Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (Papers I-III) and a prostate cancer candidate biomarker identified and verified (Papers III-V). Furthermore, a perspective on the research area affinity proteomics and its’ employment in biomarker discovery, for improved understanding and potentially improved disease diagnosis, is provided. Paper I presents the results of a comparative plasma and serum protein profiling study, with a targeted biomarker discovery approach in the context of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma samples. Minor effects were found on the detected levels of more than 300 predicted or known plasma proteins. In Paper III, the detectability of proteins in plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was observed for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several antibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich immunoassay, as presented in Papers IV and V. Furthermore, the developed sandwich immunoassay targeting multiple distinct CNDP1 epitopes in more than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V). As an outcome of the present investigations and in parallel to studies within the Biobank profiling research group, valuable lessons from study design and multiplex antibody analysis of plasma within biomarker discovery to experimental, technical and biological verifications have been collected. / <p>QC 20130821</p>

The main approach to studying the proteome is a technique called data dependent acquisition (DDA). In DDA, peptides are analyzed by mass spectrometry to determine the protein composition of a biological isolate. However, DDA is limited in its ability to analyze the proteome, in that it only selects the most abundant ions for analysis, and different protein identifications can result even if the same sample is analyzed multiple times in succession. Data independent acquisition (DIA) is a newly developed method that should be able to solve these limitations and improve our ability to analyze the proteome. We used an implementation of DIA (SWATH) to perform relative protein quantitation in the model bacterial system, Clostridium stercorarium, using two different carbohydrate sources, and found that it was able to provide precise quantitation of proteins and was overall more consistent in its ability to identify components of the proteome than DDA. Relative quantitation of proteins is an important method that can determine which proteins are important to a biochemical process of interest. How we determine which proteins are differentially regulated between different conditions is an important question in proteomic analysis. We developed a new approach to analyzing differential protein expression using variation between biological replicates to determine which proteins are being differentially regulated between two conditions. This analysis showed that a large proportion of proteins identified by quantitative proteomic analysis can be differentially regulated and that these proteins are in fact related to biological processes. Analyzing changes in protein expression is a useful tool that can pinpoint many key processes in biological systems. However, these techniques fail to take into account that enzyme activity is regulated by other factors than controlling their level of expression. Activity based protein profiling (ABPP) is a method that can determine the activity state of an enzyme in whole cell proteomes. We found that enzyme activity can change in response to a number of different conditions and that these changes do not always correspond with compositional changes. Mass spectrometry techniques were also used to identify serine hydrolases and characterize their expression in this organism. / February 2016

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

Biological systems, their physical structure and their functions, are built, maintained, and controlled by the activity of enzymes. Understanding how enzymes contribute to the regulation of various pathways and processes allows us to gain a deeper understanding of the entirety of the biological system. As changes in enzyme activity are often essential for the pathogenesis of multiple and varied diseases, identifying these changes represents a crucial step to both understanding the disease and preventing its progression within the individual. Enzymes’ functional output can be controlled by numerous different mechanisms, including control of transcription and translation, subcellular localisation, co-factor interactions, or chemical modification to specific amino acids. Activity-based protein profiling allows the potential for activity of target enzymes to be measured, thereby gaining a more accurate representation of the functional state of the biological system. In this work, profiling differential enzyme activity allows the discovery of previously unknown links between metabolic regulatory enzymes and infection by the hepatitis C virus (HCV). The novel probe wortmannin-yne is described and is shown to be able to report on the activity multiple kinases, including MAPK1, whose activity is dysregulated during HCV replication. Novel probes designed to target a smaller selection of kinases, phosphatidylinositol kinases, are reported and are shown to be capable of measuring HCV-induced changes to not only kinase activity but also regulatory protein-protein interactions with the phosphoinositide kinases. Lastly, the role of microRNA-27b in the HCV-induced dysregulation of lipid metabolic enzymes is examined. Three novel targets of microRNA-27b are identified, and their dysregulation is shown to have an effect on the life cycle of HCV. Altogether, this work has developed new tools for the study of metabolic enzymes and identified new avenues of investigation into the dysregulation of lipid metabolism.

No description available.

