA autophosphorylation sites, we determined that mobility is controlled by multiple phosphorylation sites, but the SHC binding site at Y490 may be particularly important for ligand-induced immobilization of TrkA. Therefore, two mechanisms lead to NGF- induced immobilization of TrkA - the first resulting from autophosphorylation of TrkA and the second occurring through TrkA's association with gp75.

The direct spectroscopic evidence of a conformational change observed in phosphatase and tensin homologue deleted on chromosome 10 (PTEN) bound to PI(4,5)[P.sub.2]-bearing vesicles with increased [alpha]-helicity is discussed. The results demonstrate that PTEN binds to membranes through multiple sites, but only binding of PI(4,5)[P.sub.2] to the N-terminal domain triggers a conformational change with increased [alpha]-helicity.