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Identifying SAM-binding proteins in D.melanogaster

ABSTRACT: The aim of the project is to identify proteins the ustilize S-adenosyl methionine as cofactor for performing methylation of target molecules in D. melanogaster S2 cells. For this purpose, we applied a small-molecule capturing protocol using a SAH-capture compound with UV-activated crosslinker and biotin immobilization groups. We tested the protein affinity with and without crosslinking for two different photo-activatable crosslinkers benzophenone and nitrobenzene, respectively, as well as different concentrations of the capture compound. As control we incubated the cell lysate with the streptavidin beads without adding the capture compound. Two specific capture compounds containing a S-adenosyl homocysteine as capturing functionality and either benzophenone or azo-benzene as reactive crosslinkers as well as a biotin group for affinity purification were tested in this protocol. One sample representing a HeLa cell lysate (Waters) under equal conditions was added to the dataset.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Cell Culture

SUBMITTER: Andreas Schmidt

LAB HEAD: Axel Imhof

PROVIDER: PXD005012 | Pride | 2019-03-11

REPOSITORIES: Pride

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			Action	DRS
	20140904_SV_benzophenone45uM_NE_1_5.wiff	Wiff
	20140904_SV_benzophenone45uM_NE_1_5.wiff.scan	Wiff
	20140904_SV_benzophenone45uM_NE_1_6.wiff	Wiff
	20140904_SV_benzophenone45uM_NE_1_6.wiff.scan	Wiff
	20140904_SV_benzophenone45uM_NE_1_7_2.wiff	Wiff

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Project description:In this study, the researchers isolated a colorless waxy solid from the chloroform extract of Allium stipitatum as Compound 1. They demonstrated compound 1 is a potent bactericidal agent against nonreplicating Mycobacterium tuberculosis. Using microarray technology the authors report gene expression profiles in cells treated with either 2, 5, or 10 ug/ml of compound 1 or an equivalent amount of DMSO as control for 6 h. Gene expression studies revealed that transcriptional profiles elicited in response to compound 1 were similar to the profiles generated during treatment of cells with compounds such as menadione and 8-quinolinol that result in oxidative stress. They included the thioredoxin system components encoded by trxB2 and trxC as well as several genes associated with the heat shock response such as clpB, sigH, dnaJ, dnaK, hsp, Rv0331, Rv3463, Rv3054c, and Rv1334-Rv1335. These results suggest that compound 1 possibly generates damaged proteins and other oxidative stress signals as part of its mechanism of action. The following is the full abstract of this published study. O'Donnell G, et al. (2009) J Nat Prod 72(3):360-365 From Allium stipitatum, three pyridine-N-oxide alkaloids (1-3) possessing disulfide functional groups were isolated. The structures of these natural products were elucidated by spectroscopic means as 2-(methyldithio)pyridine-N-oxide (1), 2-[(methylthiomethyl)dithio]pyridine-N-oxide (2), and 2,2'-dithio-bis-pyridine-N-oxide (3). The proposed structure of 1 was confirmed by synthetic S-methylthiolation of commercial 2-thiopyridine-N-oxide. Compounds 1 and 2 are new natural products, and 3 is reported for the first time from an Allium species. All compounds were evaluated for activity against fast-growing species of Mycobacterium, methicillin-resistant Staphylococcus aureus, and a multidrug-resistant (MDR) variants of S. aureus. Compounds 1 and 2 exhibited minimum inhibitory concentrations (MICs) of 0.5-8 mug/ml against these strains. A small series of analogues of 1 were synthesized in an attempt to optimize antibacterial activity, although the natural product had the most potent in vitro activity. In a whole-cell assay at 30 mug/ml, 1 was shown to give complete inhibition of the incorporation of (14)C-labeled acetate into soluble fatty acids, indicating that it is potentially an inhibitor of fatty acid biosynthesis. In a human cancer cell line antiproliferative assay, 1 and 2 displayed IC(50) values ranging from 0.3 to 1.8 muM with a selectivity index of 2.3 when compared to a human somatic cell line. Compound 1 was evaluated in a microarray analysis that indicated a similar mode of action to menadione and 8-quinolinol by interfering with the thioredoxin system and up-regulating the production of various heat shock proteins. This compound was also assessed in a mouse model for in vivo toxicity. A dose response design type examines the relationship between the size of the administered dose and the extent of the response of the organism(s). Compound Based Treatment: Allium stipitatum (garlic) extract dose_response_design

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Identifying SAM-binding proteins in D.melanogaster

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