Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody. Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity. Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field.

Zymomonas mobilis is a non-pathogenic, facultatively-anaerobic Gram-negative bacterium, which has historically been used for the fermentation of alcoholic beverages in many tropical/sub-tropical countries. Due to its excellent ethanol-producing capabilities, significant effort has been undertaken over recent years to utilize it for industrial ‘bioethanol’ production. Its physiological and metabolic properties indicate that it may also be an excellent organism for the bio-production of many different types of organic molecules. Consequently, the aim of my thesis was to develop new molecular methodologies that would enable Z. mobilis to be ‘engineered’ for use in future ‘bioproduction’ endeavours. In the first part of my study, I analyzed the native (cryptic) plasmids present within a variety of Z. mobilis strains, including two poorly-studied Z. mobilis strains: NCIMB 11163 and NCIMB 8227. Several plasmid libraries containing restriction-digested fragments of Z. mobilis cryptic plasmid DNA were prepared, and their inserts were sequenced. This enabled the complete DNA sequences of three small (non-integrating, double-stranded DNA) cryptic plasmids to be determined: pZMO1A and pZMO7 from NCIMB 11163, and pZMO1B from NCIMB 8227. Their DNA sequences were analyzed using bioinformatic approaches, to identify open reading frames, and regions of DNA that were putatively involved in transcription or DNA replication. In the second part of this thesis, the minimally-replicating region from plasmid pZMO7 was used to construct a series of Escherichia coli-Z. mobilis shuttle vectors. These vectors were found to be stable within several Z. mobilis strains for over 60 generations without antibiotic selective pressure. A reliable and reproducible method based on quantitative real time PCR (Q-RT-PCR) was developed to accurately determine the copy number of cryptic plasmids and shuttle-vectors present in Z. mobilis cultures. The pZMO7-based shuttle vectors exhibited good compatibility with cryptic plasmids as well as the widely-used pZM2-based shuttle vectors. Genes encoding glutathione S-transferase (GST) as well as green and red fluorescent protein (GFP and RFP) reporters were cloned into various shuttle vector constructs; placing them under the control of endogenous (Ppdc) or exogenous (Plac and Ptac) promoters. Promoter strength was evaluated by quantifying the reporter gene expression. The plasmid-based expression of GFP and RFP was visualized within planktonic and biofilm cultures using confocal laser scanning microscopy (CLSM). Shuttle vector-based GST pull-down experiments were used to study intracellular protein-protein binding interactions. In the third part of my thesis, I explored the potential use of Z. mobilis for the bioproduction of isoprenoid (terpenoid) compounds. Five predicted sesquiterpene synthases (terpene cyclases) of unknown function from the dimorphic fungus Penicillium marneffei, and several terpene cyclases from several other bacteria, fungi and plants were initially functionally-analyzed in E. coli. Several cyclase genes were cloned into E. coli-Z. mobilis shuttle vectors for expression trials within Z. mobilis cells. In summary, this thesis describes the development of a variety of novel methodologies and genetic ‘tools’ that may be used to express heterologous genes within Z. mobilis cells. These will be invaluable for future studies concerned with exploring the biology and industrial applications of this ‘microbial cell factory’. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Influenza viruses cause significant mortality and morbidity worldwide due to seasonal oubreaks as well as occassional, and sometimes devastating, pandemics. Estimates state that approximately 5% of the adult and 20% of the child population is infected yearly, leading to approximately a half-million deaths and three million severe infections in non-pandemic years. Aside from globally-circulating strains, zoonotic outbreaks caused by avian strains are a constant threat. In 1997, the first human cases of H5N1 infections occurred and since then strains of this subtype have killed approximately 700 people causing a severe disease with as high as 60% lethality rate. In March 2013, a strain of the H7N9 subtype started an epizootic in China causing a severe respiratory disease reminiscent of H5N1 infections and with a 20% case fatality rate. In this thesis, we have studied the host responses as well the viral replication kinetics of H5N1 and H7N9 strains and compared then to those of mild H1N1 seasonal and 2009 pandemic strains. During early infections of A549 cells, we have shown that the H5N1 virus induced a more profound and functional change to the host proteome. All viruses induced the NRF2-mediated oxidative stress responses and the H7N9 and H5N1 strains downregulated fibronectin, a host protein vital to infection for human strains. Using mathematical modeling and extensive growth kinetic analysis, we showed that the H5N1 and H7N9 strains had higher peak titers and faster growth kinetics. This was due to an higher infection rate for the H7N9 strain and an higher production rate for the H5N1 strain, compared to the human viruses. Conversely, the 2009 pandemic H1N1 strain had the poorest replication kinetics, longest eclipse phase and lowest infection rates. These results point towards the higher level of cellular disruption during infection with highly pathogenic strains of influenza, which may be indicative of the more profound changes required to support growth of viruses with faster kinetics to higher titers. Furthermore, the greater changes in the cellular proteome that we have characterized in vitro may be connected to the significantly greater virulence associated with infection by avian viruses in vivo, opening a novel and productive avenue of investigation to understand viral virulence mechanisms. / October 2015

