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

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

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

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes bibliographical references (p. 146-178).