Parasites Toxoplasma gondii and Plasmodium, the agent of malaria, are deadly human pathogens. These obligate intracellular parasites must invade host cells to survive, making understanding the parasite invasion machinery an important goal. At the heart of this machinery are rhomboid proteases, which catalyze the essential cleavage of parasite adhesin proteins that are required for attachment to host cells. Rhomboids are integral membrane proteins that cross the membrane seven times, and we previously deduced that they function as novel proteases; their transmembrane domains (TMDs) associate to form a serine protease active site within the membrane bilayer. Remarkably, cleavage of adhesins occurs within their TMDs. Such hydrolysis of peptide bonds within the normally hydrophobic environment of the membrane is a new paradigm in enzyme biochemistry. This paradigm is of wider importance to human health as various intramembrane proteases have recently been implicated as central players in Alzheimers Disease, hypercholesterolemia, and infection by pathogenic bacteria. However, the biochemical function of these unusual membrane enzymes is poorly understood. We seek to decipher how these enigmatic proteases function at the molecular level, with particular emphasis on their role in parasite invasion. Specifically, capitalizing on new biochemical methods for studying rhomboids that we have recently developed, we propose to investigate the following key issues: 1) physical basis of rhomboid substrate specificity compared to that of other intramembrane proteases, 2) arrangement and regulation of rhomboids in parasite membranes, 3) structural arrangement and function of rhomboid proteases, 4) development of small molecule inhibitors of rhomboid catalysis. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI066025-05
Application #
7371131
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Mcgugan, Glen C
Project Start
2005-06-16
Project End
2010-02-28
Budget Start
2008-03-01
Budget End
2009-02-28
Support Year
5
Fiscal Year
2008
Total Cost
$343,232
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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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