28 research outputs found
Differential HMG-CoA lyase expression in human tissues provides clues about 3-hydroxy-3-methylglutaric aciduria
3-Hydroxy-3-methylglutaric aciduria is a rare human autosomal recessive disorder caused by deficiency of 3-hydroxy-3-methylglutaryl CoA lyase (HL). This mitochondrial enzyme catalyzes the common final step of leucine degradation and ketogenesis. Acute symptoms include vomiting, seizures and lethargy, accompanied by metabolic acidosis and hypoketotic hypoglycaemia. Such organs as the liver, brain, pancreas, and heart can also be involved. However, the pathophysiology of this disease is only partially understood. We measured mRNA levels, protein expression and enzyme activity of human HMG-CoA lyase from liver, kidney, pancreas, testis, heart, skeletal muscle, and brain. Surprisingly, the pancreas is, after the liver, the tissue with most HL activity. However, in heart and adult brain, HL activity was not detected in the mitochondrial fraction. These findings contribute to our understanding of the enzyme function and the consequences of its deficiency and suggest the need for assessment of pancreatic damage in these patients
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Final Technical Report
Two primary technologies have been employed for analysis and measurement of the Synechocystis proteome. (1) 2D-gel electrophoresis. Currently one of the most reliable options in quantitative proteomics, typical 2D-gel experiments use isoelectric focusing (IEF) in the first dimension. In the case of membrane proteins, detergents must be added to maintain their solubility though only neutral/zwitterionic surfactants are compatible with the IEF process. We have optimized 2D gel separations for Synechocystis proteins extracted and separated into soluble and membrane subfractions. The resolution and coverage of integral membrane proteins is only marginally satisfactory and alternatives to the first dimension are being considered. Size-exclusion chromatography under non-denaturing conditions was one option that was explored but resolution was insufficient for subfractionation of the membrane-bound proteome. A more highly resolving technique, the ''Blue-native gel'' has proven excellent for Synechocystis and we plan to set up this technology in the near future. Proteins with altered expression are being identified through standard LCMSMS technologies. The analysis of PSI, PSII and SDH deficient mutants is completed, establishing the comparative aspect of the project for integration with the ultrastructural and metabolomic experiments at ASU. We are also looking forward to receiving ftsZ and VIPP1 interruption mutants to explore the effects on the proteome of cell enlargement and disruption of thylakoid biogenesis, respectively. (2) 2D liquid chromatography with mass spectrometry of intact proteins. Early experiments with total membrane protein extracts of Synechocystis showed that the spatial resolution of the reverse-phase separation used in front of the mass spectrometer limited detection to the one hundred or so most abundant proteins. The intact mass tags (IMTs) measured in this experiment represent the first of these measurements that will ultimately define the entire proteome. While some of the IMTs were matched to masses calculated from translations of genomic open-reading frames allowing reasonably confident identification of about half of them (hypothetical IMTs), we are currently validating identifications using a combination of peptide mass fingerprinting after cyanogen bromide cleavage and LC-MSMS after trypsin, of protein in fractions collected during LC-MS+. In order to gain more complete proteome coverage we are applying a liquid separation in front of the LC-MS+ experiment. Size-exclusion chromatography is the first separation technology to be employed, yielding immediate benefits, while still not satisfactory for overall resolution of complexes. Total membranes were solubilized with dodecyl maltoside (1.5%) and separated on deactivated silica (G 4000 SW). LC-MS+ analysis of less-retained chlorophyll-containing fractions, using reverse-phase and size-exclusion technologies, yielded intact protein mass spectra of the two large photosystem I subunits PsaA and PsaB as well as many other IMTs (Figures 1 & 2). These integral membrane proteins have eleven transmembrane helices and, at 81 and 83 kDa, represented one of the most significant challenges to the intact protein molecular weight approach. The identities of the proteins were confirmed by peptide mass fingerprinting and while there is good general agreement between measured and calculated masses it is noted that modest post-translational modifications are necessary to account for the measured molecular weights of the intact proteins. Whether these discrepancies are due to genuine post-translational modifications or DNA sequence errors remains to be determined. The data have been published allowing us to claim to be the first to have completed high-resolution electrospray-ionization mass spectrometry of the core subunits of Photosystem II, Photosystem I and the cytochrome b{sub 6}f complex providing effective proof-of-principle for application of the intact mass approach to the integral membrane proteome. Significantly, we reported greater integral membrane proteome coverage than a colleague studying thylakoids of Arabidopsis illustrating the benefits of the technique over sequential organic extraction of membrane proteins and 1D-gel analysis. The homogeneity of the PsaA and PsaB protein mass spectra attest to the quality of material grown at ASU and the viability of extraction and work up of the material after transport to UCLA
Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo.
Entry of monocytes into the vessel wall is an important event in atherogenesis. Previous studies from our laboratory suggest that oxidized arachidonic acid-containing phospholipids present in mildly oxidized low density lipoproteins (MM-LDL) can activate endothelial cells to bind monocytes. In this study, biologically active oxidized arachidonic acid-containing phospholipids were produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and analyzed by liquid chromatography and electrospray ionization mass spectrometry in conjuction with biochemical derivatization techniques. We have now determined the molecular structure of two of three molecules present in MM-LDL and Ox-PAPC that induce monocyte-endothelial interactions. These lipids were identified as 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/z 594.3) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (m/z 610.2). These two molecules were produced by unambiguous total synthesis and found to be identical by analytical techniques and bioactivity assays to those present in MM-LDL and Ox-PAPC. Evidence for the importance of all three oxidized phospholipids in vivo was suggested by their presence in fatty streak lesions from cholesterol-fed rabbits and by their immunoreactivity with natural antibodies present in ApoE null mice. Overall, these studies suggest that specific oxidized derivatives of arachidonic acid-containing phospholipids may be important initiators of atherogenesis
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Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo.
Entry of monocytes into the vessel wall is an important event in atherogenesis. Previous studies from our laboratory suggest that oxidized arachidonic acid-containing phospholipids present in mildly oxidized low density lipoproteins (MM-LDL) can activate endothelial cells to bind monocytes. In this study, biologically active oxidized arachidonic acid-containing phospholipids were produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and analyzed by liquid chromatography and electrospray ionization mass spectrometry in conjuction with biochemical derivatization techniques. We have now determined the molecular structure of two of three molecules present in MM-LDL and Ox-PAPC that induce monocyte-endothelial interactions. These lipids were identified as 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/z 594.3) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (m/z 610.2). These two molecules were produced by unambiguous total synthesis and found to be identical by analytical techniques and bioactivity assays to those present in MM-LDL and Ox-PAPC. Evidence for the importance of all three oxidized phospholipids in vivo was suggested by their presence in fatty streak lesions from cholesterol-fed rabbits and by their immunoreactivity with natural antibodies present in ApoE null mice. Overall, these studies suggest that specific oxidized derivatives of arachidonic acid-containing phospholipids may be important initiators of atherogenesis