Signal Transduction is mediated by protein complexes whose spatial- and temporal-distribution, composition and function within cells are often regulated by different post-translational modifications (PTM). As PTMs add or subtract a specific mass difference to a protein, mass spectrometry becomes very amenable for modification analysis. These modifications have conventionally been monitored by fragmenting the modified protein or peptide by collision induced dissociation (CID) within the mass spectrometer, and then screening for the characteristic neutral fragment or fragment ion (marker ion), which is particular to the modification in question. Unfortunately, there are two major issues with respect to the traditional mass spectrometric analysis of PTMs: (1) as there are over 300 known types of modifications, the characteristic fragmentation of only a fraction of these modifications has been studied and (2) the traditional mass spectrometric approaches can only monitor these modifications sequentially, and thus comprehensive modification analysis would be unfeasible considering the breadth of PTMs. The following work aims to address these issues by (1) analyzing PTMs that have never been characterized mass spectrometrically and (2) developing a multiplexed technique for comprehensive PTM monitoring by simultaneously screening for all known characteristic fragments. With respect to the first issue, the characteristic fragmentation of lipid modifications and HNO-induced modifications was investigated. The most prevalent indicator(s) of the modification within the mass spectra are as follows: fragmentation of N-terminal myristoylated peptides produced marker ions at 240 and 268 Th, fragmentation of cysteine farnesylated peptides produced a marker ion at 205 Th and a neutral fragment of 204 Da, and fragmentation of cysteine palmitoylated peptides produced a neutral fragment of 272 Th. For HNO-induced modifications, fragmentation of the sulfinamide- and sulfinic acid-modified peptides produced a neutral fragment of 65 Da and 66 Da, respectively. With respect to the second issue, a multiplexed technique for monitoring modifications that fragment as neutral losses, termed Multiple Neutral Loss Monitoring (MNM), has been developed, successfully validated, and then shown to be the most sensitive approach for PTM analysis. MNM, combined with a second multiplexed approach, targeted Multiple Precursor Ion Monitoring, has been used to provide a comprehensive PTM analysis. / Science, Faculty of / Chemistry, Department of / Graduate

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Diabetes is a complex disease characterised by hyperglycaemia, which often leads to microvascular complications including diabetic nephropathy and cardiomyopathy. In this thesis, I have investigated the role of Annexin-A1 (ANXA1), an endogenous anti-inflammatory peptide, in two experimental murine models of diabetes caused by streptozotocin (STZ) or high-fat high-sugar diet (HFD), which mimic type-1 (T1DM) and type-2 diabetes (T2DM) respectively. I have also investigated the levels of ANXA1 in patients with either T1DM or T2DM. Patients with T1DM have increased plasma ANXA1 levels. In a murine models of type 1 diabetes loss of endogenous ANXA1 aggravates both cardiac and renal dysfunction in mice. Specifically, I have shown that key mediators of the MAPK pathway (p38, JNK and ERK1/2) are constitutively activated in ANXA1-/- mice, and activation of these pathways is exacerbated in diabetic ANXA1-/- mice. Administration of human recombinant (hr) ANXA1 did not alter the diabetic phenotype in diabetic WT mice, but attenuated the cardiac and renal dysfunction caused by STZ. Interestingly, late administration of ANXA1 (after significant cardiac and renal dysfunction had already developed) halted the progression of both cardiac and renal dysfunction. Patients with T2DM have increased plasma ANXA1 levels. HFD-fed ANXA1-/- mice have a more severe diabetic phenotype compared to HFD-fed WT mice. Therapeutic administration of hrANXA1 prevented the development of a diabetic phenotype. Specifically, I have shown that the insulin signalling pathway is further perturbed in diabetic mice resulting in severe insulin resistance, and that these signalling abnormalities were prevented by therapeutic administration of hrANXA1. In addition, loss of endogenous ANXA1 aggravates both cardiac and renal dysfunction in mice with experimental T2DM. The GTPase RhoA is constituently activated in ANXA1-/- mice leading downstream activation of MYPT1. Feeding a HFD also activated the small GTPase RhoA, leading to increased MYPT1 activity, which could be attenuated with treatment with hrANXA1. Mice subjected to HFD for 12 weeks had a more 'leaky' blood brain barrier (BBB), which is further exacerbated in ANXA1-/- mice fed a HFD. Compared to mice fed a chow diet, mice fed a HFD had an augmented CD4+ T-cell profile; with a clear decline in CD4+FoxP3+ (anti-inflammatory) and increase in CD4+RORgt+ (pro-inflammatory) cells. Administration of hrANXA1 to mice fed on HFD restored BBB integrity and CD4+ T-cells profile similar to mice fed on normal chow diet. Mice fed a HFD also had more activated CD4+ T-cells, which adhered more readily and transmigrated through a brain endothelial mono-layer ex vivo. In contrast, administration of hrANXA1 to mice fed on HFD reduced re-activity of CD4+ T-cells, reducing the number of adherent CD4+ T-cells to the brain endothelial mono-layer.

