Structural and biochemical characterization of rhomboid intramembrane proteases
Item
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Title
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Structural and biochemical characterization of rhomboid intramembrane proteases
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Identifier
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d_2009_2013:16afd9fa8566:10988
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identifier
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11435
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Creator
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Chavez, Jose L.,
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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 | Molecular biology | Intramembrane proteases | Membrane | Proteolysis | Regulated Intramembrane Proteolysis | Rhomboid | Spitz
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Abstract
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Intramembrane proteases are involved in multiple biological processes including cell growth and development, and apoptosis. There is no conservancy between hydrosoluble and membrane proteases. However, the catalytic residues and surrounding amino acids are absolutely conserved, suggesting that they both protein families share catalytic mechanisms but with two remarkable differences. (1) The ability of intramembrane proteases to cleave their substrates in the hydrophobic interior of the lipid bilayer, and (2) to do so in regions where the substrate displays a-helical conformation. D. melanogaster rhomboid-1 cleaves within the transmembrane domain region of epidermic growth factor receptor (EGFR) ligands Gurken, Keren and Spitz, resulting in their extracellular export. We designed substrate chimeras in which the transmembrane and cytoplasmic regions of Gurken, Keren and Spitz were preserved, while their EGFR ligand ectodomain was replaced by maltose binding protein. In vitro activity assays in detergent using purified components showed that rhomboid-1, H. sapiens RHBDL2, P. aeruginosa PA3086 and E. coli GlpG display comparable activity against these substrate chimeras. Mass spectrometry analysis of the N-terminal reaction product identified a single cleavage site after Ala138 for the Spitz chimeras, after Ala122 for the Keren chimeras, and after Ala245 for the Gurken chimeras that was identical for all rhomboids tested, suggesting a conservation of proteolytic profiles among prokaryotic and eukaryotic rhomboids. The identified cleavage site was located towards the N-terminal end of the transmembrane domain of each substrate. Positions that were sensitive to alanine scanning were further studied by introducing additional mutations to show that aside of ala in position P1, amino acids with low-helical propensities are necessary in the positions P2 and P1'. Finally, a bulky hydrophobic residue with a high helical propensity is important in P2' position to control the location of cleavage. We also carried out structural work and solved the N-terminal domain of Rhomboid and showed it displays high-affinity for membranes. Our work is put in a more general context by comparison with other intramembrane proteases and future work to unravel the mechanism of substrate binding and unwinding is also discussed.
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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
