1. Regulated intracellular proteolysis

 In all cells, regulated proteolysis constantly sculpt and reshape the proteome. It is essential for regulation of cellular activities, for elimination of misfolded proteins and for amino acid recycling under starvation conditions. At the same time, proteolysis is a deleterious process that can prove deadly to the cell. Accordingly, mechanisms have evolved that allow intracellular proteolysis to occur in a carefully regulated manner. While eukaryotic proteolytic systems are highly complex, and involve hundreds of cellular components, bacterial proteolytic systems are much simpler, yet encompass similar basic principles. Accordingly, bacterial proteolytic systems are favorable as model systems for studying the basic principles that govern intracellular proteolysis. In addition, as intracellular proteolysis is essential for mycobacterial virulance and survival, mycobacterial proteases are targets for development of antibiotics against mycobacterial pathogens, such as Mycobacterium tuberculosis. We focus on two proteolytic systems:

The Pup-Proteasome System

      Pup is a small protein that is attached to a wide variety of targets via the activity of two enzymes, PafA and Dop. As Pup-tagged proteins are recognized and degraded by the multi-component bacterial proteasome, Pup serves as a degradation tag analogous to the eukaryotic ubiqtin.

         The Pup-proteasome system (PPS) was initially discovered in the pathogen Mycobacterium tuberculosis, where it was found to be important for the virulence and persistence in the host. Therefore, enzymes of the PPS are potential targets for the development of anti-tuberculosis medicine. At the same time, the conservation of the PPS in non-pathogenic actinobacterial species indicated that this system plays a fundamental role in the physiology of these bacteria. We have shown that in Mycobacterium smegmatis, a non-pathogenic mycobacterial model organism, the PPS is essential under nitrogen starvation conditions. Under such conditions, PPS activity is accelerated via the action of multiple regulatory mechanisms to support cellular functions, while carefully avoiding unnecessary protein degradation. While much exciting information has accumulated in recent years regarding the mechanism of action, regulation and physiological roles of the PPS, many intriguing questions remain unanswered. Work in our lab addresses these questions in a quest to understand the PPS molecular desig and regulation.

‍

The ClpCP AAA+ protease

          The ClpC Pprotease is a hetero-oligomeric molecular machine that couples ATP hydrolysis to protein unfolding and degradation in bacteria. In mycobacteria, ClpCP is an essential protease, and therefore a target for develeopment of antibiotics against Mycobacterium tuberculosis. Despite the ClpCP therapeuics and physiological importance, very little is known about the roles ClpCP plays in mycobacteria, and how it is regulated. Moreover, its substrate recognition and processing mechanisms are poorly understood. We study the Mycobacterium smegmatis ClpCP mechanism of action and regulation both at the genetic and biochemical levels.

‍

2. Inteins - Physiology & regulation

         Inteins (intervening proteins) are mobile genetic elements that are translated within host proteins and removed through self-protein-splicing. In this process, the two peptide bonds surrounding the intein are rearranged into one, liberating the intein from the host protein in a scarless manner. Inteins are found in all kingdoms of life, usually in essential proteins, and therefore, intein splicing is essential for cell survival. For instance, in Mycobacterium smegmatis,the DNA helicase (DnaB) is translated with two inteins, and their splicing is essential for DNA replication in this bacterium. While the intein splicing mechanism is well understood, very little is known about the physiological roles played by inteins in their cellular hosts, and how intein splicing is regulated in response to external stimuli. To address these issues, we study intein splicing, using M. smegmatis as a model organism.

‍

‍