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- Type:
- Article
- 摘抄:
- Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization–time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.
- 作者:
- Siehnel, Richard; Howard, Jeremy; Zhou, Songtao; Layh-Schmitt, Gerlinde; Greis, Kenneth D.; Curnow, Alan, and Klanke, Chuck
- 提交者:
- Kenneth Greis
- 上传日期:
- 03/03/2017
- 更改日期:
- 04/07/2017
- 创建:
- 2005-09
- 证书:
- All rights reserved
-
- Type:
- Article
- 摘抄:
- We have designed ROS-activated cytotoxic agents that are active against AML cancer cells. In this study the mechanism and synergistic effects against cells co-expressing the AML oncogenes MLL-AF9 fusion and FLT3-ITD was investigated. The agent had an IC50 value of 1.8±0.3 μM with a selectivity of 9-fold compared to untransformed cells. Treatment induced DNA strand breaks, apoptosis, and cell cycle arrest. Proteomics and transcriptomics revealed enhanced expression of the pentose phosphate pathway, DNA repair, and pathways common to cell stress. Western blotting confirmed repair by homologous recombination. Importantly, RAC1 treatment was synergistic in combination with multiple pathway targeting therapies in AML cells but less so in untransformed cells. Taken together, these results demonstrate that RAC1 can selectively target poor prognosis AML and do so by creating DNA double strand breaks that require homologous recombination.
- 作者:
- Wunderlich, Mark; Mulloy, James C.; Greis, Kenneth D.; Merino, Edward J.; Thowfeik, Fathima Shazna; Wyder, Michael; AbdulSalam, Safnas F., and Kadekaro, Ana L.
- 提交者:
- Kenneth Greis
- 上传日期:
- 03/03/2017
- 更改日期:
- 04/07/2017
- 创建:
- 2015-09
- 证书:
- All rights reserved
-
- Type:
- Article
- 摘抄:
- Cardiac myosin binding protein-C (cMyBP-C) is a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function; however, the profile of cMyBP-C degradation after myocardial infarction (MI) is unknown. We hypothesized that cMyBP-C is sensitive to proteolysis and is specifically increased in the bloodstream post-MI in rats and humans. Under these circumstances, elevated levels of degraded cMyBP-C could be used as a diagnostic tool to confirm MI. To test this hypothesis, we first established that cMyBP-C dephosphorylation is directly associated with increased degradation of this myofilament protein, leading to its release in vitro. Using neonatal rat ventricular cardiomyocytes in vitro, we were able to correlate the induction of hypoxic stress with increased cMyBP-C dephosphorylation, degradation, and the specific release of N′-fragments. Next, to define the proteolytic pattern of cMyBP-C post-MI, the left anterior descending coronary artery was ligated in adult male rats. Degradation of cMyBP-C was confirmed by a reduction in total cMyBP-C and the presence of degradation products in the infarct tissue. Phosphorylation levels of cMyBP-C were greatly reduced in ischemic areas of the MI heart compared to non-ischemic regions and sham control hearts. Post-MI plasma samples from these rats, as well as humans, were assayed for cMyBP-C and its fragments by sandwich ELISA and immunoprecipitation analyses. Results showed significantly elevated levels of cMyBP-C in the plasma of all post-MI samples. Overall, this study suggests that cMyBP-C is an easily releasable myofilament protein that is dephosphorylated, degraded and released into the circulation post-MI. The presence of elevated levels of cMyBP-C in the blood provides a promising novel biomarker able to accurately rule in MI, thus aiding in the further assessment of ischemic heart disease.
