期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：49
页码：E7947-E7956
DOI：10.1073/pnas.1617644113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe proteostasis pathway may be a source of new drug targets in Mycobacterium tuberculosis (Mtb). The conserved protein chaperone DnaK is essential in Mycobacterium smegmatis and predicted to be essential in Mtb. DnaK is regulated by cofactors, J proteins and nucleotide exchange factor GrpE. In contrast to most bacterial pathogens, Mtb has two J proteins, DnaJ1 and DnaJ2. Here, we characterize in vitro activities of Mtb DnaK, DnaJ1, DnaJ2, and GrpE, the disaggregase ClpB, and the small heat shock protein Hsp20, in reactivation of a protein aggregate. We found that DnaJ1 and DnaJ2 are individually dispensable, but collectively essential and mutations in a conserved motif of each result in cellular loss of function. These findings will help in identifying and characterizing inhibitors of Mtbs proteostasis network. 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.
