Protozoan parasites are single-celled, eukaryotic pathogens that cause among the most deadly and widespread diseases today. Combating this global scourge requires a more comprehensive understanding of parasite biology, as well as identifying and developing new targets for therapeutic intervention. Protozoan parasites deploy transmembrane adhesin proteins to mediate close and strong contact with the host cell for the purpose of invasion. Disease follows from successful invasion, making interfering with the invasion program an attractive therapeutic strategy. The adhesive contacts between parasite and host must ultimately be dismantled for parasite internalization and sealing of the host membrane. At the heart of this process are parasite rhomboid proteases that catalyze the essential cleavage of adhesin proteins. Rhomboid proteases are unusual membrane proteins with a serine protease active site assembled within the membrane. Biochemical complexity of these extraordinary enzymes has long presented obstacles to investigating their mechanism of action. We developed a pure enzyme reconstitution system for studying rhomboid catalysis directly, and identified the role for one protozoan rhomboid in host-cell invasion. Through these advances, we have recently built up a detailed structural and functional framework for understanding rhomboid catalysis, as well as developed methods to study malaria rhomboid enzymes directly. Capitalizing on these new advances, we aim to use our most informative engineered variants to delineate the enzymatic reaction, to investigate rhomboid protease conformation directly in membranes, and to decipher how protozoan rhomboid enzymes target their substrates. These insights, in turn, will be applied to the roles of parasite rhomboid enzymes in disease.

Public Health Relevance

Single-celled parasites cause many diseases including malaria, killing millions of people worldwide, and neurologic birth defects and death of AIDS patients within the US. These parasites must get inside human cells to cause disease, and in the past we discovered an unusual class of enzymes involved in cell entry. We are using innovative techniques to study how these enzymes function, and applying this knowledge to combating their roles in disease.

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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United States
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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:
Hwang, Jiwon; Ribbens, Diedre; Raychaudhuri, Sumana et al. (2016) A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP. EMBO J 35:2332-2349
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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