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ERLIN1 and ERLIN2 scaffolds bridge TMUB1 and RNF170 and restrict cholesterol esterification to regulate the secretory pathway

ABSTRACT: Complexes of ERLIN1 and ERLIN2 form large ring-like cup shaped structures on the endoplasmic reticulum (ER) membrane and serve as platform to bind cholesterol and E3-ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 and RNF170. We identify a luminal N-terminal conserved region in TMUB1 and RNF170 required for the interaction. Three-dimensional modelling using Alpha Fold Multimer shows that this conserved motif binds the SPFH domain of two adjacent ERLIN subunits at different interfaces. ERLIN1 and ERLIN2 variants that preclude these interactions are unstable and have been previously linked to hereditary spastic paraplegia (HSP), a progressive neurological disorder. Using HeLa cells that lack both ERLINs, we uncovered novel processes dependent on these scaffolds. Proteomics and lipidomics approaches demonstrated a role of ERLINs in cell adhesion, vesicular secretion, and cholesterol and sphingomyelin metabolism. Cells lacking ERLINs displayed collapsed tubular ER, a fragmented Golgi apparatus, and impaired post-Golgi trafficking. Moreover, they accumulated cholesterol esters and triacylglycerols in larger lipid droplets. We found that inhibition of sterol-O-acyltransferase 1 with avasimibe suppressed lipid droplet accumulation, Golgi fragmentation and SREBP activation. We conclude that ERLIN scaffolds maintain cholesterol levels at the ER in check by favouring trafficking to the Golgi over esterification, thereby regulating the secretory pathway.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Prerana Wagle

LAB HEAD: Elena I. Rugarli

PROVIDER: PXD048503 | Pride | 2024-05-21

REPOSITORIES: Pride

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			Action	DRS
	20200217_Q1_AWH_ColID_962_ProjID_2051_Rugarli_sample_01.raw	Raw
	20200217_Q1_AWH_ColID_962_ProjID_2051_Rugarli_sample_02.raw	Raw
	20200217_Q1_AWH_ColID_962_ProjID_2051_Rugarli_sample_03.raw	Raw
	20200217_Q1_AWH_ColID_962_ProjID_2051_Rugarli_sample_04.raw	Raw
	20200217_Q1_AWH_ColID_962_ProjID_2051_Rugarli_sample_05.raw	Raw

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Project description:Progression and relapse-free survival of ovarian carcinoma are associated with the abundance of immunosuppressed CD163highCD206high tumor-associated macrophages (TAMs) and high levels of polyunsaturated fatty acids (PUFAs), in particular arachidonic acid (AA), in the tumor microenvironment. In the present study, we have investigated whether both associations are functionally linked. Methods: The effect of PUFAs on cytokine-mediated pro-inflammatory signal transduction was studied in primary monocyte-derived macrophages (MDMs) by transcriptomics, bioinformatics, phosphoprotein analysis of signal transduction proteins and MS-based proteomics of lipid rafts. Results: Pathway analysis of transcriptional profiles revealed that high CD163 and CD206/MRC1 expression in TAMs is strongly associated with an inhibition of cytokine-triggered signal transduction. This is mirrored by an impairment of the transcriptional response to interferon-beta (IFNbeta), IFNgamma and IL-6 in monocyte-derived macrophages (MDMs) by AA. This AA-mediated inhibition of pro-inflammatory signaling is caused by dysfunctions of the cognate receptors, as indicated by the inhibition by PUFAs of JAK1, JAK2, STAT1 and STAT3 phosphorylation, with the strongest inhibitory effects exerted by AA and its non-metabolizable analog ETYA. AA/ETYA treatment of MDMs results in altered composition of lipid rafts, including reduced amounts of the interferon receptor IFNAR1, STAT1 and other immune-regulatory proteins. The AA-mediated inhibition of IFN-triggered STAT1 phosphorylation was reversed by water-soluble cholesterol, known to prevent the perturbation of lipid raft structure by PUFAs. Conclusion: Our data suggest that AA, and to a lesser degree other PUFAs, impair pro-inflammatory signaling in macrophages, at least in part by altering the structure of lipid rafts, and thereby contribute to the reeducation of tumor-associated macrophages. Our findings also suggest that the pharmacologic restoration of lipid raft functions in TAM may be a promising strategy for the development of new therapeutic approaches.

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ERLIN1 and ERLIN2 scaffolds bridge TMUB1 and RNF170 and restrict cholesterol esterification to regulate the secretory pathway

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