Structure-function analysis of the regulatory Mpa particle from the bacterial proteasome

Energy-dependent protein degradation is an essential activity in all living cells. In eukaryotes, 26S proteasomes are responsible for the majority of cellular protein turnover, and proteins destined for degradation undergo modification by covalent attachment of ubiquitin. Bacteria contain a set of architecturally related, but different degradation complexes. However, actinobacteria, including the genus Mycobacteria, represent an unusual group, as they have acquired proteasomes. The catalytic core particle is associated with a ring of ATPase subunits called Mpa thus forming a barrel-shaped structure much like other bacterial ATPases. However, substrate proteins are targeted to this proteolytic machinery through covalent attachment of a small protein called Pup to the side chain of lysine residues, thus being reminiscent of the ubiquitin tag used in eukaryotes. Importantly, Mycobacterium tuberculosis, the highly infectious pathogen causing tuberculosis, is impaired in its persistence in the lungs when it lacks components of the proteasomal degradation pathway, making this pathway a promising target for anti-tuberculosis therapeutic approaches. The overall scope of this project is a detailed examination of the unfolding and degradation activity of the mycobacterial unfoldase Mpa alone and in complex with the 20S proteasome. In order to address these questions and to gain molecular insights into the Mpa:proteasome system, a combined approach including methods from molecular biology, protein chemistry, biophysics, crystallography and electron microscopy will be employed. Structural data will be essential to put forward a model of the unfolding and degradation reaction and to design mutations whose biological importance can be tested in vitro. Structural information will be supplemented by a detailed biochemical analysis of the unfolding reaction of Mpa alone and when bound to the core particle. As there is significant sequence similarity to both eukaryotic 19S proteasome base subunits and to archaeal proteasome- activating nucleotidase (PAN), elucidation of Mpa`s molecular architecture and mode of action will likely also shed light on the general mechanism of ATP-driven unfoldases associated with proteases. Our ultimate goal aims at a better understanding of the Mpa unfoldase and its collaboration with the bacterial proteasome. We are confident that our structure-function studies will provide the necessary framework to address mechanistic aspects of the unfolding, translocation and degradation steps and to explain the physiological relevance of the corresponding processes.

The aim of this project was a detailed structural and biochemical investigation of the mycobacterial proteasome. The proteasome is a complex protease assembly, central to protein degradation in almost all living cells. Whilst the proteasomal protein degradation machinery is long known and rather well understood in higher organisms, it was only recently found that mycobacteria (including the human pathogen Mycobacerium tuberculosis) harbor a similar proteasomal complex for substrate degradation.Mycobacterial proteasomes have been thought to be composed of two components: Firstly, an unfoldase called Mpa which is responsible for both the selection of substrate proteins and the unfolding of the substrate by ATP-driven motor domains. Secondly, the protease itself, being the proteasomal core particle which eventually degrades proteins into small peptides. In analogy to known eukaryotic proteasomal assemblies, it was assumed that Mpa binds to the core particle through its very C-terminal part, although it has been obvious that the sequence is markedly different from known proteasomal activators.In our study, we put a focus on the analysis of this interplay between Mpa and core particle. We could show that both isolated Mpa and core particle are active as such, however, Mpa harbouring its native C-terminus was unable to bind and subsequently cowork with the proteasomal core particle in delivering substrate to the proteolytic active site. Even when the C-terminus of Mpa was changed to sequences analogous to those found in eukaryotic proteasomal activators, no cooperation was observed. Hence we conclude that the binding and/or activation mode of mycobacterial proteasomes must be markedly different from their eukaryotic counterparts. Interestingly, we could show that the activity of the overall Mpa:core particle complex can be restored in the context of whole cells, indicating the presence of novel, yet to be identified factors.These findings of the mycobacterial proteasome being more different from the eukaryotic one than originally anticipated renders it a highly interesting area of active research for drug development.