Sepsis is the leading cause of death in ICU patients. Patients with sepsis may develop sepsis-related cardiac dysfunction. The presence of this cardiac dysfunction can increase the mortality rate from 40% to 70%. Diabetes is a chronic disease that manifests as an elevation in blood glucose. Patients with diabetes are more susceptible to, and at increasing risk of developing, infections and subsequently sepsis. Activation of the NF-ĸB pathway plays a crucial role in the pathophysiology of sepsis-associated cardiac dysfunction and diabetic cardiomyopathy. The effect of diabetes on outcomes in patients with sepsis is highly controversial and it is still unclear whether pre-existing T2DM augments the cardiac dysfunction associated with sepsis and whether activation of NF-ĸB drives the cardiac dysfunction in T2DM/sepsis patients. In this thesis, I have first developed two models of high fat diet (HFD)-induced diabetes and diabetic cardiomyopathy in mice. Then, I developed two models of sepsis associated cardiac dysfunction using lipopolysaccharide (LPS) or caecumligation and puncture (CLP) in diabetic mice. I have demonstrated that mice with pre-existing T2DM exhibit a significantly greater cardiac (organ) dysfunction after challenge with either (low dose) LPS or mild CLP surgery. The exacerbated cardiac dysfunction was accompanied by an increase in NF-ĸB activation and reduction in Akt phosphorylation in the heart and an increase in the serum levels of proinflammatory cytokines. The increase in cardiac dysfunction, as well as the increase in the activation of NF-ĸB, caused by sepsis in animals with T2DM was largely attenuated by treatment with a selective IĸB kinase inhibitor (IKK-16) or a DPP-4 inhibitor (linagliptin). Thus, excessive activation of NF-ĸB in animals with diabetes/sepsis drives the observed excessive cardiac dysfunction, and that inhibition of NF-ĸB may be a useful target to treat the excessive inflammation and sepsis associated cardiac (organ) dysfunction in patients with T2DM and sepsis.

Vaccinia virus encodes at least eight proteins that are modified post-translationally by the addition of a 16-carbon saturated fatty acid through thioester linkage to cysteine residues. This is referred to as palmitylation of proteins. The purpose of this work was to gain an understanding of palmitylation, focusing on what defined the substrate for the modification, and the biological relevance of protein palmitylation in the vaccinia virus life cycle. A systematic approach was taken to identify the genes in vaccinia virus that encode these proteins. We found that vaccinia virus palmitylproteins are of the "late" temporal class, associate with intracellular membranes, and are specific for a particular form of the infectious virion. These criteria were used to narrow the number of genes expressed by vaccinia virus that potentially encode palmitylproteins. The "candidate" palmitylprotein genes were cloned and transiently expressed in mammalian tissue culture cells and analyzed for incorporation of palmitic acid. In addition to three previously identified vaccinia virus palmitylproteins, three new palmitylproteins were identified. The six known palmitylprotein genes were mutated to determine the site of modification, leading to the identification of the modification site for four of the six proteins. One of the proteins, p37, was analyzed further for biological significance of the palmitate modification. A recombinant vaccinia virus was constructed that did not express the wild-type palmitylated form of p37, but expressed a nonpalmitylated form of the protein instead. This virus was severely inhibited from proceeding past a particular morphogenetic stage, leading to an attenuated phenotype in tissue culture systems. Although the expression of the nonpalmitylated protein appeared normal compared to the wild-type protein, the lack of the palmityl moiety resulted in the loss of a targeting signal that directed the protein to its normal intracellular location. By this work, significant contributions have been made toward understanding the process of protein palmitylation. We have identified, at least for vaccinia virus, primary structural determinants specifying the modification, leading to the identification of a palmitylation motif. Considering the attenuated phenotype of the mutant virus, our conclusion is that palmitylation is necessary for biological function, at least for p37. / Graduation date: 1999

