Infection by protozoan parasites causes some of the most prevalent and devastating diseases worldwide. With a sparse yet dwindling arsenal of useful drugs and a paucity of effective vaccines, identifying new targets for therapeutic intervention has become imperative. Despite their enormous diversity, many protozoan parasites rely upon a common strategy centered on a repertoire of transmembrane adhesins to gain close contact with host cells. Severing enzymes are then often deployed to modulate these initial interactions. At the core of this strategy are parasite-encoded rhomboid proteases that cleave many different families of adhesins in diverse intracellular and extracellular parasites. Recent advances in genetic manipulation have validated these enzymes as promising therapeutic targets for remarkably diverse parasitic diseases. Yet the promise of this exciting progress rests on deciphering the enzymatic mechanism of rhomboid proteases at sufficient clarity to guide therapeutic targeting. Rhomboid proteases pose a challenge to existing enzyme paradigms: their conserved six transmembrane segments fold into a helical bundle inside the cell membrane with a novel protease active site at its core. Understanding the principles underlying such membrane-immersed catalysis is lacking due to a paucity of tools with which to study it in quantitative and unbiased ways. We recently developed the first quantitative assays for interrogating rhomboid architectural stability, reaction kinetics directly within the membrane, and transmembrane protein dynamics using spectroscopic methods. Our preliminary studies suggest an unanticipated new mechanism of action for membrane-immersed proteolysis, a means for it's direct regulation by cellular ions, and directly observed the first catalytic comple between enzyme and substrate. Capitalizing on these advances, we propose to leverage our newly developed methods to delineate rhomboid enzyme mechanism in quantitative terms. The outcome of these lines of investigation have direct and immediate implication on targeting these key enzymes for the treatment of various parasitic diseases that kill millions of people worldwide.

Public Health Relevance

Single-celled parasites infect much of the world's population, causing millions of deaths and billions of dollars in lost revenue each year. Even in advanced countries like the United States, these devastating microbes remain a leading cause of neurological birth defects and death of AIDS patients. In response to a dwindling supply of useful drugs for treating these infections, we are studying an enzyme class that many parasites rely upon to cause infection, and, if we understand its inner workings precisely, could be targeted as a new line of therapeutics.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Mcgugan, Glen C
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Johns Hopkins University
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Baker, R P; Urban, S (2017) An Inducible Reconstitution System for the Real-Time Kinetic Analysis of Protease Activity and Inhibition Inside the Membrane. Methods Enzymol 584:229-253
Cho, Sangwoo; Dickey, Seth W; Urban, Siniša (2016) Crystal Structures and Inhibition Kinetics Reveal a Two-Stage Catalytic Mechanism with Drug Design Implications for Rhomboid Proteolysis. Mol Cell 61:329-340
Krishnamurthy, Shruthi; Deng, Bin; Del Rio, Roxana et al. (2016) Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail. MBio 7:
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Urban, Siniša (2016) Nicastrin guards Alzheimer's gate. Proc Natl Acad Sci U S A 113:1112-4
Riestra, Angelica M; Gandhi, Shiv; Sweredoski, Michael J et al. (2015) A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells. PLoS Pathog 11:e1005294
Baker, Rosanna P; Urban, Siniša (2015) Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating. Nature 523:101-5
Urban, Siniša; Moin, Syed M (2014) A subset of membrane-altering agents and ?-secretase modulators provoke nonsubstrate cleavage by rhomboid proteases. Cell Rep 8:1241-7
Dickey, Seth W; Baker, Rosanna P; Cho, Sangwoo et al. (2013) Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity. Cell 155:1270-81
Zhou, Yanzi; Moin, Syed M; Urban, Sinisa et al. (2012) An internal water-retention site in the rhomboid intramembrane protease GlpG ensures catalytic efficiency. Structure 20:1255-63

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