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.

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The aim of this thesis is to in various ways explore protein expression in human normal tissue and in cancer and to apply that knowledge in biomarker discovery. In Paper I the prognostic significance of RNA-binding motif protein 3 (RBM3) is explored in malignant melanoma. To further evaluate the prognostic significance of RBM3 expression was assessed in 226 incident cases of malignant melanoma from the prospective populationbased cohort study Malmö Diet and Cancer Study using tissue microarray technique (TMA). RBM3 was shown to be down regulated in metastatic melanoma and high nuclear expression in the primary tumor was an independent marker of prolonged over all survival. As a tool to facilitate clinical biomarker studies the Human Protein Atlas has created a tissue dictionary as an introduction to human histology and histopathology. In Paper II this work is introduced. A cancer diagnosis can be a complex process with difficulties of establishing tumor type in localized disease or organ of origin in generalized disease. Immunohistochemically assisted diagnosis of cancer is common practice among pathologists where its application combined with known protein expression profiles of different cancer types, can strengthen or help dismiss a suspected diagnosis. In Paper III the diagnostic performance of 27 commonly used antibodies are tested in a predominantly metastatic, multicancer cohort using TMA technique. Overall these 27 diagnostic markers showed a low sensitivity and specificity for its intended use, highlighting the need for novel, more specific markers. Breast, ovarian, endometrial and ovarian cancers affect predominantly women. Differential diagnostics between these cancer types can be challenging. In Paper IV an algorithm, based on six different IHC markers, to differentiate between these cancer types is presented. A new diagnostic marker for breast cancer, namely ZAG is also introduced. In Paper V the transcriptomic landscape of the adrenal gland is explored by combining a transcriptomic approach with a immunohistochemistry based proteomic approach. In the adrenal gland we were able to detect 253 genes with an elevated pattern of expression in the adrenal gland, as compared to 31 other normal human tissue types analyzed. This combination of a transcriptomic and immunohistochemical approach provides a foundation for a deeper understanding of the adrenal glands function and physiology.

Die von der caprotec bioanalytics GmbH entwickelte und vermarktete Capture Compounds Mass SpectrometryTM (CCMS-Technologie) ermöglicht die spezifische Isolierung und Identifizierung von Proteinen, basierend auf ihre gezielte Wechselwirkung mit kleinen trifunktionalen Moleküle (sog. Capture CompoundsTM). Dadurch wird sowohl eine Reduzierung der Komplexität zellulärer Proteingemische als auch eine Anreicherung niedrig abundanter Proteine erreicht. In der vorliegenden Arbeit erfolgte die Synthese von neuartigen Capture Compounds, die dazu dienen sollen, unterschiedliche biochemische Fragestellungen zu beantworten und das Anwendungsspektrum der CCMS-Technologie stark erweitern. / The Capture Compound Mass SpectrometryTM (CCMS-Technology) is a novel technology developed and marketed by caprotec bioanalytics GmbH. CCMS allows the isolation of sub-proteomes based on specific interactions of target proteins with synthetic small molecules, called Capture Compounds (CCs). In this way, CCMS affords the functional reduction of complex protein mixtures that derived from e.g. cell lysates, and the enrichment and identification of low abundant proteins. In the present work, the synthesis of different novel Capture Compounds that helps studying different biological problems and overcoming present technological limitations of CCMS is described.

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.

<p>Type 2 diabetes mellitus (T2DM) is a heterogeneous disease affecting millions of people worldwide. Both genetic and environmental factors contribute to the pathogenesis. The disease is characterized by alterations in many genes and their products. Historically, genomic alterations have mainly been studied at the transcriptional level in diabetes research. However, transcriptional changes do not always lead to altered translation, which makes it important to measure changes at the protein level. Proteomic techniques offer the possibility of measuring multiple protein alterations simultaneously.</p><p>In this thesis, the proteomic technique surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) has been applied and evaluated in the context of T2DM research. Protocols for pancreatic islet and serum/plasma protein profiling and identification have been developed. In addition, the technique was used to analyze the influence of genetic background versus diabetic environment by determining serum protein profiles of individuals with normal glucose tolerance (NGT) and T2DM with or without family history of diabetes. In total thirteen serum proteins displayed different levels in serum from persons with NGT versus patients with T2DM. Among these proteins, apolipoprotein CIII, albumin and one yet unidentified protein could be classified as being changed because of different genetic backgrounds. On the other hand, ten proteins for instance transthyretin, differed as a result of the diabetic environment.</p><p>When plasma protein patterns of NGT and T2DM individuals characterized by differences in early insulin responses (EIR) were compared, nine proteins were found to be varying between the two groups. Of these proteins five were identified, namely two forms of transthyretin, hemoglobin α-chain, hemoglobin β-chain and apolipoprotein H. However no individual protein alone could explain the differences in EIR. In conclusion, SELDI-TOF MS has been successfully used in the context of T2DM research to identify proteins associated with family history of diabetes and β-bell function. </p>

