However, it is possible that PknH is cleaved by two separate proteases at the transmembrane interface in a fashion similar to RseB in E. coli. This process, termed Regulated Intramembrane Proteolysis, involves the activities of an HtrA-family protease, DegS, and a metalloprotease, RseP. Because the extracytoplasmic side of the transmembrane domain of PknH contains an arginine and lysine, it is conceivable that PepD cleaves in this area and produces a peptide that was missed during our semi-tryptic mass spectrometric analysis. Alternatively, the peptide identified may be the product of a cleavage event mediated by another protease, as PepD was able to co-immunoprecipitate multiple proteases in both M. tuberculosis and M. smegmatis. Regardless, the identified binding proteins and substrates provide a starting point for further investigations into the physiological role of PepD in M. tuberculosis. Based on this data, we postulate that PepD functions to proteolytically regulate Rv2744c levels to help maintain cell wall/ cell envelope homeostasis in M. tuberculosis. A model is also proposed that builds upon observations previously reported by Barik et al and others concerning interactions between the SigE and MprAB signalling pathways in M. tuberculosis following exposure to extracytoplasmic stress. The serine/ threonine protein kinase, PknB, contains PASTA domains that have been postulated to bind peptidoglycan and may serve as cell wall sensors. As the peptidoglycan becomes disordered due to extracellular stress, PknB activates and phosphorylates RseA, the anti-sigma factor of SigE. Phosporylation of RseA leads to proteolytic degradation of this protein by ClpC1P2, releasing SigE and inducing expression of components of the SigE regulon including mprA and clgR. MprA and ClgR in turn upregulate gene products within their cognate regulons including clgR itself, clpC1, clpP2, ppk1, pepD, and sigE. Upregulation of clp genes initiates a positive feedback loop through SigE by enhancing degradation of RseA. Similarly, upregulation of ppk1 encoding polyphosphate kinase increases polyphosphate levels and enhances activation of the MprAB two-component system, mediating a positive feedback loop through SigE. The Rv2744c generated following upregulation of clgR is secreted extracytoplasmically, where it functions in an as-of-yet undefined role to help mediate resistance to the recognized stress. In Escherichia coli and other bacterial species, PspA forms higher order oligomers where the protein is thought to function as a structural scaffold to help maintain proton motive force. While it is currently unclear if higher order oligomers are formed by Rv2744c in M. tuberculosis, Rv2744c can interact with GSI-IX Gamma-secretase inhibitor itself in bacterial two-hybrid assays Navitoclax carried out in E. coli. Overproduction of Rv2744c and/or exposure of this protein to stress that perturbs the cell wall, including that mediated through peptidoglycan-disrupting agents, may lead to unstructured regions of Rv2744c that become recognized by PepD.