Peptidases 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. / Chemistry and Chemical Biology

Prolyl endopeptidase SmPEP from the blood fluke Schistosoma mansoni is investigated here for the first time. This enzyme is potentially interesting as a drug target for the treatment of schistosomiasis. SmPEP was detected in the extract of adult worms by enzyme activity and immunoreactivity. Enzymatically active SmPEP was produced in the E. coli expression system and was chromatographically purified. The pH optimum of recombinant SmPEP was about 8. Substrate specificity analysis revealed that SmPEP cleaved peptide substrates by endopeptidase activity, however, macromolecular substrates were not fragmented. The residue preferences in the positions P3 to P1' were determined using synthetic fluorogenic peptide substrates. SmPEP was found to be highly sensitive to the inhibition by Z-Ala-Pro-CMK and Z-Arg-Pro-CHO. Primary screening of crystallization conditions for recombinant SmPEP was performed. " (In Czech)"

Celiac Disease (CD) is an autoimmune disorder that emerges due to the ingestion of gluten, a protein found in a variety of common grains such as wheat, rye, and barley. Approximately 1 in 100 individuals in the US suffer from CD, making it the most commonly diagnosed gastrointestinal disorder (Ciclitira et. al., 2005). These proline-rich gluten peptides are resistant to proteolysis and accumulate in the duodenum of the small intestine. Once in the duodenum, these peptides illicit an autoimmune response resulting in villous atrophy. Current treatment for CD requires a rigorous adherence to a gluten-free diet. Nevertheless, gluten-containing grains are ubiquitous in the western diet, so accidental exposure to gluten remains as a persistent threat. The approach of this project centers on genetically engineering a strain Lactobacillus reuteri to secrete a Myxococcus xanthus prolyl endopeptidase (PEP), an enzyme that hydrolyzes a peptide bond adjacent to an internal proline residue. The data from this study revealed that recombinant M. xanthus PEP purified from E. coli was effective in degrading Suc-Ala-Pro-pNA, a chromogenic substrate containing an internal proline residue. When introduced into a L. reuteri expression vector, mutations accumulated in the vector over the course of 5 days. These data suggested that toxicity was possibly associated with M. xanthus PEP and the amyl signal peptide.