Proteases work in crowded intracellular environments occupied by thousands of potential protein substrates. Thus, knowing how these destructive enzymes choose the ?right? proteins for degradation is critical for understanding both their biological functions and those of specific substrates. Intracellular proteases play important roles in eliminating damaged or harmful proteins, in resculpting the proteome following changes in gene expression, in cell-cycle control, and as sensor and regulatory components in stress-response pathways. Our primary goal is to determine the molecular mechanisms that allow peptide signals in substrates, adaptors, and other regulatory molecules to interact with proteases to control protein degradation in bacteria. A second practical goal is to develop synthetic systems of targeted degradation that test basic principles and provide community tools for studying protein function. ATP-dependent degradation of cytoplasmic proteins by AAA+ proteases occurs in all organisms. Biochemical, genetic, and structural studies will elucidate fundamental molecular mechanisms of substrate recognition for three Escherichia coli AAA+ proteases (ClpXP, HslUV, and Lon), and provide paradigms for understanding how orthologs of these enzymes identify the correct intracellular substrates in other bacteria and eukaryotes. Regulated intramembrane proteolysis (RIP) is a method of signal transduction. We will determine the detailed molecular mechanisms that allow a PDZ-protease (DegS) to sense envelope stress in the periplasm of E. coli and initiate a proteolytic cascade that relays information across the inner membrane to a second PDZ-protease (RseP). The molecular logic of this signaling system and its control by input signals and interactions of the PDZ- proteases with two regulatory proteins (RseA and RseB) will be elucidated. We will also dissect a related RIP system (AlgW-MucA-MucB) that controls alginate biosynthesis in Pseudomonas aeruginosa. Understanding intracellular degradation is a key goal of basic research, with applications in biotechnology and medicine. For example, knowing how substrates are identified will enable improved bacterial expression of recombinant proteins, and controlled degradation systems will permit validation of new antibiotic targets. AAA+ proteases often play roles in the virulence of bacterial pathogens and thus can be antibiotic targets. Moreover, mutations in the MucA and MucB proteins of the P. aeruginosa RIP system increase mortality and morbidity in patients with cystic fibrosis. Finally, understanding DegS function will be relevant to studies of its human homolog, HtrA2/Omi, which contributes to caspase- independent apoptosis and cancer prevention. Relevance Understanding intracellular degradation is a key goal of basic research, with applications in biotechnology and medicine. For example, knowing how substrates are identified would allow improved bacterial expression of recombinant proteins, and controlled-degradation systems would permit validation of novel antibiotic targets. AAA+ proteases often play roles in the virulence of bacterial pathogens and thus can be antibiotic targets. Moreover, mutations in the MucA and MucB proteins of the P. aeruginosa RIP system increase mortality and morbidity in patients with cystic fibrosis. Finally, understanding DegS function will be relevant to studies of its human homolog, HtrA2/Omi, which contributes to caspase-independent apoptosis and cancer prevention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI016892-33
Application #
8238386
Study Section
Special Emphasis Panel (ZRG1-IDM-A (02))
Program Officer
Korpela, Jukka K
Project Start
1980-04-01
Project End
2013-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
33
Fiscal Year
2012
Total Cost
$744,514
Indirect Cost
$301,351
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Stein, Benjamin J; Grant, Robert A; Sauer, Robert T et al. (2016) Structural Basis of an N-Degron Adaptor with More Stringent Specificity. Structure 24:232-42
Olivares, Adrian O; Baker, Tania A; Sauer, Robert T (2016) Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines. Nat Rev Microbiol 14:33-44
Barthelme, Dominik; Sauer, Robert T (2016) Origin and Functional Evolution of the Cdc48/p97/VCP AAA+ Protein Unfolding and Remodeling Machine. J Mol Biol 428:1861-9
Hari, Sanjay B; Sauer, Robert T (2016) The AAA+ FtsH Protease Degrades an ssrA-Tagged Model Protein in the Inner Membrane of Escherichia coli. Biochemistry 55:5649-5652
Baytshtok, Vladimir; Fei, Xue; Grant, Robert A et al. (2016) A Structurally Dynamic Region of the HslU Intermediate Domain Controls Protein Degradation and ATP Hydrolysis. Structure 24:1766-1777
de Regt, Anna K; Kim, Seokhee; Sohn, Jungsan et al. (2015) A conserved activation cluster is required for allosteric communication in HtrA-family proteases. Structure 23:517-26
Totaro, Kyle A; Barthelme, Dominik; Simpson, Peter T et al. (2015) Substrate-guided optimization of the syringolins yields potent proteasome inhibitors with activity against leukemia cell lines. Bioorg Med Chem 23:6218-22
de Regt, Anna K; Baker, Tania A; Sauer, Robert T (2015) Steric clashes with bound OMP peptides activate the DegS stress-response protease. Proc Natl Acad Sci U S A 112:3326-31
Barthelme, Dominik; Jauregui, Ruben; Sauer, Robert T (2015) An ALS disease mutation in Cdc48/p97 impairs 20S proteasome binding and proteolytic communication. Protein Sci 24:1521-7
Baytshtok, Vladimir; Baker, Tania A; Sauer, Robert T (2015) Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases. Proc Natl Acad Sci U S A 112:5377-82

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