Functions of alternative ClpP subunits in Pseudomonas aeruginosa

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, May, 2020Cataloged from the official PDF of thesis.Includes bibliographical references.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.by Gina D. Mawla.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Biolog

A Novel Approach for Studying Histone H1 Function in Vivo

In this report, we investigate the mechanisms that regulate Drosophila histone H1 expression and its association with chromatin in vivo. We show that histone H1 is subject to negative autoregulation and exploit this result to examine the effects of mutations of the main phosphorylation site of histone H1

ClpP1P2 peptidase activity promotes biofilm formation in Pseudomonas aeruginosa

Caseinolytic proteases (Clp) are central to bacterial proteolysis and control cellular physiology and stress responses. They are composed of a double-ring compartmentalized peptidase (ClpP) and a AAA+ unfoldase (ClpX or ClpA/ClpC). Unlike many bacteria, the opportunistic pathogen P. aeruginosa contains two ClpP homologs: ClpP1 and ClpP2. The specific functions of these homologs, however, are largely elusive. Here, we report that the active form of PaClpP2 is a part of a heteromeric PaClpP1(7)P2(7) tetradecamer that is required for proper biofilm development. PaClpP1(14) and PaClpP1(7)P2(7) complexes exhibit distinct peptide cleavage specificities and interact differentially with P. aeruginosa ClpX and ClpA. Crystal structures reveal that PaClpP2 has non-canonical features in its N- and C-terminal regions that explain its poor interaction with unfoldases. However, experiments in vivo indicate that the PaClpP2 peptidase active site uniquely contributes to biofilm development. These data strongly suggest that the specificity of different classes of ClpP peptidase subunits contributes to the biological outcome of proteolysis. This specialized role of PaClpP2 highlights it as an attractive target for developing antimicrobial agents that interfere specifically with late-stage P. aeruginosa development