title: Reconstitution of a Mycobacterium tuberculosis proteostasis network highlights essential cofactor interactions with chaperone DnaK.
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.
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY 10021.
authors.forename: Tania J
authors.initials: TJ
authors.lastname: Lupoli
authors.affiliation: Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065.
authors.forename: Allison
authors.initials: A
authors.lastname: Fay
authors.affiliation: High-Throughput Screening and Spectroscopy Resource Center, The Rockefeller University, New York, NY 10065.
authors.forename: Carolina
authors.initials: C
authors.lastname: Adura
authors.affiliation: Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065.|Division of Infectious Diseases, Memorial Sloan-Kettering Cancer Center, New York, NY 10065.
authors.forename: Michael S
authors.identifier: 0000-0001-7918-5164
authors.initials: MS
authors.lastname: Glickman
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY 10021; cnathan@med.cornell.edu.
authors.forename: Carl F
authors.initials: CF
authors.lastname: Nathan
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000254:growth & development / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D000074702:Proteostasis
chemical_list: D001426:Bacterial Proteins; C000595121:DnaK protein, Mycobacterium smegmatis; C068113:GrpE protein, Bacteria; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D000251:Adenosine Triphosphatases
keywords: DnaJ proteins; DnaK; M. tuberculosis; chaperones; proteostasis