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dc.contributor.advisorSaghatelian, Alan
dc.contributor.authorLone, Anna Mari
dc.date.accessioned2013-03-14T20:27:52Z
dash.embargo.terms2015-02-06en_US
dc.date.issued2013-03-14
dc.date.submitted2012
dc.identifier.citationLone, Anna Mari. 2012. The Biochemistry and Physiology of Peptidases. Doctoral dissertation, Harvard University.en_US
dc.identifier.otherhttp://dissertations.umi.com/gsas.harvard:10693en
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:10406350
dc.description.abstractPeptidases regulate important physiological processes by controlling levels of bioactive peptides and occasionally through noncatalytic processes. This thesis presents a study of prolyl endopeptidase-like (PREPL), which is a peptidase involved in several human deletion syndromes, including hypotonia-cystinuria syndrome (HCS). Phenotypes tentatively attributed to PREPL deletion include hypotonia and decreased growth hormone (GH) levels. However, little is known about the mechanisms by which PREPL deletion causes these phenotypes. To better understand PREPL catalytic activity, we used an activity-based protein profiling fluorescence polarization screen to identify the first specific PREPL inhibitors. We proceeded to demonstrate the activity of these inhibitors in cells and discovered several classes of cell-active PREPL inhibitors. Further, one of these inhibitors, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile, was able to enter mouse brains. To characterize PREPL substrate specificity, we performed several substrate profiling experiments, but no substrates could be identified, in line with reports from other groups who used related approaches to attempt to identify PREPL substrates. To characterize any noncatalytic functions of PREPL, we used an affinity purification-mass spectrometry approach (AP-MS) to search for any protein-protein interactions of PREPL. We identified brain-expressed X-linked 2 (BEX2) as a novel interactor of PREPL, and confirmed this interaction by immunoblot. Several other proteins identified in the AP-MS experiment, including several members of the STRIPAK complex are being further investigated for possible PREPL interaction. To determine whether HCS phenotypes are in fact due to PREPL deletion and to delineate the molecular pathways involved, we generated a conditional PREPL knockout mouse. These mice were visibly smaller than wildtypes and growth curve analysis verified that from week three of life, there was a significant difference in weight between wildtype and knockout mice. Initial surface righting task experiments also indicate that PREPL knockout pups may have a hypotonia phenotype. In summary, we have developed several new tools for studying PREPL catalytic and noncatalytic function, demonstrated that PREPL deletion causes a GH-related growth deficiency and possible hypotonia and thus moved several steps closer to understanding the molecular mechanisms underlying PREPL deletion phenotypes.en_US
dc.description.sponsorshipChemistry and Chemical Biologyen_US
dc.language.isoen_USen_US
dash.licenseMETA_ONLY
dc.subjectBiochemistryen_US
dc.subjectCalcitonin Gene Related Peptideen_US
dc.subjectHypotonia-Cystinuria Syndromeen_US
dc.subjectMass spectrometryen_US
dc.subjectPeptidasesen_US
dc.subjectPeptide Profilingen_US
dc.subjectProlyl endopeptidase-likeen_US
dc.titleThe Biochemistry and Physiology of Peptidasesen_US
dc.typeThesis or Dissertationen_US
dash.embargo.until10000-01-01
thesis.degree.date2012en_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorHarvard Universityen_US
thesis.degree.leveldoctoralen_US
thesis.degree.namePh.D.en_US
dc.contributor.committeeMemberKahne, Danielen_US
dc.contributor.committeeMemberGray, Nathanaelen_US
dash.contributor.affiliatedLone, Anna Mari


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