Plants, green algae and cyanobacteria perform photosynthetic conversion of sunlight into chemical energy in the permanently changing natural environment. For successful survival and growth photosynthetic organisms have developed complex sensing and signaling acclimation mechanisms. The environmentally dependent protein phosphorylation in photosynthetic membranes is implied in the adaptive responses; however, the molecular mechanisms of this regulation are still largely unknown. We used a mass spectrometry-based approach to achieve a comprehensive mapping of the in vivo protein phosphorylation sites within photosynthetic membranes from the green alga Chlamydomonas reinhardtii subjected to distinct environmental conditions known to affect the photosynthetic machinery. The state transitions process regulating the energy distribution between two photosystems, involves the temporal functional coupling of phosphorylated light-harvesting complexes II (LHCII) to photosystem I (PSI). During state transitions several of the thylakoid proteins undergo redox-controlled phosphorylation-dephosphorylation cycles. This work provided evidences suggesting that redox-dependent phosphorylation-induced structural changes of the minor LHCII antenna protein CP29 determine the affinity of LHCII for either of the two photosystems. In state 1 the doubly phosphorylated CP29 acts as a linker between the photosystem II (PSII) core and the trimeric LHCII whereas in state 2 this quadruply phosphorylated CP29 would migrate to PSI on the PsaH side and provide the docking of LHCII trimers to the PSI complex. Moreover, this study revealed that exposure of Chlamydomonas cells to high light stress caused hyperphosphorylation of CP29 at seven distinct residues and suggested that high light-induced hyperphosphorylation of CP29 may uncouple this protein together with LHCII from both photosystems to minimize the damaging effects of excess light. Reversible phosphorylation of the PSII reaction center proteins was shown to be essential for the maintenance of active PSII under high light stress. Particularly dephosphorylation of the light-damaged D1 protein, a central functional subunit of the PSII reaction center, is required for its degradation and replacement. We found in the alga the reversible D1 protein phosphorylation, which until our work, has been considered as plant-specific. We also discovered specific induction of thylakoid protein phosphorylation during adaptation of alga to limiting environmental CO2. One of the phosphorylated proteins has five phosphorylation sites at both serine and treonine residues. The discovered specific low-CO2- and redox-dependent protein phosphorylation may be an early adaptive and signalling response of the green alga to limitation in inorganic carbon. This work provides the first comprehensive insight into the network of environmentally regulated protein phosphorylation in algal photosynthetic membranes.

The most universal abiotic influence is temperature, and thus, thermotolerance, adaptations and response to thermal variation, is a fundamental factor shaping evolution. Prokaryotic life may have an upper thermal limit near \(150^{\circ}C\); however, eukaryotic survival is limited to \(50^{\circ}C\) – the thermal maximum for sustained biosynthesis and homeostasis. My research focuses on understanding the physiological and biochemical factors that limit eukaryotic thermotolerance, by studying an organism near the upper limit of all eukaryotes: Paralvinella sulfincola. P. sulfincola, a hydrothermal vent polychaete, has the broadest known thermal range of any metazoan: \((5-48^{\circ}C)\). This species, along with the mesotolerant congener with Paralvinella palmiformis, is found at vents along the Juan de Fuca Ridge, Washington, USA. Making an ideal study system, both species are found in similar habitats, genetically comparable, and amenable to recovery and shipboard experimentation. Here, I present data from a series of high pressure in vivo experiments that investigate stress response to variations in temperature, pH, sulfide concentration, and duration. Field work was coupled with a suite of biomolecular techniques including pyrosequencing, comparative proteomics, enzyme assays, and quantitative PCR. From this research, the first to quantify global protein and antioxidant responses to temperature in an extremely thermotolerant eukaryote, three primary conclusions can be reached. 1) Pronounced thermal tolerance in P. sulfincola is likely enabled by its constitutive expression of heat shock proteins and limited by its ability to quickly and appropriately respond to the commensurate increase in oxidative stress. 2) Thermal tolerance limits are likely negatively affected by synergistic multistress effects. 3) Antioxidant gene expression response differs significantly between chronically and acutely stressed treatments, supporting the theory that oxidative stress is limiting in this system.

