Identification of a novel member of mouse hsp70 family. Its association with cellular mortal phenotype.

Aging Process Research Laboratory, Tsukuba Life Science Center, Ibaraki, Japan.
Journal of Biological Chemistry (Impact Factor: 4.6). 04/1993; 268(9):6615-21.
Source: PubMed

ABSTRACT A novel 66-kDa protein, named p66mot-1, is identified to be associated with cellular mortality by virtue of its presence in cytosolic fractions of serially passaged mouse embryonic fibroblasts (MEF) and the mortal hybrids obtained by the fusion of mortal (MEF) and immortal (MN48-1, derivative of NIH 3T3) cells. Immortal cells lack this protein in their cytosolic fractions. cDNA cloning and homology search placed it in the heat shock protein 70 (hsp70) family. Microinjection of anti-p66 antibody to senescent MEF could transiently stimulate their cell division, which supports the cellular mortality-related function of p66mot-1.

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    ABSTRACT: The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with age-related diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor compound screenings.
    PLoS ONE 01/2015; 10:e0117170. DOI:10.1371/journal.pone.0117170 · 3.53 Impact Factor
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    ABSTRACT: Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to posses pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the carboxy-terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson (PD) patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins, and identified ribosomal protein L-7 (RPL-7) and elongation factor-1 alpha (EF-1α) that differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis such as, p53 inactivation and hTERT/hnRNP-K activation, (ii) causes increased level of endogenous oxidative stress, (iii) results in decreased tolerance of cells to exogenous oxidative stress and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to premature aging/anti-proliferative effect of mutants, and hence have significance in cellular aging, PD pathology and prognosis. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 02/2015; DOI:10.1074/jbc.M114.627463 · 4.60 Impact Factor


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