Mycobacterium tuberculosis (Mtb) is the etiologic agent of the disease tuberculosis, which kills approximately 1.4 million people each year. Mtb's arduous treatment regimen and the recent emergence of increasingly drug-resistant Mtb strains have given rise to an urgent need for the development of new anti- tubercular drugs. One promising therapeutic target is the mycobacterial proteasome, a large proteolytic complex that is essential for both Mtb virulence and resistance to the host's antimicrobial nitric oxide response. Recent work has shown that proteins can be targeted for proteasomal degradation in Mtb by the pupylation pathway, a process that is functionally analogous to eukaryotic ubiquitination. Pupylation centers around the small protein modifier Pup (prokaryotic ubiquitin-like protein), which is covalently ligated to a protein to target it for proteolysis. Despite advances in our understanding of the Pup-proteasome system, no group has yet recapitulated robust degradation of a native substrate by the Mtb proteasome in vitro, implying that additional unknown factors are necessary for proteasomal activity. To search for such factors, we used a catalytically inactive """"""""proteasome trap"""""""" that stabilizes proteasomal interactions to identify novel proteasome-associated proteins. A protein we are now referring to as PafE (proteasome accessory factor E) copurified with the proteasome trap in high abundance and co-occurs evolutionarily with the prokaryotic 20S core particle. Preliminary studies demonstrated that PafE resembles known proteasomal cofactors, as it forms multimeric rings and interacts with the 20S through a conserved carboxy-terminal motif. Thus, in an effort to enhance our understanding of the prokaryotic proteasome complex, the proposed research seeks to determine PafE's role in proteasome function through two specific aims. First, we will determine if PafE plays a role in proteasomal degradation in vivo. Through immunoblotting we will determine if PafE is required for the degradation of known pupylated proteasome substrates. In addition, we will utilize the proteasome trap to search for non-pupylated PafE-dependent substrates that copurify with the proteasome only in the presence of PafE. We will then determine if PafE is required for Mtb virulence in a mouse infection model. Second, we will determine if PafE has a direct role in proteasomal degradation in vitro. We will use recombinant 20S and PafE purified from E. coli to validate that binding occurs independent of any other factors. We will then perform in vitro reactions using purified components to test if PafE influences the proteasomal degradation rate of either a peptide substrate or the native Mtb proteasome substrate Pup~FabD. These studies will substantially enhance our understanding of the prokaryotic proteasome and the mechanisms that regulate its function. Because the Mtb proteasome is essential for normal growth and virulence, this work has strong implications for the elucidation of new therapeutic avenues to treat tuberculosis.

Public Health Relevance

The mycobacterial proteasome is essential for full virulence of M. tuberculosis, making it an attractive therapeutic target for the treatment of tuberculosis. Identification of new factors that are necessary for proteasomal function will therefore reveal novel potential drug targets, facilitating the development of anti- tubercular therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
1F30AI110067-01A1
Application #
8781505
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Jacobs, Gail G
Project Start
2014-09-01
Project End
2017-10-01
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
New York University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10016
Jastrab, Jordan B; Samanovic, Marie I; Copin, Richard et al. (2017) Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E (pafE) Mutant. J Bacteriol 199:
Bai, Lin; Jastrab, Jordan B; Isasa, Marta et al. (2017) Structural Analysis of Mycobacterium tuberculosis Homologues of the Eukaryotic Proteasome Assembly Chaperone 2 (PAC2). J Bacteriol 199:
Wu, Yujie; Hu, Kuan; Li, Defeng et al. (2017) Mycobacterium tuberculosis proteasomal ATPase Mpa has a ?-grasp domain that hinders docking with the proteasome core protease. Mol Microbiol 105:227-241
Bai, Lin; Hu, Kuan; Wang, Tong et al. (2016) Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 113:E1983-92
Jastrab, Jordan B; Wang, Tong; Murphy, J Patrick et al. (2015) An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 112:E1763-72
Jastrab, Jordan B; Darwin, K Heran (2015) Bacterial Proteasomes. Annu Rev Microbiol 69:109-27