- 作者:
- Muthusamy, Saminathan; Nair, Nandini; McElligott, Andrew; Martin, Jody L.; Govindan, Suresh; Gongora, Enrique; henderson, Kyle K.; Greis, Kenneth D.; Barefield, David; Luther, Pradeep K.; Sadayappan, Sakthivel, and Winegrad, Saul
- 提交者:
- Kenneth Greis
- 上传日期:
- 03/03/2017
- 更改日期:
- 04/07/2017
- 创建:
- 2012-01
- 证书:
- All rights reserved
-
- Type:
- Article
- 摘抄:
- Cardiolipin (CL) is a mitochondrial phospholipid essential for electron transport chain (ETC) integrity. CL-deficiency in humans is caused by mutations in the tafazzin (Taz) gene and results in a multisystem pediatric disorder, Barth syndrome (BTHS). It has been reported that tafazzin deficiency destabilizes mitochondrial respiratory chain complexes and affects supercomplex assembly. The aim of this study was to investigate the impact of Taz-knockdown on the mitochondrial proteomic landscape and metabolic processes, such as stability of respiratory chain supercomplexes and their interactions with fatty acid oxidation enzymes in cardiac muscle. Proteomic analysis demonstrated reduction of several polypeptides of the mitochondrial respiratory chain, including Rieske and cytochrome c1 subunits of complex III, NADH dehydrogenase alpha subunit 5 of complex I and the catalytic core-forming subunit of F0F1-ATP synthase. Taz gene knockdown resulted in upregulation of enzymes of folate and amino acid metabolic pathways in heart mitochondria, demonstrating that Tazdeficiency causes substantive metabolic remodeling in cardiac muscle. Mitochondrial respiratory chain supercomplexes are destabilized in CL-depleted mitochondria from Taz knockdown hearts resulting in disruption of the interactions between ETC and the fatty acid oxidation enzymes, very long-chain acyl-CoA dehydrogenase and long-chain 3-hydroxyacylCoA dehydrogenase, potentially affecting the metabolic channeling of reducing equivalents between these two metabolic pathways. Mitochondria-bound myoglobin was significantly reduced in Taz-knockdown hearts, potentially disrupting intracellular oxygen delivery to the oxidative phosphorylation system. Our results identify the critical pathways affected by the Taz-deficiency in mitochondria and establish a future framework for development of therapeutic options for BTHS.
- 作者:
- Haffey, Wendy D.; Mandala, Satish K.; Purevjav, Enkhsalkhan; Huang, Yan; Javadov, Sabzali; Greis, Kenneth D.; Powers, Corey; Strauss, Arnold W.; Towbin, Jeffrey A., and Khuchua, Zaza
- 提交者:
- Kenneth Greis
- 上传日期:
- 03/03/2017
- 更改日期:
- 04/07/2017
- 创建:
- 2015-06
- 证书:
- All rights reserved
-
- Type:
- Article
- 摘抄:
- Organovanadium compounds have been shown to be insulin sensitizers in vitro and in vivo. One potential biochemical mechanism for insulin sensitization by these compounds is that they inhibit protein tyrosine phosphatases (PTPs) that negatively regulate insulin receptor activation and signaling. In this study, bismaltolato oxovanadium (BMOV), a potent insulin sensitizer, was shown to be a reversible, competitive phosphatase inhibitor that inhibited phosphatase activity in cultured cells and enhanced insulin receptor activation in vivo. NMR and X-ray crystallographic studies of the interaction of BMOV with two different phosphatases, HCPTPA (human low molecular weight cytoplasmic protein tyrosine phosphatase) and PTP1B (protein tyrosine phosphatase 1B), demonstrated uncomplexed vanadium (VO ) in the active site. Taken together, these findings support phosphatase inhibition as a mechanism for insulin sensitization by BMOV 4 and other organovanadium compounds and strongly suggest that uncomplexed vanadium is the active component of these compounds.
- 作者:
- Diven, Conrad; Peters, Kevin G.; Evdokimov, Artem; Howard, Brian W.; Soper, Shari; Genbauffe, Frank; Greis, Kenneth D.; Rastogi, Vinit; Eby-Wilkens, Elaine; Pokross, Matthew, and Maier, Matthew
- 提交者:
- Kenneth Greis
- 上传日期:
- 03/01/2017
- 更改日期:
- 04/07/2017
- 创建:
- 2003-09
- 证书:
- All rights reserved
-
- Type:
- Article
- 摘抄:
- Background: Role of apolipoprotein (apo) A-II on metabolism of high density lipoproteins (HDLs) is unknown. Results: Conformational changes of apoA-I, the major apolipoprotein of HDL, caused by apoA-II in discoidal HDL are confined to two regions of apoA-I. Conclusion: Interactions between the two major apolipoproteins in discoidal HDL are site specific. Significance: Functional implications of HDL complexes will significantly benefit from such structural information.
- 作者:
- Gauthamadasa, Kekulawalage; Vaitinadin, Nataraja Sarma; Homan, Reyn; Macha, Stephen; Dresman, James L.; D. Silva, R. A. Gangani, and Greis, Kenneth D.
