Functions of alternative ClpP subunits in Pseudomonas aeruginosa

Abstract

Proteolysis is the process by which proteins are broken down, or hydrolyzed, into small peptides or amino acids by enzymes. Cells from all forms of life carry out regulated protein degradation as a way to control cellular physiology and regulate stress responses. Clp proteases, containing a AAA+ (A̲TPases A̲ssociated with various cellular A̲ctivities) unfoldase stacked with a compartmentalized peptidase, are central to bacterial proteolysis, and use the energy of ATP hydrolysis to unfold and translocate protein substrates into the peptidase chamber for their destruction. The opportunistic pathogen Pseudomonas aeruginosa is unusual in that it contains two isoforms of the subunits that form the ClpP peptidase chamber. These isoforms, PaClpP1 and PaClpP2, have not been well characterized previously and their specific functions are largely elusive. This work examines the structures and functions of PaClpP1 and PaClpP2 and proposes a model for functional peptides generated by these enzymes in P. aeruginosa development. Biochemical analysis establishes that PaClpP2 is only active as a peptidase when it is part of a PaClpP1₇P2₇ heterocomplex. Furthermore, multiple lines of evidence support that P. aeruginosa cells have two distinct ClpP peptidase assemblies: PaClpP1₁₄ and PaClpP1₇P2₇. Importantly, peptidase and protease analyses establish that these two ClpP assemblies exhibit distinct peptide cleavage specificities and interact differentially with the AAA+ unfoldases, ClpX and ClpA. Finally, the PaClpP2 peptide-cleavage active site uniquely contributes to P. aeruginosa biofilm development. Therefore, results presented in this thesis suggest that within AAA+ proteases, the specificity of the peptidase subunits, not only the recognition properties of the AAA+ unfoldase, control the biological outcome(s) of proteolysis.