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 possess 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 C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease 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 then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which 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 (heterogeneous nuclear ribonucleoprotein 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 the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.

Highlights

  • Mortalin, an essential chaperone, is enriched in cancers; it possesses pro-proliferative and anti-apoptotic functions and has been found mutated in some Parkinson patients

  • 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 ␣ (EF-1␣), which 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-1␣ proteins

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Summary

Background

An essential chaperone, is enriched in cancers; it possesses pro-proliferative and anti-apoptotic functions and has been found mutated in some Parkinson patients. 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 (heterogeneous nuclear ribonucleoprotein 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-1␣ proteins These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to. Knocking in extra copies of a Caenorhabditis elegans homologue of mot-2 caused increase in their lifespan (19) These effects have been ascribed, in part, to the ability of mot-2 to (i) inactivate wild-type p53 functions including transcriptional activation (20, 21), control of centrosome duplication (22), and deregulation of apoptosis in cancer cells (23–25); (ii) activate telomerase and hnRNP-K (26); (iii) and regulate oxidative stress (27, 28) and mitochondrial structure (29, 30). We demonstrate that in contrast to the role of mot-2 in cancer phenotype, these proteins cause growth arrest of cells by different signaling pathway

EXPERIMENTAL PROCEDURES
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AND DISCUSSION
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