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dc.contributor.authorStoller, Timothy J.
dc.date.accessioned2018-07-12T18:28:02Z
dc.date.available2018-07-12T18:28:02Z
dc.date.issued1988
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 49-10, Section: B, page: 4163.;Advisors: Dennis Shields.
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:8819448
dc.identifier.urihttps://hdl.handle.net/20.500.12202/3218
dc.description.abstractSomatostatin (SRIF) is a 14 amino acid peptide hormone that is synthesized as a larger precursor: preproSRIF. The goals of this project were: (i) To determine if cells which synthesize hormones that are not posttranslationally cleaved have processing enzymes capable of efficiently cleaving foreign prohormones; (ii) Investigate the role of the SRIF proregion in mediating ER to Golgi transport and targeting to the regulated secretory pathway; (iii) Determine the specificity of endoproteolytic cleavage using site-directed mutagenesis to alter the paired-basic residues in the processing site.;cDNA encoding preproSRIF was introduced into two mammalian cell lines, GH{dollar}\sb3{dollar} (rat anterior pituitary cell which synthesized growth hormone) and 3T3 (mouse fibroblasts) using a recombinant retrovirus. Clonal lines expressing preproSRIF were established and the proteolytic processing efficiency, specificity and kinetics of secretion determined. In GH{dollar}\sb3{dollar} cells, preproSRIF was efficiently processed to the mature hormone as determined by HPLC analysis and microsequencing. Surprisingly, mature SRIF was stored more efficiently than the endogenous growth hormone, 55% versus 5%.;Using in vitro site-directed mutagenesis, the paired-basic amino acids were changed to ArgArg and LysArg. Microsequencing showed that the mutant cleavage sites were accurately processed in GH{dollar}\sb3{dollar} cells, indicating either a relative lack of cleavage specificity or the presence of multiple amino acid-specific processing enzymes.;The role of the proregion in mediating intracellular transport and sorting was tested directly by expressing two fusion proteins: a chimera of the SRIF preproregion and ape {dollar}\alpha{dollar}-globin and ape {dollar}\alpha{dollar}-globin fused to an N-terminal signal sequence. The signal peptide of the latter chimera was accurately cleaved but globin was not secreted and rapidly turned over intracellularly (t{dollar}\sb{lcub}1/2{rcub}{dollar} = 5 min). In contrast, 30% of the newly synthesized proregion-globin was transported through the secretory pathway. Most significantly, 40% of the proSRIF-globin was proteolytically processed at the native cleavage site generating mature globin which was secreted. These results demonstrate that the propeptide can act as a universal transport sequence. (Abstract shortened with permission of author.).
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.titleThe role of prohormones in mediating intracellular sorting, processing and secretion
dc.typeDissertation


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