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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI066025-09
Application #
8635274
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Mcgugan, Glen C
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Urban, SiniĊĦa; 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
Parussini, Fabiola; Tang, Qing; Moin, Syed M et al. (2012) Intramembrane proteolysis of Toxoplasma apical membrane antigen 1 facilitates host-cell invasion but is dispensable for replication. Proc Natl Acad Sci U S A 109:7463-8
Urban, Sinisa (2010) Taking the plunge: integrating structural, enzymatic and computational insights into a unified model for membrane-immersed rhomboid proteolysis. Biochem J 425:501-12
Urban, Sinisa (2009) Making the cut: central roles of intramembrane proteolysis in pathogenic microorganisms. Nat Rev Microbiol 7:411-23
Urban, Sinisa; Baker, Rosanna P (2008) In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells. Biol Chem 389:1107-15
Urban, Sinisa; Shi, Yigong (2008) Core principles of intramembrane proteolysis: comparison of rhomboid and site-2 family proteases. Curr Opin Struct Biol 18:432-41
Urban, Sinisa; Baker, Rosanna P (2008) In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells. Biol Chem :
Baxt, Leigh A; Baker, Rosanna P; Singh, Upinder et al. (2008) An Entamoeba histolytica rhomboid protease with atypical specificity cleaves a surface lectin involved in phagocytosis and immune evasion. Genes Dev 22:1636-46
Baker, Rosanna P; Young, Keith; Feng, Liang et al. (2007) Enzymatic analysis of a rhomboid intramembrane protease implicates transmembrane helix 5 as the lateral substrate gate. Proc Natl Acad Sci U S A 104:8257-62

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