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Effect of leucine and pyruvate on the proteomic profile of cysteine treated wt and ncl1 deleted strain

ABSTRACT: Cell requires optimal amount of nutrients for the synthesis of macromolecules like lipid, protein and nucleotides. There is a very limited knowledge about how cells maintain homeostasis in excess nutrients. By using Yeast as a model system we have shown that some aminoacids are toxic at higher concentration. Using cysteine as a reference molecule we delineated different biological pathways/processes required for survival. Leucine and pyruvate are two molecules which can rescue cysteine induced toxicity.Through a comprehensive genetic screen we show that leucine mediated effect depends on a tRNA methyltransferase (Ncl1), absence of which decouples transcription and translation in the cell. Using proteomics approach we have determined the altered pathway in both wild type and ncl1 deletion strain and the effect of leucine and pyruvate on the proteome of cysteine treated cells.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

DISEASE(S): Cardiovascular System Disease

SUBMITTER: Rahul Chakraborty

LAB HEAD: Shantanu Sengupta

PROVIDER: PXD014880 | Pride | 2019-08-13

REPOSITORIES: Pride

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Source:

			Action	DRS
	R1-iTRQ-YEAST-125mM-10ul.mgf	Mgf
	R1-iTRQ-YEAST-125mM-10ul.wiff	Wiff
	R1-iTRQ-YEAST-125mM-10ul.wiff.scan	Wiff
	R1-iTRQ-YEAST-150mM-10ul.mgf	Mgf
	R1-iTRQ-YEAST-150mM-10ul.wiff	Wiff

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Project description:Cysteine occupies a central position in plant metabolism due to its biochemical functions. Arabidopsis thaliana cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes that catalyze the biosynthesis of cysteine. Because they are localized in the cytosol, plastids and mitochondria, this results in multiple subcellular cysteine pools. Much progress has been made on the most abundant OASTL enzymes; however, information on the less abundant OASTL-like proteins has been scarce. To unequivocally establish the enzymatic reaction catalyzed by the minor cytosolic OASTL isoform CS-LIKE (AT5G28030), we expressed this enzyme in bacteria and characterized the purified recombinant protein. Our results demonstrate that CS-LIKE catalyzes the desulfuration of L-cysteine to sulfide plus ammonia and pyruvate. Thus, CS-LIKE is a novel L-cysteine desulfhydrase (EC 4.4.1.1), and we propose to designate it DES1. The impact and functionality of DES1 in cysteine metabolism was revealed by the phenotype of the T-DNA insertion mutants des1-1 and des1-2. Mutation of the DES1 gene leads to premature leaf senescence, as demonstrated by the increased expression of senescence-associated genes and transcription factors. Also, the absence of DES1 significantly reduces the total cysteine desulfuration activity in leaves, and there is a concomitant increase in the total cysteine content. As a consequence, the expression levels of sulfur-responsive genes are de-regulated, and the mutant plants show enhanced antioxidant defenses and tolerance to conditions that promote oxidative stress. Our results suggest that DES1 from Arabidopsis is an L-cysteine desulfhydrase involved in maintaining cysteine homeostasis, mainly at late developmental stages or under environmental perturbations. Using the Affymetrix ATH1 GeneChips, we performed a comparative transcriptomic analysis on leaves of the des1-1 and wild type plants grown on soil for three weeks. Three biological replicates were performed for each sample and hybridized to the chips. We made the comparison of des1-1 leaves versus wild-type leaves to classify the differently expressed genes in the mutant plant.

Project description:Data analysis. Spot detection and matching were performed with a comparative cross analysis of all the gels using DeCyder software v.6.5 (GE Healthcare). 178 spots were selected based on 1.15-fold for protein ratio cut-off, allowing for the appearance of the spots in 23 out of 28 gels (69 out of 84 total images). Data from 95 spots were submitted. 93 spots were identified with high confidence. Spot picking and Trypsin digestion. The spots of interest were picked up by Ettan Spot Picker (GE Healthcare) based on the in-gel analysis and spot picking design by DeCyder software. The gel spots were washed a few times then digested in-gel with modified porcine trypsin protease (Promega, Fitchburg, WI). The digested tryptic peptides were desalted using a Zip-tip C18 (Millipore, Billerica, MA). Peptides were eluted from the Zip-tip with 0.5 uL of matrix solution (alpha-cyano-4-hydroxycinnamic acid, 5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid, 25mM ammonium bicarbonate) and spotted on a MALDI plate. Mass Spectrometry. MALDI-TOF MS and TOF/TOF tandem MS/MS were performed on AB SCIEX TOF/TOF 5800 System (AB SCIEX). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 4000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 4000 laser shots per fragmentation spectrum on each of the 7-10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions). Database search. Both the resulting peptide mass and the associated fragmentation spectra were submitted to GPS Explorer workstation equipped with MASCOT search engine (Matrix Science, Boston, MA) to search the Swiss-Prot database. Searches were performed without constraining protein molecular weight or isoelectric point, with variable carbamidomethylation of cysteine and oxidation of methionine residues, and with one missed cleavage also allowed in the search parameters. Candidates with either protein score C.I.% or Ion C.I.% greater than 95 were considered significant. When multiple IDs were significant for a given spot, the selection was made by evaluating apparent molecular weight, isoelectric point, the location of the spot in the gel, and the presence of strips of multiple protein isoforms in the adjacent spots.

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Effect of leucine and pyruvate on the proteomic profile of cysteine treated wt and ncl1 deleted strain

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