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.
Highlights
IntroductionHuman mortalin ( named mtHsp, GRP75, HspA9 and PBP74) [1,2,3,4] is a highly conserved molecular chaperone of the Hsp family that is primarily found in the mitochondria
Human mortalin [1,2,3,4] is a highly conserved molecular chaperone of the Hsp70 family that is primarily found in the mitochondria
Recombinant human mortalin was produced in soluble form and purified until homogeneity The recombinant human mortalin was produced in a soluble state through its co-expression with hHep1, as previously shown [34]
Summary
Human mortalin ( named mtHsp, GRP75, HspA9 and PBP74) [1,2,3,4] is a highly conserved molecular chaperone of the Hsp family that is primarily found in the mitochondria. Mortalin is the import motor that drives the preprotein import process and helps the folding of these proteins in the mitochondrial matrix [11, 19] Due to their importance for protein homeostasis, Hsp proteins have been considered targets for the drug-based treatments for cancers [7, 20,21,22], misfolding diseases and protein folding disorders [23]. Mortalin presents similar structural elements as other Hsp proteins: an N-terminal ATPase domain (NBD) and a C-terminal peptide-binding domain (PBD). These two domains should be reciprocally controlled by a bidirectional heterotrophic allostery dependent on the presence of ATP/ADP on the NBD and a client protein bound to the PBD [22, 24]. The mammalian mitochondria presents the main Hsp co-chaperones: 1) J-proteins (Hsp40), which should stimulate Hsp ATPase activity, and 2) two GrpE orthologous proteins, which should act as nucleotide exchange factors controlling the rate cycle of Hsp70 [22]
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