Protein degradation or proteolysis is a process in which proteins are naturally destroyed in a cell to maintain protein homeostasis. For this purpose, different kinds of directed protein degradation machinery, i.e., proteases, proteasomes are present in addition to the lysosomal degradation.1 Mycobacterium tuberculosis is a type of actinobacteria that causes tuberculosis and has both bacteria-like proteases and eukarya-like proteasomes. Like other proteasomal complexes, the Mtb proteasomes are composed of two independent compartments named the ATPase compartment (Mpa) and the Peptidase compartment (20S CP)2. A hexameric ATPase compartment, Mpa (mycobacterial proteasomal activator), is a member of the AAA+ (ATPase Associated with various cellular Activities) ATPase superfamily that recognises, unfolds, and translocates the protein substrate having a degron tag called Pup (prokaryotic ubiquitin-like protein) by using ATP as an energy source. In the current talk. I will discuss the detailed current literature on Mpa-20CP proteasomal complex and propose my doctoral research work on ensemble and single molecule studies of Mpa. The initial part of my talk will be on structural and biophysical properties of the enzyme. We have studied the chemical and thermal stability of hexameric Mpa to probe its unfolding and de-oligomerization models. In addition, we have performed ensemble degradation assays using the Mpa-20SCP complex to determine the kinetics of degradation. The motor properties of Mpa such as translocation velocity, stepping kinetics, and processivity are not known, and optical tweezers based single-molecule study will provide insights into these properties. In addition, they will also highlight the difference between proteases and proteasomes3,4. I will present our strategies and initial results for performing these experiments.
References:
1. De Mot, R., Nagy, I., Walz, J., and Baumeister, W. (1999). Proteasomes and other self-compartmentalizing proteases in prokaryotes. Trends Microbiol 7, 88–92. 10.1016/S0966-842X(98)01432-2.
2. Kavalchuk, M., Jomaa, A., Muller, A.U., and Weber-Ban, E. (2022). Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex. Nat Commun 13, 276. 10.1038/s41467-021-27787-3.
3. Mukherjee, S., Mepperi, J., Sahu, P., Barman, D.K., and Kotamarthi, H.C. (2023). Single-Molecule Optical Tweezers As a Tool for Delineating the Mechanisms of Protein-Processing Mechanoenzymes. ACS Omega 8, 87–97. 10.1021/acsomega.2c06044.
4. Snoberger, A., Anderson, R.T., and Smith, D.M. (2017). The Proteasomal ATPases Use a Slow but Highly Processive Strategy to Unfold Proteins. Front Mol Biosci 4. 10.3389/fmolb.2017.00018.