As obligatory intracellular parasites, viruses are in a constant battle with their host to establish infection. They can facilitate their propagation by modulating host immune or metabolic pathways. This modulation involves targeting various molecular factors such as microRNAs (miRNA), enzymes, or small molecules. Understanding how viruses alter the chemical makeup of a cell is crucial to identifying what pathways are being targeted, furthering our understanding of the virus life cycle, and may aid in identifying biomarkers of disease. Here, we examine host-virus interactions in the context of two viruses, hepatitis c virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). First, the modulation of serine hydrolases by a pro-viral microRNA, miRNA-122, is investigated using activity-based protein profiling (ABPP). This study identifies a downstream target of miRNA-122 that is differentially activated during HCV infection which can be targeted pharmacologically to reduce HCV infectivity. Second, we apply similar techniques to identify serine hydrolase changes associated with SARS-CoV-2 infection. Results point towards enrichment of endocannabinoid metabolism which may offer an alternative therapeutic avenue for combating SARS-CoV-2 infection. Together, the work presented in this thesis provides avenues for further investigation into miRNA-122 interactions during HCV infection and endocannabinoid metabolism in SARS-CoV-2 infection.

The serine hydrolase (SH) enzyme superfamily is one of the largest and most diverse enzyme classes in eukaryotes and prokaryotes. The most virulent human malaria parasite Plasmodium falciparum has over 40 predicted serine hydrolases (SH). Prior investigation on a few of these have suggested their critical role in parasite biology. The majority of the SHs in P. falciparum have not been functionally characterized. Investigation of these uncharacterized SHs will provide new insights into essential features of parasite metabolism and possibly lead to new antimalarial targets. In this study, we have employed activity-based protein profiling (ABPP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to functionally characterize SHs. In our effort to profile plasmodial SHs using ABPP, we have identified a human erythrocyte SH, acylpeptide hydrolase (APEH) in the developing parasites. This finding is the first report of internalization of host hydrolytic enzyme by the parasite. Treatment of parasites with an APEH specific triazole urea inhibitor, AA74-1, caused growth inhibition in parasites with poor potency in the first replication cycle, however, the potency dramatically increased in the second cycle. We show that this unique growth inhibition profile is due to the inability of AA74-1 to inhibit parasite-internalized APEH in vivo. These findings suggest that internalization of active APEH by the parasite is essential for parasite survival. Lipases catalyze the hydrolysis of ester bonds of lipid species such as neutral lipids and phospholipids. Although roles of lipases in propagation, as well as virulence in various organisms, have been acknowledged, in P. falciparum lipases remain understudied. We combined LC-MS/MS with the SH-directed ABPP to identify lipases of SH superfamily in P. falciparum. We have identified 16 plasmodial SHs with putative lipase activity. Bioinformatics analysis of our identified lipases is consistent with our findings. We have screened a panel of various classes of SH inhibitors in a competitive ABPP. A plasmodial putative lipase was potently and specifically inhibited by human monoacylglycerol lipase inhibitor. This inhibition profile suggests it as a monoacylglycerol lipase which plays a role in releasing fatty acids from neutral lipid. This finding shows that how inhibitor screening can aid in building hypotheses on biological roles of an enzyme. Altogether, in this dissertation, we have presented a robust strategy of identifying and functionally characterizing SHs in P. falciparum, which opens the door to the discovery of new biological processes. / Doctor of Philosophy / Malaria contributed to nearly a half a million deaths in 2017. The vast majority of malaria-related deaths are due to the parasite Plasmodium falciparum. This parasite resides inside human red blood cells (erythrocytes) and grows rapidly during a 48 hour cycle. There are over 40 serine hydrolase (SH) superfamily proteins in the parasite. Biological functions of the majority of SHs in the parasite remains unknown. Study on these SHs will provide new insights into parasite biology, and possibly present new antimalarial drug targets. We used chemical biology techniques to identify and functionally characterize parasite SHs. In one study, we show the parasite intenalized a human erythrocyte SH, acylpeptide hydrolase (APEH). We used an APEH-specific inhibitor to investigate the biological significance of internalized APEH in parasite biology. Treatment of the parasite with the inhibitor resulted in parasite growth inhibition suggesting internalization of APEH is essential for parasite survival. Lipases are enzymes that aid in break down of lipids and have shown to be crucial for growth and pathogenicity in various organisms. Lipases and lipid catabolism remain understudied in the malaria parasite. We used mass spectrometry in our approach to identify 16 lipases in asexual parasites. We have also shown that screening with highly specific inhibitors can help in predicting biological function of a particular enzyme. In summary, in this body of work, we have presented an approach of studying SHs in the malaria parasite, which will provide new insights into parasite biology.