The attachment of myristic acid to the N-terminal glycine residue of many eukaryotic and viral proteins is often essential for the acquisition of the protein's biological activity. Vaccinia virus (VV), the prototype member of the Poxviridae, expresses several myristylated proteins during the course of infection. Only one of these proteins, L1R, has been identified and characterized. Experiments were performed to identify and analyze four additional VV myristylproteins. These proteins were identified as the A-type inclusion protein (92 kDa), G9R (39 kDa), A16L (36 kDa), and E7R (17 kDa). The latter three proteins were shown to be myristylated on an N-terminal glycine residue. Additional studies demonstrated that both A16L and E7R are soluble proteins, unlike L1R, which is a constituent of the viral envelope. Furthermore, A16L could not be detected in either purified extracellular enveloped virus (EEV) or in intracellular mature virus (IMV). These are the two predominant forms of infectious virions produced during a VV infection. E7R was detected in EEV and, to a lesser extent, in IMV. Unlike the other proteins, the amino terminal sequence of the A-type inclusion protein did not fit the consensus sequence for N-myristylation (M-G-X-X-X-S/T/A/C/N), suggesting that it was internally myristylated. A combination of studies revealed that the protein is both myristylated and palmitylated. Addition of each acyl group could be separated temporally: myristylation occured co-translationally, while palmitylation occurred post-translationally. Genetic analyses of lysine doublets and arginine/lysine doublets within the A-type inclusion protein indicated that these sites are not utilized for myristylation. This is in contrast to the precursors of TNFoc and Ilia which are internally-myristylated on a lysine doublet. It is not clear why this protein would be both myristylated and palmitylated. Only class four palmitylproteins, such as the Src family of proteins, have been shown to be both myristylated and palmitylated. The A-type inclusion protein expressed by cowpox virus forms a large symmetrical matix in the cytoplasm of infected cells and generally contains mature virions. It is possible, therefore, that the function of acylation may be to stabilize the protein matrix or to assist in occlusion of enveloped virus particles. / Graduation date: 1998

Thesis (Ph. D)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Aug 16

<p>Stroke is the leading cause of morbidity and the third leading cause of death in the United States. Over 80% of strokes are ischemic in nature, produced by a thrombus occluding the cerebral circulation. Currently, there is only one pharmacologic treatment FDA approved for ischemic stroke; recombinant tissue-type plasminogen activator (rtPA). Unfortunately, thrombolysis with rtPA is underutilized, as it must be administered within three hours of symptom onset and it is not uncommon for treatment to result in intracranial hemorrhage. For these reasons, safe and effective treatments of stroke are a medical necessity. </p><p>Aptamers are an attractive emerging class of therapeutic agents that offer additional safety because their activity can be reversed with administration of a complimentary oligonucleotide. Accordingly, I hypothesized that aptamers could be used to treat acute ischemic stroke. First, an antithrombotic aptamer previously generated against coagulation factor IXa was used in a murine model of middle cerebral artery occlusion. Upon factor IXa aptamer administration following stroke, neurological function and inflammatory profiles were improved. Moreover, mice previously treated with the aptamer, followed by induction of subarachnoid hemorrhage, had severe mortality levels and hemorrhage grades that were mitigated by administration of the aptamer's matched antidote.</p><p>Second, I generated aptamers against the antifibrinolytic protein plasminogen activator inhibitor-1 (PAI-1), under the hypothesis that aptamer inhibition of PAI-1 would result in a reversible thrombolytic agent. However, after further testing, the aptamers were not found to disrupt the interaction between PAI-1 and its target proteases. Instead, the aptamers were shown to prevent PAI-1 binding to vitronectin, which translated to restoration of breast cancer cell adhesion in an environment of PAI-1 mediated detachment. </p><p>Therefore, aptamer inhibition of factor IXa has demonstrated efficacy in improving outcome following stroke, and should life-threatening hemorrhage arise, an antidote specific to the interventional agent is able to decrease not only hemorrhage grade, but also mortality. This may result in a safer stroke therapy, while a novel aptamer generated against PAI-1 may have application as an antimetastatic agent, which could be used as adjuvant therapy to traditional breast cancer treatment.</p> / Dissertation

Thesis (Ph.D.) -- University of Texas at Arlington, 2008.