Genetic mapping of complex traits has been successful over the last decade, with over 2,000 regions in the genome associated to disease. Yet, the translation of these findings into a better understanding of disease biology is not straightforward. The true promise of human genetics lies in its ability to explain disease etiology, and the need to translate genetic findings into a better understanding of biological processes is of great relevance to the community. We hypothesized that integrating genetics and protein- protein interaction (PPI) networks would shed light on the relationship among genes associated to complex traits, ultimately to help guide understanding of disease biology. First, we discuss the design, testing and implementation of a novel in silico approach (“DAPPLE”) to rigorously ask whether loci associated to complex traits code for proteins that form significantly connected networks. Using a high-confidence set of publically available physical interactions, we show that loci associated to autoimmune diseases code for proteins that assemble into significantly connected networks and that these networks are predictive of new genetic variants associated to the phenotypes in question. Next, we study variation in the electrocardiographic QT-interval, a heritable phenotype that when prolonged is a risk factor for cardiac arrhythmia and sudden cardiac death. We show that a large proportion of QT-associated loci encode proteins that are members of complexes identified by immunoprecipitations in mouse cardiac tissue of proteins known to be causal of Mendelian long-QT syndrome. For several of the identified proteins, we show they affect cardiac ion channel currents in model organisms. Using replication genotyping in 17,500 individuals, we use the complexes to identify genome-wide significant loci that would have otherwise been missed. Finally, we consider whether PPIs can be used to interpret rare and de novo variation discovered through recent technological advances in exome-sequencing. We report a highly connected network underlying de novo variants discovered in an autism trio exome-sequencing effort, and we design, test and implement a novel statistical framework (“DAPPLE/SEQ”) to analyze rare inherited variants in the context of PPIs in a way that significantly boosts power to detect association.

HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunnelling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. In this dissertation, we developed and characterized a lentiviral vector-based system to express Nef in T-cell lines and primary human peripheral blood mononuclear cells, and then used this system to perform a proteomic screen to identify Nef-associated host cell factors and better understand how Nef hijacks the T-cell machinery to maximize HIV production and dissemination. Bioinformatic and cell-based analysis of the resulting host factors revealed a mechanism by which Nef enhances nanotube formation. To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3). Wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Together, our findings identify the exocyst complex as a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Furthermore, linkages revealed between Nef and the exocyst complex suggest a new paradigm of exocyst involvement in polarized targeting for intercellular transfer of viral proteins and viruses.

Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) has become the preferred method for large-scale peptide and phosphopeptide identification and quantification. The dominance of LC-MS/MS is the result of improved chromatographic, mass spectrometry and bioinformatic technologies. The applications of these technological improvements drive biological innovation by expanding the realm of possible experimentation, facilitating the creation and evaluation of novel hypotheses. Such improvements are the focus of this dissertation. New technologies are presented and their proteome wide applications in biological systems are demonstrated. A comparison of common phosphopeptide enrichment methods is presented in chapter two, which demonstrates that a combination of methods provides non-overlapping data sets. This comparison was performed in mitotically arrested fission yeast, a previously unstudied system by phosphoproteomic methods. This chapter remarks upon phosphorylation site conservation between lower and higher eukaryotes, as a means of predicting potentially relevant phosphorylation events in mammals. A new protocol for tissue based peptide quantification is presented in chapter three. The large-scale application of this method is detailed in a system of mouse liver phosphorylation, between fasted and re-fed states. The effect of peptide and protein level false discovery rates on the accuracy of phosphorylation site quantification is highlighted. This method is a cost-effective alternative to available techniques, such as metabolic labeling, and expands the application of proteomics to include larger animals. Finally, an in depth analysis of quantitative LC-MS/MS based multiplexing is the subject of the last chapter. New techniques for peptide pre-fractionation and ion quantification are discussed, which improve proteome coverage and quantitative accuracy. This proteome-wide multiplexing is applied to an analysis of the budding yeast environmental stress response. Applicable methods of data processing and a means of obtaining biologically relevant information out of multidimensional proteomic data sets are discussed. In all chapters, the data presented represent the largest analyses of their kind. This dissertation provides a solid guide for future proteome-wide studies, focused on the identification and quantification of peptides and their posttranslational modifications.