- 提交者:
- Kenneth Greis
- 上传日期:
- 03/01/2017
- 更改日期:
- 04/07/2017
- 创建:
- 2012-03
- 证书:
- All rights reserved
-
- Type:
- Article
- 摘抄:
- The primary structure of the major surface glycoconjugate of Leishmania donovani parasites, a lipophosphoglycan, has been further characterized. The repeating PO4-6Galp beta 1-4Man disaccharide units, which are a salient feature of the molecule, are shown to terminate with one of several neutral structures, the most abundant of which is the branched trisaccharide Galp beta 1-4(Manp alpha 1-2)Man. The phosphosaccharide core of lipophosphoglycan, which links the disaccharide repeats to a lipid anchor, contains 2 phosphate residues. One of the core phosphates has previously been localized on O-6 of the galactosyl residue distal to the lipid anchor; the second phosphate is now shown to be on O-6 of the mannosyl residue distal to the anchor and to bear an alpha-linked glucopyranosyl residue. Also, the anomeric configuration of the unusual 3-substituted Galf residue in the phosphosaccharide core is established as beta. The complete structure of the core is thus PO4-6Galp alpha 1-6Galp alpha 1-3Galf beta 1-3[Glcp alpha 1-PO4-6]Manp alpha 1-3Manp alpha 1-4GlcN alpha 1-. This further clarification of the structure of lipophosphoglycan may prove beneficial in determining the structure-function relationships of this highly unusual glycoconjugate.
- 作者:
- Ferguson, Michael A. J.; Turco, Salvatore J.; Thomas, Jerry R.; Gorin, Philip A. J.; Homans, Steven W.; McConville, Malcom J.; Greis, Kenneth D., and Thomas-Oates, Jane E.
- 提交者:
- Kenneth Greis
- 上传日期:
- 03/01/2017
- 更改日期:
- 04/07/2017
- 创建:
- 1992-05
- 证书:
- All rights reserved
-
- Type:
- Article
- 摘抄:
- An extracellular phosphoglycan (exPG), present in the culturem edium of the promastigote form L oefi shmania donovani, was purified and structurally characterized. The purification scheme included ethanol precipitation of the culture medium, anion exchange chromatography, hydrophobic chromatography on phenyl-Sepharose, and preparative polyacrylamgeild e electrophoresis. Structural analysis by ‘H-’H NMR, methylation linkage analysis, and glycosidase digestion revealed that the exPG consisted of thfoel lowing structure: (CAP)+[P04-6Galp@1-4Manpal]lo-11-POr6GalpB1-4Man. The capw as found to be ones eovf eral small, neutral oligosaccharides, the most abundant of which was the trisaccharide Galp@l-4(Manpal-2)Man. The results indicated structural analogy to the cellular-derived lipophosphoglycan (LPG) from L. donovani. The important exceptions are a lacko f the lipid anchor, the entire phosphosaccharide core, and several of the repeating disaccharide units. Although the function of exPGis presently unknowni,t may play a protective role for the promastigote in the insect vector or during infection of a mammalian host
- 作者:
- Turco, Salvatore J.; Thomas, Jerry R.; Homans, Steven W.; Ferguson, Michael A.; McConville, Malcolm J., and Greis, Kenneth D.
- 提交者:
- Kenneth Greis
- 上传日期:
- 03/01/2017
- 更改日期:
- 04/07/2017
- 创建:
- 1992-04
- 证书:
- All rights reserved
-
- Type:
- Article
- 摘抄:
- Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes.
- 作者:
- Wang, Feng; Jarrold, Bradley; Burt, Thomas; DeMuth, Jeffrey, and Greis, Kenneth D.
- 提交者:
- Kenneth Greis
- 上传日期:
- 03/01/2017
- 更改日期:
- 04/07/2017
- 创建:
- 2005-06
- 证书:
- All rights reserved
-
- Type:
- Image
- 摘抄:
- Lamprometra palmata, at top, with arms of Capillaster sp cf C. multiradiatus below it, at night
- 作者:
- Meyer, David L.
- 提交者:
- David L. Meyer
- 上传日期:
- 02/21/2017
- 更改日期:
- 02/21/2017
- 创建:
- 2017-02-21
- 证书:
- Attribution-NonCommercial 4.0 International