Type 2 diabetes mellitus (T2DM) is a heterogeneous disease affecting millions of people worldwide. Both genetic and environmental factors contribute to the pathogenesis. The disease is characterized by alterations in many genes and their products. Historically, genomic alterations have mainly been studied at the transcriptional level in diabetes research. However, transcriptional changes do not always lead to altered translation, which makes it important to measure changes at the protein level. Proteomic techniques offer the possibility of measuring multiple protein alterations simultaneously. In this thesis, the proteomic technique surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) has been applied and evaluated in the context of T2DM research. Protocols for pancreatic islet and serum/plasma protein profiling and identification have been developed. In addition, the technique was used to analyze the influence of genetic background versus diabetic environment by determining serum protein profiles of individuals with normal glucose tolerance (NGT) and T2DM with or without family history of diabetes. In total thirteen serum proteins displayed different levels in serum from persons with NGT versus patients with T2DM. Among these proteins, apolipoprotein CIII, albumin and one yet unidentified protein could be classified as being changed because of different genetic backgrounds. On the other hand, ten proteins for instance transthyretin, differed as a result of the diabetic environment. When plasma protein patterns of NGT and T2DM individuals characterized by differences in early insulin responses (EIR) were compared, nine proteins were found to be varying between the two groups. Of these proteins five were identified, namely two forms of transthyretin, hemoglobin α-chain, hemoglobin β-chain and apolipoprotein H. However no individual protein alone could explain the differences in EIR. In conclusion, SELDI-TOF MS has been successfully used in the context of T2DM research to identify proteins associated with family history of diabetes and β-bell function.

The field of proteomics has rapidly expanded due to the completion of the human genome sequence. This thesis validates affinity-purified monospecific antibodies of polyclonal origin, for protein profiling in a broad spectrum of normal tissues and cells. Validation of antibodies is crucial for development of reliable binders for target proteins and this thesis evaluates the generation and application of large sets of msAbs in different settings. MsAbs were generated towards recombinant Protein Epitope Signature Tag (PrEST) antigens using a stringent affinity-purification strategy, presented in the first study. The specificity of msAbs was studied using reverse phase protein arrays and immunohistochemistry (IHC), and results presented over 90% success rate in the protein array analysis. In IHC, 81% of the msAbs displayed apparent specific staining in normal tissues. MsAbs were also compared with commercial analogs (cAbs) using IHC and Western blot. Results presented similar outcome between msAbs and cAbs in both applications, although interpretation suggested more extensive IHC staining patterns with msAbs than with monoclonal analogs. For antibody validation, an approach called paired antibodies was presented and involved the generation of two msAbs towards non-overlapping epitopes on the same protein. Similarities in protein detection between paired antibodies were studied using three different antibody-based methods. Similar results were observed in several applications, indicating that this strategy can be a useful tool for studying known and unknown proteins. Given the reliability of msAbs, they were also applied in a study investigating the impact of tissue fixatives on protein detection. The study showed that different fixation mechanisms appeared to affect protein recognition by indicating that aldehyde-based fixation, e.g. induced by neutral buffered formalin, was preferred for tissues used in IHC and non-aldehyde based fixation was applicable for tissues used in protein extraction analysis and Western blotting. Conclusively, validation results suggest that msAbs are reliable affinity binders that can be used as valuable tools for proteome-wide protein profiling in tissues and cells.

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.