N-CoR (nuclear hormone receptor corepressor) was identified originally as a corepressor that binds to, and mediates transcriptional repression by, nuclear hormone receptors (Hö rlein et al. 1995). Thyroid-hormone and retinoic-acid receptors (TR and RAR) of the nuclear hormone receptor family actively repress the transcription of target genes in the absence of ligand (Chambon 1994;Mangelsdorf et al. 1995). Transcriptional repression is mediated by a conserved region in the aminoterminal part of the ligand-binding domain of TR (Baniahmad et al. 1995). N-CoR binds to the ligand-binding domain, termed the Co-R box, and, thereby, mediates transcriptional repression (Hö rlein et al. 1995). N-CoR is a large protein with a molecular mass of 270,000 (Mr 270K), and contains three repressor domains in its amino-terminal region (Hö rlein et al. 1995). Another corepressor, SMRT, which also binds to the Co-R box, shows striking homology to N-CoR (Chen and Evans 1995). N-CoR also forms a complex with mammalian Sin3 orthologs (mSin3A and mSin3B), which bind to another repressor, Mad (Alland et al. 1997;Hassing et al. 1997;Heinzel et al. 1997;Laherty et al. 1997;Nagy et al. 1997). The basic helix-loop-helix (bHLH) proteins of the Mad family act as transcriptional repressors after heterodimerization with Max (Ayer et al. 1993). N-CoR is required for Mad-induced transcriptional repression. The same target sequence of Mad/Max, the so-called E-box, is also recognized by a heterodimer of Myc/Max that activates transcription. It is believed that transcriptional activation of a group of target genes by Myc/Max enhances cellular proliferation or transformation, whereas transcriptional repression of the same target genes by Mad/Max leads to suppression of proliferation or induction of terminal differentiation in a wide range of cell types Chin et al. 1995;Roussel et al. 1996)

Mortalin, also known as mthsp70/GRP75/PBP74, interacts with the tumor suppressor protein p53 and inactivates its transcriptional activation and apoptotic functions. Here, we examined the level of mortalin expression in a large variety of tumor tissues, tumor-derived and in vitro immortalized human cells. It was elevated in many human tumors, and in all of the tumor-derived and in vitro immortalized cells. In human embryonic fibroblasts immortalized with an expression plasmid for hTERT, the telomerase catalytic subunit, with or without human papillomavirus E6 and E7 genes, we found that subclones with spontaneously increased mortalin expression levels became anchorage-independent and acquired the ability to form tumors in nude mice. Furthermore, overexpression of mortalin was sufficient to increase the malignancy of breast carcinoma cells. The study demonstrates that upregulation of mortalin contributes significantly to tumorigenesis, and thus is a good candidate target for cancer therapy. ' 2006 Wiley-Liss, Inc.

Functional impairment of mitochondria and proteasomes and increased oxidative damage comprise the main pathological phenotypes of Parkinson disease (PD). Using an unbiased quantitative proteomic approach, we compared nigral mitochondrial proteins of PD patients with those from age-matched controls. 119 of 842 identified proteins displayed significant differences in their relative abundance (increase/decrease) between the two groups. We confirmed that one of these, mortalin (mthsp70/GRP75, a mitochondrial stress protein), is substantially decreased in PD brains as well as in a cellular model of PD. In addition, nine candidate mortalin-binding partners were identified as potential mediators of PD pathology. Parkinson disease (PD)1 is characterized by preferential dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta (SNpc) with subsequent DA loss in the nigrostriatal pathway and the presence of Lewy bodies in the remaining nigral neurons (1). Although the mechanisms underlying PD development remain elusive in both genetic and sporadic PD, mitochondrial and proteasomal dysfunction and oxidative stress are recognized as major contributors (2, 3).The pivotal roles of these pathways are further substantiated by the fact that all chemically induced parkinsonian models, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (4), rotenone (5), and more recently epoxomicin (6), lead to mitochondrial and proteasomal dysfunction as well as increased oxidative stress. Nonetheless despite decades of research, identification of molecules involved in these processes in the setting of DAergic neurodegeneration has yielded limited success. Consequently current clinical treatment of PD is at a standstill with the replacement of DA or with DAergic agonistic approaches (7,8).In this study, we used an unbiased state-of-the-art proteomic technique called shotgun proteomics multidimensional protein identification technology (MudPIT) to quantitatively profile mitochondrial proteins from pathologically verified PD patients and normal age-matched controls as well as in a cellular model of PD, i.e. DAergic cells treated with parkinsonian toxicant rotenone. MudPIT uses multidimensional LC and tandem mass spectrometry to separate and fragment peptides for protein identification (9) as well as for quantification when used in combination with ICAT and stable isotope labeling by amino acids in cell culture (SILAC) techniques (10, 11). With these approaches, we identified many novel proteins with quantitative expression differences in the SNpc of PD patients as compared with controls. One of these proteins, mortalin/mthsp70/GRP75, decreased significantly in many PD brain samples and in the cellular model of PD. Several mortalin-binding proteins likely participating in rotenone-mediated toxicity were also identified. Furthermore overexpression and silencing of mortalin expression in the cellular model of PD significantly influenced PD type pathologies. Thus, we report for the first time that a mitochondrial stress protei...

