Studying the mycobacterial Pup-proteasome system
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. For instance, AAA+ proteases, the enzymes that catalyze most regulated proteolysis in the cell, are well designed to carefully chose and degrade their substrates while avoiding uncontrolled degradation of other cellular proteins. In eukaryotes, the 26S proteasome is the only AAA+ protease acting in the cytoplasm and nucleus, where it degrades predominantly ubiquitin-tagged proteins. In analogy, the bacterial proteasome relies on a prokaryotic ubiquitin-like protein (Pup) for protein tagging.
The Pup proteasome system is a protein tagging and degradation system. Pup, a 64-residue unstructured protein, is attached via its C-terminal glutamate to a wide variety of targets, thereby tagging them to degradation by the bacterial proteasome. A single promiscuous ligase, PafA, forms an isopeptide bond between the side chain of the Pup C-terminal glutamate and lysine side chains in target proteins. In many bacterial species, Pup is translated with a C-terminal glutamine rather than a glutamate, and an enzyme termed Dop must convert this terminal glutamine to a glutamate before PafA can act. By the same enzymatic mechanism, Dop can also hydrolyze the isopeptide bond linking Pup to a target, thereby detaching Pup from an already pupylated protein. Pupylated proteins are recognized and degraded by the multi-component bacterial proteasome. One component of this proteolytic machine is the 20S core particle (CP), which contains a degradation chamber typically enclosed by two homo-heptameric beta rings stacked between two homo-heptameric alpha rings. The second critical component is Mpa, a homo-hexameric AAA+ enzyme that acts to recognize, unfold, and translocate pupylated proteins into the 20S CP chamber for degradation.
The 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 molecular design of a complex proteolytic pathway.
We focus on two main research directions:
Regulation of the Pup-proteasome system
The PPS responds to external stimuli in a carefully regulated manner. Indeed, both pupylation and degradation of pupylated proteins are accelerated under conditions of nitrogen starvation. Our lab studies how the PPS senses external stimuli, as well as the mechanisms that allow for control of the cytoplasmic levels and activity of the PPS components.
For instance, we ask the following questions:
- How is nitrogen starvation sensed by the PPS?
- To which stimuli, other than nitrogen starvation, does the PPS respond?
- How are the levels and activities of the PPS enzymes controled?
- What feedback loops and kinetic constrains direct PPS regulation?
PPS mechanism of action
The role of most PPS components is known and their mechanism of action has been studied in recent years. Yet, many questions regarding the mechanism of protein pupylation and proteasomal degradation still await answers. For instance, it is currently unclear how PafA chooses is substrates and whether this activity is regulated at the enzyme level. Likewise, cooperation and feedback between PPS enzymes is poorly understood. Our lab combines genetic, biochemical and structural approaches to answer questions such as:
- How structure-function relationships allow the protesome to act in a highly controlled manner?
- What are the structure-function relationships that define PafA and Dop activities?
- How is substrate recognition by PafA determined at the molecular level?
- Does coupling between pupylation and degradation in vivo exist?
- How are the activities of the PPS enzymes regulated at the biochemical level?