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Regulators of proteostasis are translationally repressed in fibroblasts from sporadic and LRRK2-G2019S Parkinson’s patients

ABSTRACT: Here we used non-canonical amino acid labeling and mass-spectrometry based proteomics to perform a quantitative comparison of nascent proteins in fibroblasts from sporadic and G2019S mutation PD patients compared to healthy individuals. We found that several proteins were under-represented among nascent proteins in cells from PD patients. Among these were regulators endo-lysosomal sorting, mRNA processing and translation itself.

INSTRUMENT(S): LTQ Orbitrap, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin, Fibroblast

SUBMITTER: Dani Flinkman

LAB HEAD: Eleanor Coffey

PROVIDER: PXD031144 | Pride | 2022-02-21

REPOSITORIES: Pride

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			Action	DRS
	170929_Dani_212011A_01.RAW	Raw
	170929_Dani_212011M_01.RAW	Raw
	170929_Dani_372011A_01.RAW	Raw
	170929_Dani_372011M_01.RAW	Raw
	170929_Dani_432012A_01.RAW	Raw

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Project description:Most patients with triple negative breast cancer (TNBC) fail to respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression remain elusive. Here, we identify aberrant B7H3 glycosylation and found N-glycosylation of B7H3 at NXT motif sites are responsible for its protein stability and immunosuppression in TNBC tumors. Mechanistically, fucosyltransferase FUT8 catalyzes B7H3 core fucosylation at N-glycans to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7H3-mediated immunosuppressive function in TNBC cells. Abnormal B7H3 glycosylation mediated by FUT8 overexpression could be physiologically significant and clinically relevant in TNBC patients. Notably, combination of core fucosylation inhibitor 2F-Fuc and anti-PDL1 results in enhanced therapeutic efficacy in B7H3-positive TNBC tumors. These suggest targeting FUT8-B7H3 axis can be a promising strategy for improving anti-tumor immune responses in TNBC patients. To obtain the direct evidence that B7H3 is N-glycosylated in TNBC cells, we analysed the peptides of purified human B7H3 protein from B7H3-WT re-expressed and B7H3-8NQ re-expressed MDA-MB-231 cell lines by Nanoscale liquid chromatography coupled to tandem MS (nano LC-MS/MS). The result showed that there were eight N-glycosylation sites (Asn positions 91, 104, 189, 215, 309, 322, 407, and 433) in B7H3-WT cells, but not in B7H3-8NQ cells, as determined by Asn to Asp conversion after PNGase F treatment. As B7H3 contains a nearly exact tandem duplication of the IgV-IgC domain, there were four pairs of N-glycosylation sites identified through identical peptide sequence, including N91 and N309, N104 and N322, N189 and N407, and N215 and N433, in each of the IgV-IgC domains. Together, the results indicate that B7H3 is exclusively N-glycosylated at these four pairs of glycosylation sites in TNBC cells.

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Regulators of proteostasis are translationally repressed in fibroblasts from sporadic and LRRK2-G2019S Parkinson’s patients

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