Biochemical and structural characterization of di-aspartyl intramembrane proteases
Item
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Title
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Biochemical and structural characterization of di-aspartyl intramembrane proteases
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Identifier
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d_2009_2013:436f68766751:10800
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identifier
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11013
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Creator
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Torres Arancivia, Celia Mirtha,
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Contributor
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Iban Ubarretxena
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Date
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2011
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biochemistry | gamma secretase | intramembrane protease | presenilin
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Abstract
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A new era in the study of proteases was initiated with the discovery of intramembrane-cleaving proteases (I-CLiPs). In an unusual mechanism, I-CLiPs are able to catalyze peptide bond cleavage of substrates within the membrane bilayer, an environment poorly permeable to water. Here we will focus on gamma-secretase and presenilin(PS)-like proteases, membrane proteases that use two aspartic acids as catalytic residues and constitute the most relevant family of I-CLiPs. We first describe the purification of gamma-secretase from a mammalian expression system. We used this preparation for negative-stain single-particle electron microscopy and determined the structure of a native-like catalytically active gamma-secretase complex at a resolution of 25 A. Antibody labeling of the extracellular domain of Nicastrin was employed to ascertain the topology of the reconstruction. In addition, active site labeling with a gold-coupled inhibitor demonstrated that gamma-secretase contains a single active site facing a large conical internal cavity.;Secondly, we used the yeast P. pastoris as an expression system to isolate milligram quantities of active and fully mature gamma-secretase. The higher yield of gamma-secretase obtained from this system allowed the addition of a size exclusion chromatography step to the purification procedure to reduce the structural heterogeneity of the preparation and thereby improve the structural analyses.;Finally, we provide novel proof for the existence of a presenilin-like protease in the euryarchaeota M. marisnigri (MCMJR1). We tested in vitro protease activity on chimeric protein substrates containing transmembrane domain (TMD) regions and found that MCMJR1 was indeed able to generate a proteolytic product. Mutagenesis confirmed that two aspartic acid residues were essential for the activity of the protease. Also, the activity of MCMJR1 was blocked when an inhibitor of gamma-secretase was incubated with the enzyme. Mass spectrometry analysis revealed that MCMJR1 cleaves the chimeric substrates close to the middle of the TMD with promiscuous peptide bond selectivity.;In conclusion, our study has enhanced the understanding of PS/gamma-secretase and provided new routes for functional and structural studies on di-aspartyl intramembrane proteases, both of which are pivotal in unraveling the mechanistic details of intramembrane proteolysis in biology and disease.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Biochemistry