The mortalin genes, mot-1 and mot-2, are hsp70 family members that were originally cloned from normal and immortal murine cells, respectively. Their proteins differ by only two amino acid residues but exhibit different subcellular localizations, arise from two distinct genes, and have contrasting biological activities. We report here that the two proteins also differ in their interactions with the tumor suppressor protein p53. The pancytosolic mot-1 protein in normal cells did not show colocalization with p53; in contrast, nonpancytosolic mot-2 and p53 overlapped significantly in immortal cells. Transfection of mot-2 but not mot-1 resulted in the repression of p53-mediated transactivation in p53-responsive reporter assays. Inactivation of p53 by mot-2 was supported by the down-regulation of p53-responsive genes p21 WAF-1 and mdm-2 in mot-2-transfected cells only. Furthermore, NIH 3T3 cells transfected with expression plasmid encoding green fluorescent proteintagged mot-2 but not mot-1 showed an abrogation of nuclear translocation of wild-type p53. These results demonstrate a novel mechanism of p53 inactivation by mot-2 protein.Evidence has been accumulating that inactivation of p53, a tumor suppressor and cellular transcription factor (1), is involved in cellular transformation and immortalization (2-5). Extensive analyses of p53 have defined at least four functional domains, including an amino terminus transactivation domain (amino acids 1-44), a sequence-specific DNA-binding domain (amino acids 100 -300), a carboxyl terminus oligomerization domain, and a regulatory domain (amino acids Ref. 6), and shown that the conformation of p53 and its interactions with other proteins have key roles in its various cellular activities (7,8). Several cellular proteins, including some of the hsp70 family members, have been shown to interact with p53 (9 -12). Although mutational or mdm-2-mediated inactivation of p53 is a common event involved in cellular transformation (1), p53 is inactivated in a considerable number of tumors and transformed cells by an unknown mechanism(s).We initially cloned mortalins mot-1 and mot-2, which code for pancytosolically and perinuclearly distributed members of the hsp70 family of proteins, from normal and immortal murine cells, respectively (13,14). The open reading frames of the two types of murine mortalins differ in two nucleotides, encode proteins differing in two amino acids, arise from distinct genes, and have contrasting biological activities (13-16). RNA in situ hybridization and immunohistochemical studies on mortalin in normal murine tissues showed a higher level of expression in nondividing cell populations than in dividing cells. However, tumor tissues were seen to have a high intensity of mortalin staining by an antibody that reacts with both the mot-1 and mot-2 proteins (17, 18). Mortalin was also identified as PBP-74, mtHSP70, and Grp75 and has been assigned roles in antigen processing, in vivo nephrotoxicity, and radioresistance in independent studies from other groups (19,20)...

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Mortalin/mthsp70/PBP74/Grp75 (called mortalin hereafter), a member of the Hsp70 family of chaperones, was shown to have different subcellular localizations in normal and immortal cells. It has been assigned to multiple subcellular sites and implicated in multiple functions ranging from stress response, intracellular trafficking, antigen processing, control of cell proliferation, differentiation, and tumorigenesis. The present article compiles and reviews information on the multiple sites and functions of mortalin in different organisms. The relevance of its differential distributions and functions in normal and immortal cell phenotypes is discussed.

