Dataset Information

Analysis of fluorophore-labeled human cytochrome b5

ABSTRACT: Cytochrome b5 (b5) is a small accessory protein involved in several reactions. The interaction of b5 with its partner proteins, e.g., cytochrome P450 enzymes, has received much attention and has been the subject of numerous studies. In order to obtain further insights into protein-protein interactions of b5, we modified it with a green fluorophore, Alexa 488, which has a maleimide linker. The labeling was applied to both WT (without Cys) and the T70C mutant. This study revealed labeled sites in WT b5 and confirmed the Cys-selective labeling in the T70C mutant.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Kristie Lindsey Rose

LAB HEAD: Kristie Lindsey Rose

PROVIDER: PXD047758 | Pride | 2024-06-22

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	6049_S3_PeptideTable_SearchResults.xlsx	Xlsx
	6251_PeptideTable_SearchResults.xlsx	Xlsx
	pEX1_ml_6251_20230317_S1_T70C_b5_Alexa488_2uL_1.raw	Raw
	pEX1_pp_6049_20220131_S3_gel_2ul_3.raw	Raw

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Publications

Proteomics, modeling, and fluorescence assays delineate cytochrome b5 residues involved in binding and stimulation of cytochrome P450 17A1 17,20-lyase.

Tateishi Yasuhiro Y Webb Stephany N SN Li Bian B Liu Lu L Lindsey Rose Kristie K Leser Micheal M Patel Purvi P Guengerich F Peter FP

The Journal of biological chemistry 20240126 3

Cytochrome b5 (b5) is known to stimulate some catalytic activities of cytochrome P450 (P450, CYP) enzymes, although mechanisms still need to be defined. The reactions most strongly enhanced by b5 are the 17,20-lyase reactions of P450 17A1 involved in steroid biosynthesis. We had previously used a fluorescently labeled human b5 variant (Alexa 488-T70C-b5) to characterize human P450 17A1-b5 interactions, but subsequent proteomic ana ...[more]

PMID: 38280431

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Project description:Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in Cys biosynthesis and located in different subcellular compartments. These enzymes are made up of a complex variety of isoforms resulting in different subcellular Cys pools. To unravel the contribution of cytosolic Cys to plant metabolism, we characterized the knockout oas-a1.1 and osa-a1.2 mutants, deficient in the most abundant cytosolic OASTL isoform in Arabidposis thaliana. Total intracellular Cys and glutathione concentrations were reduced, and the glutathione redox state was shifted in favour of its oxidized form. Interestingly, the capability of the mutants to chelate heavy metals did not differ from that of the wild type, but the mutants have an enhanced sensitivity to Cd. With the aim of establishing the metabolic network most influenced by the cytosolic Cys pool, we used the ATH1 GeneChip for evaluation of differentially expressed genes in the oas-a1.1 mutant grown under non-stress conditions. The transcriptomic footprints of mutant plants had predicted functions associated with various physiological responses that are dependent on reactive oxygen species and suggested that the mutant was oxidatively stressed. To further elucidate the specific function(s) of the OAS-A1 isoform in the adaptation response to cadmium we extended the trasncriptome experiment to the wild type and oas-a1.1 mutant plants exposed to Cd. The comparison of transcriptomic profiles showed a higher proportion of genes with altered expression in the mutant than in the wild type, highlighting up-regulated genes identified as of the general oxidative stress response rather than metal-responsive genes. Wild type and oas-a1.1 mutant plants were grown hydroponically and, after a two-week acclimation period, the roots and shoots were harvested separately. Total RNA was then prepared and analyzed using the Affymetrix-Arabidopsis ATH1GeneChip array. Three biological replicates were performed for each sample. We made two different comparisons to classify the differently expressed genes in the mutant plant: oas-a1.1 roots versus wild-type roots and oas-a1.1 shoots versus wild-type shoots. Hydroponically-grown wild type and oas-a1.1 mutant plants were further treated with 50µM CdCl2 and 18h-treated-roots and 24h-treated-shoots were harvested. Total RNA was then prepared and analyzed using the Affymetrix-Arabidopsis ATH1GeneChip array. Three biological replicates were performed for each sample. Different comparisons were performed as follows: 18h Cd-treated wild type roots versus untreated wild type roots; 24h Cd-treated wild type shoots versus untreated wild type shoots; 18h Cd-treated oas-a1.1 roots versus untreated oas-a1.1 roots; 24h Cd-treated oas-a1.1 shoots versus untreated oas-a1.1 shoots; 18h Cd-treated oas-a1.1 roots versus 18h Cd-treated wild type roots; 24h Cd-treated oas-a1.1 shoots versus 24h Cd-treated wild type shoots

Dataset Information

Analysis of fluorophore-labeled human cytochrome b5

Dataset's files

Publications

Proteomics, modeling, and fluorescence assays delineate cytochrome b<sub>5</sub> residues involved in binding and stimulation of cytochrome P450 17A1 17,20-lyase.

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Dataset Information

Analysis of fluorophore-labeled human cytochrome b5

Dataset's files

Publications

Proteomics, modeling, and fluorescence assays delineate cytochrome b&lt;sub&gt;5&lt;/sub&gt; residues involved in binding and stimulation of cytochrome P450 17A1 17,20-lyase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Proteomics, modeling, and fluorescence assays delineate cytochrome b<sub>5</sub> residues involved in binding and stimulation of cytochrome P450 17A1 17,20-lyase.