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Cunjie_SLC3A2_TripleTOF6600_Fig4_5

ABSTRACT: This dataset consists of 40 raw MS files, acquired on a TripleTOF 6600 (AB SCIEX, Concord, Ontario, Canada) mass spectrometer operated in data dependent acquisition (DDA) and SWATH mode. Code for variants with additional NXS/T sites: 255I WTseq: (N365, N381, N424, N506) 256G N273A (+1 site) 257A N273A; N355D (+2 sites) 258F N355D; N492D (+2 sites) 259J N273A; N355D; N492D (+3 sites) CJ1 D365N (-1 site) CJ2 D381N (-1 site) Cunjie Zhang did all the sample preparation, mass spectrometric acquisition and data analysis. The files are associated with a manuscript submitted for publication by Cunjie Zhang et al. SLC3A2 (4F2hc, CD98) is a single-pass transmembrane glycoprotein and acts as a disulfide-linked adaptor to the SLC7A5 and SLC7A11 exchangers which import essential amino acids and cystine while exporting Gln and Glu, respectively. Cancer cells often over-express SLC3A2, SLC7A5, SLC7A11 and the N-glycans branching, which support tumor progression by increasing mTOR signaling and mitigation of oxidative stress (ox-stress). Branched chain amino acids and mitigation of oxidative stress are also central to aging. Furthermore, the evolution of N-glycosylation sites on SLC3A adaptors and SLC7A transporters suggests a critical but poorly understood role for N-glycans. We have profiled the N-glycan structures at each site SLC3A2 (4F2hc, CD98) and analyzed their impact on trafficking, protein interactions, cellular AA balance and sensitivity to ox-stress.] James W. Dennis is the corresponding author of the manuscript; James W. Dennis should be contacted for questions on this dataset DENNIS@lunenfeld.ca This submission is associated with: Figure 4 Figure 5 Table S4

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Anne-Claude Gingras

PROVIDER: MSV000092989 | MassIVE |

REPOSITORIES: MassIVE

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Project description:BACKGROUND & AIMS- More frequent interaction of bacteria with the colonic epithelium is associated with ulcerative colitis (UC). The identities of all proteins which promote bacterial clearance in colonic epithelial cells are unknown. Previously, we discovered that dCAP-D3 (Chromosome Associated Protein-D3), regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS- Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing CAP-D3 shRNA. CAP-D3 levels in colonic epithelial cells from healthy and UC patient tissues were analyzed by immunoblot. RNA-sequencing identified bacterially-induced CAP-D3 target genes. The role of CAP-D3 target genes in bacterial clearance was analyzed by gentamycin protection assays, immunofluorescent staining, and by using pharmacologic inhibitors. RESULTS- CAP-D3 expression was reduced in colonic epithelial cells from UC patients with active disease. Reduction of CAP-D3 expression inhibited autophagy and decreased intracellular bacterial clearance. The components of the heterodimeric SLC7A5/SLC3A2 amino acid transporter were identified as CAP-D3 target genes; their levels increased in infected, CAP-D3 deficient cell lines and in cells from UC patients. In HT-29 cells, this resulted in earlier SLC7A5 recruitment to Salmonella-containing vacuoles, increased mTOR activity, and enhanced bacterial survival. Inhibition of SLC7A5/SLC3A2 or mTOR activity rescued the bacterial clearance defect in CAP-D3 deficient cells. CONCLUSIONS- CAP-D3 attenuates amino acid transporter transcription to promote bacterial autophagy in colon epithelial cells. CAP-D3 protein levels are decreased in patients with active UC, suggesting that CAP-D3 is a potential therapeutic target to restore mucosal homeostasis in UC patients. Three RNA samples from 3 independent experiments including timepoints taken at 0, 0.5 and 7 hours post-infection were analyzed on a bioanalyzer for quality; one of the 0.5 hour post-infection samples was excluded at this time due to poor RNA purity. Directional, cDNA libraries made from cellular mRNAs were generated from the other 8 samples and sequenced (paired-end sequencing of 100 bp reads) in the Genomics Core at the University of Chicago on an Illumina HiSeq2000.

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Cunjie_SLC3A2_TripleTOF6600_Fig4_5

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