Intracellular proteases of the AAA+ and HtrA families remove toxic proteins from cells and participate in cellular processes that promote health and prevent disease. Because protein synthesis is energetically expensive and proteolysis is irreversible, protein degradation must be carefully regulated to conserve precious cellular resources. Growing evidence suggests that some AAA+ and HtrA proteases can also help proteins refold and/or prevent their aggregation, but how a single enzyme can carry out these activities in addition to the seemingly antagonistic activity of protein degradation is unknown and represents the major goal of this proposal. Building on our experience and results from the previous funding period, we will use a combination of structural biology, biochemistry, protein-engineering, and molecular genetics to determine how protease versus chaperone activities are mediated by changes in protein conformation or assembly and are regulated by the binding of substrates and adaptor proteins. For example, we will determine the regulatory events and features that allow the archaeal AAA+ Cdc48 enzyme to function either as a disassembly chaperone or as a part of the Cdc48?20S proteasome. We will also test if this alternative proteasome operates in eukaryotic cells. Notably, inhibitors of the eukaryotic 20S peptidase are used to treat multiple myeloma and are active in animal models of autoimmune and inflammatory disease, and knowledge of which proteasomes are the actual targets would be an important advance. Our studies will identify mutations that eliminate just chaperone or just protease activity, allowing the importance of each activity to be quantified, identify new substrates and adaptors, reveal connections between enzyme function and biological mechanism, and allow the rational design of highly selective inhibitors for use in basic science and medicine. In pathogenic bacteria, enzymes of the AAA+ and HtrA families are required for virulent infection and are potential antibiotic targets. Changes in expression levels or malfunction of AAA+ and HtrA enzymes in humans have been linked to arthritis, cancer, vascular disease, macular degeneration, myopathy, diabetes, dementia, retardation, Parkinson's disease, and Alzheimer's disease. Although the molecular mechanisms are largely unknown, proteases and chaperones of the AAA+ and HtrA families have also been shown to play critical roles in a large number of diverse cellular pathways, including antigen presentation, cell-cycle progression, DNA repair/recombination, Golgi and nuclear membrane reassembly, autophagy, mitochondrial function, apoptosis, and sensing and combating stress, mutation, infection, and aging. Our studies will establish or clarify mechanistic connections between AAA+ and HtrA enzymes and biological function and dysfunction.

Public Health Relevance

Understanding how cellular enzymes help proteins to fold, avoid aggregation, and ultimately destroy proteins that are damaged and dangerous is an important goal of basic research, with applications in medicine and biotechnology. Enzymes of the AAA+ and HtrA families clear cells of toxic proteins, help some proteins to refold or avoid aggregation, and play important roles in many other cellular processes needed to promote health and prevent disease. Our studies will determine the molecular mechanisms that allow these important enzyme families to perform seemingly antagonistic functions and establish how these activities help to maintain the health of cells and organisms.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Korpela, Jukka K
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Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
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Hari, Sanjay B; Grant, Robert A; Sauer, Robert T (2018) Structural and Functional Analysis of E. coli Cyclopropane Fatty Acid Synthase. Structure 26:1251-1258.e3
Brown, Breann L; Kardon, Julia R; Sauer, Robert T et al. (2018) Structure of the Mitochondrial Aminolevulinic Acid Synthase, a Key Heme Biosynthetic Enzyme. Structure 26:580-589.e4
Amberg-Johnson, Katherine; Hari, Sanjay B; Ganesan, Suresh M et al. (2017) Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens. Elife 6:
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Baytshtok, Vladimir; Chen, Jiejin; Glynn, Steven E et al. (2017) Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation. J Biol Chem 292:5695-5704
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
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
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
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
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

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