Introduction: Persistent pulmonary hypertension of the newborn (PPHN) is associated with an elevated thromboxane to prostacyclin ratio, pulmonary artery (PA) hyperreactivity and hypersensitivity. Thromboxane receptor (TP), coupling with G-protein Gαq causes pulmonary vasoconstriction; whereas prostacyclin receptor (IP), coupling with Gαs, causes vasodilation and TP phosphorylation via adenylyl cyclase (AC)-cAMP-protein kinase A (PKA), desensitizes TP. Both TP phosphorylation and Gαq palmitoylation play major roles in regulation of signaling through the TP-Gαq complex. We hypothesized that increased Gαq palmitoylation and decreased AC activity could cause hypoxic TP hyperresponsiveness. We studied the impact of hypoxia on selected post-translational modifications of the receptor-G-protein complex, determining TP vasoconstriction: Gαq palmitoylation, TP phosphorylation and upstream AC activity. Methods: Force responses to thromboxane mimetic U46619, palmitoylation inhibition by 2-bromopalmitate (2-BP) and AC activation (forskolin) were studied by myography in hypoxic PPHN and control newborn swine pulmonary artery. Ca2+ mobilization was studied by fluorescent calcium indicators fura-2AM in pulmonary myocytes (PASMC), and fluo-4NW in HEK293 cells. Effects of hypoxia on Gαq palmitoylation were studied by metabolic labeling. Gαq cysteines and TP serines were mutated to determine sites of post-translational modifications. Protein expression and receptor-G-protein coupling were studied by Western blot and co-immunoprecipitation. PKA activity was assayed; and AC activity quantified. Results: Hypoxia increases Gαq palmitoylation, without increasing total palmitate uptake. Palmitoylation inhibition decreases U46619-stimulated force generation as well as Ca2+ mobilization in PPHN PA rings and hypoxic PASMC. Mutation of palmitoylable cysteine and palmitoylation inhibition proportionately decrease U46619-mediated Ca2+ mobilization in HEK293 cells. TP serine phosphorylation is decreased by hypoxia due to decreased PKA activity; this causes TP hypersensitivity and hyper-reactivity. Serine 324 of TPα is the target of PKA-mediated desensitization. AC activator-induced relaxation is reduced in PPHN PA. Basal and receptor-stimulated AC activity are decreased in hypoxic PASMC. Decreased AC activity is not due to decreased AC expression, ATP availability nor increased Gαi activation. Conclusion: Increased Gαq palmitoylation plays a role in TPα hyper-responsiveness in hypoxic PPHN. Hypoxia also reduces responses to agents acting through AC, unleashing TP-mediated vasoconstriction. Reactivation of pulmonary AC might be useful therapeutically to promote vasodilation and TP desensitization. / October 2016

Bcl11b (B-cell lymphoma/leukemia 11b), also known as Ctip2 (Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2), is a C2H2 zinc finger transcriptional regulatory protein, which is an essential protein for post-natal life in the mouse and plays crucial roles in the development, and presumably function, of several organ systems, including the central nervous, immune, craniofacial formation and cutaneous/skin systems. Moreover, inactivation of Bcl11b has been implicated in the etiology of lymphoid malignancies, suggesting that Bcl11b may function as a tumor suppressor. Bcl11b was originally identified as a protein that interacted directly with the orphan nuclear receptor COUP-TF2. Later studies revealed that this C2H2 zinc finger protein can bind DNA directly in a COUP-independent manner, and it has been studied mostly as a transcription repressor. In T cells, gene repression mediated by Bcl11b involves the recruitment of class I HDACs, HDAC1 and HDAC2, within the context of the Nu It has become evident that post-translational modifications (PTMs) play essential roles in modulating activity of transcription regulators. By sensing extracellular signals, cells initiate a series of signaling cascades, which eventually transduce to transcription factors by PTMs, leading to changes of gene expression profile. cleosome Remodeling and Deacetylation (NuRD) complex. The hypothesis that Bcl11b functions as a transcriptional repressor has been supported by transcriptome analyses in mouse T cells and human neuroblastoma cells. However, approximately one-third of the genes that were dysregulated in the double positive (DP) cells of Bcl11b-null mice were down-regulated relative to control T cells, suggesting that Bcl11b may act as a transcriptional activator in some promoter and/or cell contexts. We have also found that Bcl11b functions as a transcriptional activator in a promoter context-dependent manner. However, how Bcl11b and its transcription regulatory activity is regulated still remain largely unknown. Here, we study the reversible, covalent modification of Bcl11b by phosphorylation and small ubiquitin-like modifier (SUMO). We have identifiedK679 and K877 as the two major Bcl11b SUMOylation sites by mutagenesis study. We have shown that phosphorylation and SUMOylation of Bcl11b are likely mutually exclusive processes, and phosphorylation of Bcl11b inhibits its SUMOylation by promoting the recruitment of SUMO specific protease SENP1. To study the function of Bcl11b SUMOylation, we fused SUMO1 to the amino terminus of Bcl11b. This generated a form of Bcl11b that was constitutively sumoylated without the complications of indirect effects associated with overexpression of SUMO1. Our data presented using the constitutive SUMO-Bcl11b demonstrated that SUMOylation compromises the transcription repression mediated by Bcl11b. Interestingly, when Bcl11b is fused to a cleavable form of SUMO, Bcl11b is targeted to ubiquitination pathway and it is degraded through proteasome machinery, suggesting that SUMOylation targets Bcl11b to the ubiquitination-proteasome machinery and deSUMOylation of SUMO conjugated Bcl11b is required for this process. These results described herein provide a framework for understanding the mechanisms underlying the transcription regulatory activities of Bcl11b, and how Bcl11b is regulated by post-translational modifications, including phosphorylation and SUMOylation. These studies may contribute to a better understanding of the molecular and cellular basis for Bcl11b function in vivo. / Graduation date: 2011 / Access restricted to the OSU Community at author's request from June 9, 2011 - June 9, 2012

