Dataset Information

Identification of LC3-binding proteins on lipid droplets

ABSTRACT: In the search for LD-localized proteins that may mediate lipophagy, we purified LDs from HeLa cells treated with oleic acid (OA), which stimulates LD biogenesis and lipophagy, together with chloroquine (CQ), which inhibits lysosomal degradation. We incubated the LD lysates with purified GST-LC3B, and then pulled down GST-LC3B for mass spectrometry analysis

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: maomao pu

LAB HEAD: Wei Liu

PROVIDER: PXD036200 | Pride | 2023-10-24

REPOSITORIES: Pride

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Publications

ORP8 acts as a lipophagy receptor to mediate lipid droplet turnover.

Pu Maomao M Zheng Wenhui W Zhang Hongtao H Wan Wei W Peng Chao C Chen Xuebo X Liu Xinchang X Xu Zizhen Z Zhou Tianhua T Sun Qiming Q Neculai Dante D Liu Wei W

Protein & cell 20230901 9

Lipophagy, the selective engulfment of lipid droplets (LDs) by autophagosomes for lysosomal degradation, is critical to lipid and energy homeostasis. Here we show that the lipid transfer protein ORP8 is located on LDs and mediates the encapsulation of LDs by autophagosomal membranes. This function of ORP8 is independent of its lipid transporter activity and is achieved through direct interaction with phagophore-anchored LC3/GABARAPs. Upon lipophagy induction, ORP8 has increased localization on L ...[more]

PMID: 37707322

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Project description:Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway. For microarray screening, Invitrogen Protoarray v4.0 protein microarrays were used. Human NEMO expressed as a C-terminal GST fusion was purified from E. coli lysates and labelled with biotin. NEMO or biotinylated GST were applied to the microarrays and binding partners detected using streptavidin-Alexa Fluor 647. Significant interactors on both arrays were detected using Invitrogen Protoarray Prospector software and a Z-score cutoff of 3.0. Following subtraction of interactors present on the GST control array, a final set of significant NEMO interactors was derived. Full experimental details are supplied in Fenner, B. J., Scannell, M. & Prehn, J. H. M. (2010). Expanding the substantial interactome of NEMO using protein microarrays. PLoS ONE (in press).

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