Stress protein mortalin is a multifunctional protein and is highly expressed in cancers. It has been shown to interact with tumor suppressor protein-p53 (both wild and mutant types) and inactivates its transcriptional activation and apoptotic functions in cancer cells. In the present study, we found that, unlike most of the cancer cells, HepG2 hepatoma lacked mortalin-p53 interaction. We demonstrate that the mortalin-p53 interaction exists in cancer cells that are either physiologically stressed (frequently associated with p53 mutations) or treated with stress-inducing chemicals. Targeting mortalin-p53 interaction with either mortalin small hairpin RNA or a chemical or peptide inhibitor could induce p53-mediated tumor cell-specific apoptosis in hepatocellular carcinoma; p53-null hepatoma or normal hepatocytes remain unaffected. Cell Death and Differentiation (2011) 18, 1046-1056 doi:10.1038/cdd.2010; published online 14 January 2011The wild-type p53 is a key tumor suppressor protein that eliminates genetically unstable cells by inducing either cell cycle arrest or apoptosis through transcriptional regulation or direct interaction with apoptotic proteins. 1,2 Functional inactivation of p53, a frequent event in cancer cells, occurs by three main mechanisms: (i) mutations, (ii) post-translational modifications and (iii) cytoplasmic sequestration 3-5 by its binding proteins. Although several cellular proteins are shown to interact with p53, the mechanism of its inactivation still remains unclear. Furthermore, interaction of p53 with its binding partners is context dependent, and influenced by both intracellular and extracellular environment. Cellular stress response (intrinsic and extrinsic) has been shown to evoke p53 signaling 6 through its modifications, including phosphorylation (at serine and/or threonine), 7 acetylation, 8 sumoylation, 9 glycosylation, 10 ribosylation 11 or ubiquitylation. 12 Furthermore, it has been shown that p53 protein interacts with several stress proteins, including Hsp40, Hsp70, Hsp84, Hsp90, DnaK, DnaJ and GrpE in vivo, 13-15 that potentially modulate p53 activities. However, their roles in development and progression of cancer, physiologically a stressed condition, remain unclear.Mortalin/mthsp70/GRP75/PBP74, a member of the heat shock protein (Hsp) 70 family, is enriched in human cancer cells. [16][17][18] Overexpression of mortalin was sufficient to increase the malignancy of breast cancer cells in both in vitro and in vivo models. The underlying mechanism was shown to be the sequestration of wild-type p53 in the cytoplasm, leading to inhibition of its transcriptional activation and control of centrosome duplication functions. [19][20][21][22] A cationic inhibitor (MKT-077) of mortalin that releases p53 from mortalin-p53 complex was shown to cause activation of p53 and growth arrest of cancer cells. 23 Similarly, mortalin-binding p53 peptides caused nuclear translocation and activation of p53. 19 Mortalin was identified as a marker for hepatocellular carcinoma (HCC) metastasis and r...

Purpose: Ashwagandha is regarded as a wonder shrub of India and is commonly used in Ayurvedic medicine and health tonics that claim its variety of health-promoting effects. Surprisingly, these claims are not well supported by adequate studies, and the molecular mechanisms of its action remain largely unexplored to date. We undertook a study to identify and characterize the antitumor activity of the leaf extract of ashwagandha. Experimental Design: Selective tumor-inhibitory activity of the leaf extract (i-Extract) was identified by in vivo tumor formation assays in nude mice and by in vitro growth assays of normal and human transformed cells. To investigate the cellular targets of i-Extract, we adopted a gene silencing approach using a selected small hairpin RNA library and found that p53 is required for the killing activity of i-Extract. Results: By molecular analysis of p53 function in normal and a variety of tumor cells, we found that it is selectively activated in tumor cells, causing either their growth arrest or apoptosis. By fractionation, purification, and structural analysis of the i-Extract constituents, we have identified its p53-activating tumor-inhibiting factor as withanone. Conclusion: We provide the first molecular evidence that the leaf extract of ashwagandha selectively kills tumor cells and, thus, is a natural source for safe anticancer medicine.