Background and objectives: Endothelial dysfunction and cancer are two of the important aspects of obesity-related medical complications, the prevalence of which is reaching an epidemic level worldwide. The protein kinase LKB1 has been shown to play opposite roles in these two metabolic diseases by promoting cellular senescence and inhibiting cell proliferation through regulating a series of its downstream targets. However, the molecular mechanisms wherebyLKB1 itself is regulated by its upstream molecules remains poorly understood. The major objectives of this study are to identify novel upstream regulators of LKB1 and to investigate how these upstream regulators modulate the subcellular localization and physiological functions of LKB1 by post-translational modifications. Key findings: 1. Our proteomic analysis demonstrated that LKB1 was modified by both acetylation and phosphorylation. The acetylation sites of mouseLKB1 include Lys48, Lys64and Lys312. The phosphorylation sites of mouseLKB1 include: Ser31, Thr32,Tyr36, Ser69, Thr71, Ser334and Thr336. 2. In both human embryonic kidney 293 (HEK293)cells and primary porcine aortic endothelial cells (PAECs), the nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase SirT1 attenuated the acetylation levels of LKB1,which consequently resulted in enhancedLKB1ubiquitination, thereby leading to the proteasome-mediated degradation of LKB1. 3. In primary PAECs, overexpression of SirT1 protected cells from cell cycle arrest and cellular senescence, whereas overexpression of LKB1 exhibited the opposite effects.SirT1 antagonizedLKB1-induced G1 phase arrest and cellular senescence by promoting the deacetylation and protein degradation of LKB1. 4. The in vitro phosphorylation assay and mass spectrometry analysis demonstrated that LKB1 could be phosphorylated by the Akt kinase at Ser334which was critical for the interaction between LKB1 and 14-3-3. The enhanced association between LKB1 and 14-3-3 subsequently attenuated the interaction between LKB1 and Ste20 related adaptor α(STRADα), which further promoted the nuclear accumulation of LKB1. 5. The cell proliferation and cell cycle distribution analysis of the stably-transfected MDA-MB-231 breast cancer cells demonstrated that overexpression of the LKB1 mutant S334D, which mimicked Ser334 phosphorylation and localized exclusively in the nucleus, completely lost its anti-tumor activities. On the other hand, the S334A mutation enhanced the tumor suppressive functions of LKB1. 6. Nude mice inoculated with the LKB1 S334A stably-transfected MDA-MB-231 cells exhibited delayed tumor onset, decreased tumor growth rate and tumor weight. By contrast, inoculation of nude mice with the MDA-MB-231 cells overexpressing LKB1 S334D mutation showed the opposite effects on these parameters. Conclusions: These results collectively suggest that the deacetylase SirT1 and the protein kinase Aktare the two important upstream regulators of LKB1. SirT1 prevents LKB1-induced cellular senescence and protect endothelial ageing by promoting proteasome-mediated degradation of LKB1. Akt inhibits the tumor-suppressive activity of LKB1 by enhancing the phosphorylation-dependent nuclear translocation. Further investigations on the precise mechanisms whereby SirT1 and Akt regulate LKB1 functions may help to design novel therapeutic strategies for treating obesity-related diseases, such as diabetes, cardiovascular disease and cancer. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

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.

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.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (sites viewed on August 10, 2006). PDF text of dissertation: 98 p. : ill. ; 1.16Mb. UMI publication number: AAT 3208087. Includes bibliographical references. Also available in microfilm, microfiche and paper format.

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

No description available.

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