Thesis advisor: Eranthie Weerapana / Activity-based protein profiling (ABPP) is a chemical proteomic technique that allows for selective labeling, visualization, and enrichment of the subset of active enzymes in a complex proteome. Given the dominant role of posttranslational modifications in regulating protein function in vivo, ABPP provides a direct readout of activity that is not attained through traditional proteomic methods. The first application of chemical proteomics in C. elegans was used to identify dysregulated serine hydrolase and cysteine-mediated protein activities in the long-lived daf-2 mutant, revealing LBP-3, K02D7.1, and C23H4.2 as novel regulators of lifespan and dauer formation. The tools of ABPP were also utilized in studying protein interactions at the host-pathogen interface of V. cholerae infection, discovering four pathogen-secreted proteases that alter the biochemical composition of the host, decrease the activity of host serine hydrolases, and inhibit bacterial binding by a host-secreted lectin. Lastly, ABPP was used to study the targets of protein arginine deiminases (PADs) using a citrulline-specific activity-based probe (ABP), highlighting its utility in detecting biologically relevant PAD substrates as well as identifying mRNA processing factors as previously unknown targets of PAD. Taken together, these studies demonstrate the ability of ABPP to discover novel protein regulators of physiological and pathological processes. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Hepatitis C virus (HCV) is a growing health concern in Canada and around the world, as it currently infects 3% of the global population. While there is no vaccine available against this virus, novel and effective treatment regimens have improved prospects for the cure of HCV. Complications caused by HCV can lead to severe liver disease and even death. The limited viral proteome forces HCV to rely heavily on various host factors for its replication. Additionally HCV modulates the host physiology to facilitate its pathogenesis; consequently, the in dept study of essential host-virus interactions expands our understandingof how the virus and related species commandere host cell machinery. This understanding can help create new therapeutic strategies, which may have applications towards HCV and other related RNA viruses. While numerous studies have demonstrated that HCV modulates the abundance of various host proteins, the systematic study of the virus’s effect on the enzymatic activity has been relatively unexplored. For this reason, activity-based protein profiling (ABPP) was applied to study the changes in the activity of host enzymes during HCV replication. ABPP is a functional proteomics technique that employs active site-directed probe (ABP) to report on the activity of enzymes within complex proteomes, such as living cells. Herein, directed and non-directed ABPs were employed for specific as well as global profiling of the alterations in the activity of cellular enzymes during HCV replication. As a result, essential host enzymes that are differentially active during HCV infection were identified. Furthermore, I have developed a quantitative ABPP method for relative quantification of the cellular enzymes activity during HCV infection. These results contribute to the discovery of disease-associated biomarkers, with diagnostic significance, and aid in the identification of potential targets for therapeutic interventions. In addition to developing protein-based tools to study host-virus interactions, I employed a novel technique to investigate the interactions of micro-RNA 122 (miR-122), an essential HCV host factor, with the viral RNA genome. This in vitro screening approach, interrogates the folding of HCV RNA using viral RNA-coated magnetic bead (VRB) to determine target site accessibility for RNA silencing. This method predicts the relative affinity of small RNAs towards HCV genomic RNA that are not easily predicted by informatic means, and led to discovery of potent miR-122 interaction site within the large, highly-structured HCV RNA genome. For that reason, VRB assay may represent an attractive tool for the examination of target site accessibility for RNA silencing.

Thesis advisor: Eranthie Weerapana / Cysteine residues on proteins play important catalytic and regulatory roles in complex proteomes. These functional residues can be modified under physiological conditions by posttranslational modifications (PTMs) to regulate protein activities and modulate cysteine reactivity. Many PTMs are highly labile and dynamic, rendering it difficult to detect modified proteins within complex systems. To contribute to the chemical-proteomic methods currently available, chemical probe-Mass Spectrometry (MS) platforms were developed to study oxidative cysteine modifications. A MS platform for the assessment of S-nitrosation in vitro identified Cys329 of Cathepsin D (CTSD) as highly sensitive to S-nitrosothiol formation. To achieve a more physiological relevant representation of S-nitrosation, this platform was later adapted for study in live cells using a caged electrophile, Caged BK. Additionally, oscillation of cysteine oxidation as a function of circadian rhythm in Drosophila melanogaster and human samples was explored. As a compliment to these MS platforms, a 4-aminopiperidine-based cysteine-reactive probe library was developed. These probes have been used to target specific reactive cysteines as an alternate way to regulate protein function and can be used as tools to provide insight into the roles of these residues in protein activities. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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.

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.

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.