Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-04T20:46:25Z No. of bitstreams: 1 MuhammadFaheemTese2016.pdf: 102451329 bytes, checksum: 190df4ef7dc2a9c25576fabb32681db8 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-04T20:46:36Z (GMT) No. of bitstreams: 1 MuhammadFaheemTese2016.pdf: 102451329 bytes, checksum: 190df4ef7dc2a9c25576fabb32681db8 (MD5) / Made available in DSpace on 2017-09-04T20:46:36Z (GMT). No. of bitstreams: 1 MuhammadFaheemTese2016.pdf: 102451329 bytes, checksum: 190df4ef7dc2a9c25576fabb32681db8 (MD5) Previous issue date: 2016-02-12 / Metagenomics techniques are now widely used for the search of new valuable enzymes of interest and other biotechnological products. Sophistication in the second-generation sequencing has significantly facilitated metagenomics technique for collection of huge amount of microbial genomic data. One of the current focuses in science is to seek the interpretation and transformation of the collected genomic data into functional proteomics data. Combination of structural biology and genomic data is one way to achieve such goal. In this study we have assessed a novel bacterial protein selected on a screen for activity on carbohydrates in a microbial metagenomic library from the gut of Capra hircus. Initial sequence analysis of the open reading frame (ORF) for this selected novel bacterial protein indicated that it could be annotated as an uncharacterized novel bacterial cell wall modifying enzyme. Sequence analysis of the protein has shown that it carries three domains: an N-terminus cysteine protease, a peptidoglycan binding (PGBD) and a C-terminus Src-Homology 3 (SH3) bacterial domain. Later with homology modeling we have observed that it carries an additional N-terminus domain with LCI fold. We have successfully cloned, expressed and purified this Capra hircus putative cysteine protease (PCP). Autoproteolytic activity has been observed for PCP, which was inhibited with protease inhibitors cocktail. We have observed that the autoproteolytic activity is carried either by the second or third domain of PCP. This protein has shown cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most of bacterial cell wall modifying enzymes. Ampicillin binding to PCP was further evaluated with fluorimetric analysis. PCP structure was modeled by homology modeling with good validation statistics and in agreement with circular dichroism data. The domains of PCP have conserved LCI, Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), PGBD and SH3 folds. It has a conserved active site dyad, Cys100 and His161, which is a signature of cysteine proteases. Furthermore, the overall architecture of the model was assembled in SAXS generated density map. Initial protein crystals are also obtained for the last two domains, which diffracted to very low resolution. / ***

Sperm protein, enterokinase and agrin (SEA) modules are small folds within large heavily glycosylated modular proteins. Because decreased expression of SEA-containing proteins such as perlecan (PLN) can lead to diseases such as Schwartz-Jampel syndrome (SJS), characteristics of the PLN SEA module including folding, potential for autocleavage, and protein binding were studied. Sequence analyses, recombinant protein evaluation, and a yeast two-hybrid screen were used to study the PLN SEA module and compare it to the mucin (MUC) 1 SEA module. In silico modeling of the PLN SEA module demonstrated a well conserved α/β sandwich fold. Experiments with expressed proteins showed that unlike MUC1, the PLN SEA module does not autocleave. Two-hybrid screening identified four “high confidence” proteins as potential binding partners which were explored in preliminary experiments. Together, these results demonstrate that PLN SEA module is unique and its properties cannot be generalized with other SEA module proteins such as MUC1.

Serine beta-lactamase-like protein (LACTB) is a tumour suppressor that modulates mitochondrial lipid metabolism and induces differentiation of breast cancer cells. This is achieved by the LACTB-dependent downregulation of phosphatidylserine- decarboxylase (PISD) which subsequently leads to decreases in the amounts of phosphatidylethanolamines and lysophosphatidylethanolamines in mitochondrial membranes. However, PISD was shown to not be a direct substrate of the LACTB enzyme what leaves the identity of the LACTB substrate an open question. To fill this important gap in the mechanism of the LACTB tumour suppressive pathway, this diploma thesis was focused on finding a physiological substrate of LACTB via Proteomic Identification of protease Cleavage Sites (PICS) assay. For this purpose, the other sub-aims of this project were to isolate recombinant wild-type LACTB and its catalytic mutant, to reveal ideal in vitro conditions for LACTB activity and to find out the requirements needed for LACTB multimerization. My results show that in vitro activity of LACTB is increased in the presence of higher pH and calcium ions. I also show that higher LACTB multimeric forms are bound together via disulfide bonds as they disintegrate after treatment with dithiothreitol. Furthermore, and most importantly, I show...