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Assesing Orthogonality of ER-HRP and cyt-APEX proximity labeling

ABSTRACT: This project was evaluating the orthogonality of proximity labeling peroxidases that are used to determine partitioning of proteins between the ER and the cytosol.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Joseph Genereux

LAB HEAD: Joseph Genereux

PROVIDER: PXD033764 | Pride | 2022-05-08

REPOSITORIES: Pride

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Project description:Mycolactone is a diffusible macrolide produced by the skin pathogen Mycobacterium ulcerans that suppresses the development of protective immunity against Buruli ulcer disease. In this proteomics study we aimed to identify proteins of which the expression levels changed upon mycolactone treatment. Jurkat T cells were labeled light or heavy by stable isotope labeling by amino acids in cell culture (SILAC), then treated with mycolactone (light) or vehicle (heavy) for 1h prior to activation with PMA/IO for 6h. After mixing light- and heavy-labeled cultures, cells were lysed and extracted proteins were digested with trypsin. The resulting peptide mixture was analyzed by LC-MS/MS and proteins were identified by the MaxQuant software and quantified based on the intensity of the light and heavy signals in the MS spectra of their peptides. The analysis was repeated with reversed labeling, leading to a total of 4,636 proteins that were quantified in both analyses. Interestingly, 52 proteins were down-regulated in mycolactone-treated cells (mycolactone/control ratio < 1.4), while only two proteins were up-regulated (mycolactone/control ratio > 1.4). Gene ontology analysis further revealed that the down-regulated proteins were significantly enriched in proteins located in the plasma membrane and endoplasmic reticulum and we could show that this enrichment was not due to a bias in protein extraction, since the distribution of all identified proteins across different subcellular compartments was similar to those of all human proteins. Analysis of SP-PIR Family-Domains keywords in the UniProt database confirmed this observation and showed an additional enrichment in glycoproteins, proteins with an immunoglobulin domain and proteins involved in immune responses. Furthermore, we observed that the down-regulated proteins were significantly enriched in single-pass type I/II membrane proteins. In contrast, the fraction of multi-pass membrane proteins was comparable among the down-regulated and all identified proteins, suggesting that multi-pass membrane proteins may at least partially resist mycolactone-mediated down-regulation.

			Action	DRS
	MB-I-88-1_peptide.tsv	Tabular
	MB-I-88-1_protein.fas	Other
	MB-I-88-1_protein.tsv	Tabular
	MB-I-88-1_psm.tsv	Tabular
	MB-I-88-2_peptide.tsv	Tabular

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Assesing Orthogonality of ER-HRP and cyt-APEX proximity labeling

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