Maize is the staple food for a majority of people in Southern Africa, but plant diseases are responsible for at least 10% of crop production losses. Bacterial leaf streak (BLS) of maize was first reported in South Africa in 1949 and has not been reported elsewhere. Very little is known about the pathogen involved and therefore it is deemed necessary to compile a characteristic profile for the pathogen to prevent the possibility of major crop losses as a result of this disease. This study aimed to use biochemical and phenotypic methods to determine the specific characteristics of the causal agent of BLS. Diseased plant material showing symptoms of BLS were collected during the maize production seasons of 2012 and 2013 within South Africa’s maize production regions namely the North West, Free State, Gauteng and Northern Cape provinces. To prevent contamination, maize leaves were surface sterilised thoroughly before bacterial isolation commenced. Sections of the infected maize leaves were placed on GYC agar plates on which yellow, mucoid bacterial colonies after incubation for 24 to 48 hrs. The isolated bacteria were purified and the molecular identification of the bacteria was conducted in a related study. Although literature indicates that Xanthomonas campestris pv. zeae is the causal agent of BLS, pure cultures obtained from maize leaves showing characteristic symptoms of BLS were identified as species of Xanthomonas, Pantoea, and Enterobacter. To elucidate the pathogenicity of the isolated strains, pathogenicity tests based on Koch’s postulates were performed. Results from the pathogenicity tests confirmed that only the isolate Xanthomonas species was capable of inducing the characteristic BLS symptoms when healthy maize plants were inoculated with the suspected pathogens. It is important to inoculate the maize seedlings at the correct age (four-leaf stage) and the spray method is recommended. Re-isolation was repeated from the same plant material used during the initial isolation process but the isolation method was amended. The optimised isolation method involved the use of a dilution range and spread plate method. Colonies from this isolation technique grew as bright yellow colonies that were identified as Xanthomonas spp. This outcome indicates the importance of surface sterilisation, pulverisation and subsequent dilution of plant materials for isolation of bacterial pathogens from diseases plants. These isolates were used to create protein profiles with SDS-PAGE electrophoresis and carbon utilisation patterns with the Biolog® GN2 system. Protein profiling banding patterns was assessed based on presence/absence criteria. Highly similar protein profiles were observed among the X. campestris pv. zeae isolates but groupings of different protein profiles were determined when minor differences in the protein profiles was taken into account. Xanthomonas campestris pv. zeae was successfully distinguished from the X. axonopodis pv. vasculorum reference strain through unique SDS banding patterns. Banding patterns obtained from cultures grown in a liquid medium (tryptic soy broth) were of a higher quality than the banding patterns obtained from bacteria harvested from solid media (CYG agar plates). Carbon source utilisation data was used to evaluate the average well colour development obtained from each isolate. Statistically significant differences were found among some of the isolates, with some isolates being metabolically more active than other isolates. Substrate utilisation patterns produced by the isolates corresponded to previously published studies on various Xanthomonas species. The cell count of the samples used during carbon utilisation patterns must be standardised in order to obtain reliable results. During this study, the application of Koch’s postulates and two inoculation techniques confirmed that Xanthomonas campestris pv. zeae is the pathogen responsible for bacterial leaf streak of maize. Members of the Pantoea and Enterobacter genera were found on the leaf surface of maize plants infected with BLS but inoculations of healthy maize plants with these bacteria did not result in bacterial leaf streak symptoms on the maize plants. These bacteria were not pathogenic and were considered endophytes. The identified pathogen was characterised through protein and metabolic profiling. The protein profiles of the pathogen obtained through analysis of the major bands of the SDS-PAGE gels were highly similar and distinguishable from the Xanthomonas reference culture. Groupings within the X. campestris pv. zeae group was found when major and minor bands were considered, this may however be altered when the intensities of the bands are used during analysis. Carbon utilisation patterns were assessed using Biolog® GN2 plates. A metabolic fingerprint was created for the pathogen of BLS, it was possible to distinguish between X. campestris pv. zeae and other Xanthomonas strains based on the fingerprint. This fingerprint could be used to identify the pathogen. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015