Research Projects

We have established previously that Leptospira caseinolytic protease (ClpP) isoforms mixture is intrinsically active and its oligomerization is distinct from those of other ClpP orthologs. Therefore, the role of other ClpP regulatory proteins (ClpA or ClpC) and its cognate adaptor proteins (ClpS1 and ClpS2) in modulating specific proteolytic activity in ClpP1P2 peptidase complex is an interesting subject to understand proteostasis in Leptospira. In this project we propose to clone, express, purify and biochemically characterize the two predicted ClpS adaptor proteins (LIC11356 and LIC11815) and its cognate ATPase chaperone ClpA (LIC11814) or ClpC (LIC10339). Since the efficiency of genetic manipulation in pathogenic Leptospira has very low success rate and very difficult, understanding of these unprecedented regulation of proteolytic complexes in association with its multi-chaperone and adaptor proteins under in vitro condition may assist the biochemist in developing novel drugs.

We have PCR-amplified the full-length ORFs of clpA, clpC, clpS1, and clpS2 from Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 and cloned them into the pET23a bacterial expression vector. LinClpA, LinClpC, LinClpS1, and LinClpS2 were purified under native conditions, yielding 1–2.5 mg/L. Biochemical characterization of LinClpC revealed its ATP-dependent oligomerization and interaction with both LinClpP isoforms (LinClpP1 and LinClpP2). The LinClpC in association with LinClp1P2 heterocomplex forms LinClpCP1P2 machinery which imparts protease activity. We have also studied the role of natural antibiotic, acyldepsipeptide (ADEP1) on stimulation of protease activity of LinClpP1P2 complex. The study also investigates the structural and functional characteristics of chaperone, LinClpA and adaptor proteins, LinClpS1, and LinClpS2 from Leptospira interrogans. LinClpA, a 740-amino acid protein, features an N domain and two AAA+ ATPase domains (D1 and D2). The N-domain deletion mutant (LinClpAΔN) displayed increased ATPase activity and nucleotide-induced oligomerization. LinClpA’s ATPase activity was enhanced by LinClpP and inhibited by LinClpS isoforms, while LinClpAΔN’s activity remained unchanged in the presence of LinClpS1 and LinClpS2. Additionally, we confirmed the functionality of the predicted SsrA-tag sequence (ANNELALAA) in Leptospira. The LinClpAP1P2 complex recognized and degraded an eGFP-SsrA-tagged protein substrate in vitro.

Leptospira, Fluorescent Spectrophotometer, Department of Science and Technology (DBT), Insights to the proteolytic processing and regulation of Clp protease in Leptospira by its ATPase chaperone and adaptor proteins, Medical Sciences, Prof. Manish Kumar, Indian Institute of Technology (IIT), Guwahati, Assam, Dr. Shankar Kanaujia