Parkinson's disease (PD) is a progressive neurological disorder resulting from loss of dopaminergic neurons of the substantia nigra, in part due to mitochondrial dysfunction. The E3 ubiquitin ligase, PARKIN, and mitochondrial kinase, PINK1, found mutated in familial early onset recessive forms of PD play central roles in mitochondrial homeostasis, thus maintaining control of a diversity of cellular processes, including energy metabolism, calcium buffering, and cell death. Together, PARKIN and PINK1 control mitochondrial homeostasis via a signaling cascade in which depolarization-induced PINK1 stabilization and activation on the mitochondrial outer membrane (MOM) promotes recruitment of PARKIN. Consequently, the outer mitochondrial membrane is extensively decorated with ubiquitin, ultimately resulting in removal of the damaged organelles via mitophagy, the selective autophagic removal of mitochondria. While PARKIN has been demonstrated to ubiquitylate Porin, Mitofusin, and Miro proteins on the MOM, the full repertoire of PARKIN substrates - the PARKIN-dependent ubiquitylome - remains poorly defined. Here, large-scale quantitative diGlycine (diGly) capture proteomics was used to identify PARKIN-dependent ubiquitylation on lysine residues in proteins modified upon mitochondrial depolarization. Hundreds of ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN activity has the capacity to dramatically alter the ubiquitylation status of the mitochondrial proteome. Complementary interaction proteomics identified physical association of PARKIN with a cohort of MOM ubiquitylation targets, autophagy receptors, and the proteasome, interactions which were completely dependent upon mitochondrial damage and drastically reduced upon mutation of the active site residue, C431, found mutated in PD patients. Furthermore, structural and evolutionary analysis of PARKIN-dependent ubiquitylation events revealed extensive conservation of target sites on cytoplasmic domains in vertebrate and D. melanogaster MOM proteins. Parallel PINK1 interaction proteomics identified numerous subunits of the translocase of the outer mitochondrial membrane (TOMM) and a novel interactor, CLU1, shown to regulate mitochondrial morphology in lower eukaryotes. Positive genetic interaction between CLU1, PINK1, and PARKIN suggests the potential of a newly identified node of regulation for the PINK1/PARKIN pathway. These studies define how PARKIN and PINK1 re-sculpt the proteome to support mitochondrial homeostasis, ultimately contributing toward an improved understanding of their role in the progression of disease.

Bovine papillomavirus type 1 (BPV1) induces fibropapillomas in cattle, and has long served as a useful model virus to study the molecular biology of the papillomaviruses. The cellular transforming activity of BPV1 maps to its E5, E6 and E7 genes. While the cellular transformation function of BPV1 E5 is well elucidated, the biological functions of the BPV1 E6 and E7 oncoproteins are still largely unknown. To further our understanding of the cellular functions of BPV1 proteins, I performed an unbiased mass spectrometry-based proteomic analysis to identify their cellular interacting partners. I subsequently focused on characterizing the interactions of the BPV1 E6 and E7 proteins with some of their cellular interactors. I discovered Mastermind-like 1 (MAML1) and the other components of the Notch transcription complex as novel cellular interacting partners of BPV1 E6. A parallel proteomic screen performed in the laboratory using the HPV E6 proteins as baits identified MAML1 and the Notch transcription complex as interactors of the E6 proteins from beta-genus HPVs, but not those from the alpha-genus HPVs. Further investigation revealed that the beta-genus HPV E6 proteins repress the basal expression and transcriptional activation of endogenous Notch target genes in keratinocytes. For the BPV1 E7 protein, I confirmed a previously reported interaction with UBR4, and showed that this interaction is dependent on the UBR box of UBR4 and the N- terminal of E7. Since little is known about the biological function of UBR4, I performed a proteomic screen to identify its interactors. I identified the E2 ubiquitin-conjugating enzyme UBE2A as an interactor of UBR4, and showed that UBR4 is able to discharge ubiquitin from UBE2A in an in vitro discharge assay. Together with the UBR4 auto-ubiquitylation observed in an in vitro ubiquitylation assay, these results suggest that UBR4 can function as an E3 ubiquitin ligase. In summary, these studies lay the groundwork for further systems-level studies of the biological functions of papillomavirus proteins, identify the Notch signaling pathway as a novel target of cutaneous papillomaviruses such as BPV1 and beta-genus HPVs, and provide evidence that the N-recognin UBR4 can act as an E3 ubiquitin ligase.