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Reconstitution of a Mycobacterium tuberculosis proteostasis network highlights essential cofactor interactions with chaperone DnaK.

TitleReconstitution of a Mycobacterium tuberculosis proteostasis network highlights essential cofactor interactions with chaperone DnaK.
Publication TypeJournal Article
Year of Publication2016
AuthorsLupoli TJ, Fay A, Adura C, Glickman MS, Nathan C
JournalProc Natl Acad Sci U S A
Volume113
Issue49
PaginationE7947-E7956
Date Published2016 Dec 06
ISSN1091-6490
Abstract

During host infection, Mycobacterium tuberculosis (Mtb) encounters several types of stress that impair protein integrity, including reactive oxygen and nitrogen species and chemotherapy. The resulting protein aggregates can be resolved or degraded by molecular machinery conserved from bacteria to eukaryotes. Eukaryotic Hsp104/Hsp70 and their bacterial homologs ClpB/DnaK are ATP-powered chaperones that restore toxic protein aggregates to a native folded state. DnaK is essential in Mycobacterium smegmatis, and ClpB is involved in asymmetrically distributing damaged proteins during cell division as a mechanism of survival in Mtb, commending both proteins as potential drug targets. However, their molecular partners in protein reactivation have not been characterized in mycobacteria. Here, we reconstituted the activities of the Mtb ClpB/DnaK bichaperone system with the cofactors DnaJ1, DnaJ2, and GrpE and the small heat shock protein Hsp20. We found that DnaJ1 and DnaJ2 activate the ATPase activity of DnaK differently. A point mutation in the highly conserved HPD motif of the DnaJ proteins abrogates their ability to activate DnaK, although the DnaJ2 mutant still binds to DnaK. The purified Mtb ClpB/DnaK system reactivated a heat-denatured model substrate, but the DnaJ HPD mutants inhibited the reaction. Finally, either DnaJ1 or DnaJ2 is required for mycobacterial viability, as is the DnaK-activating activity of a DnaJ protein. These studies lay the groundwork for strategies to target essential chaperone-protein interactions in Mtb, the leading cause of death from a bacterial infection.

DOI10.1073/pnas.1617644113
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID27872278
PubMed Central IDPMC5150378
Grant ListP30 CA008748 / CA / NCI NIH HHS / United States
T32 CA009149 / CA / NCI NIH HHS / United States
U19 AI111143 / AI / NIAID NIH HHS / United States