Dataset Information

LILRA3-GFP and GFP only Transfected HEK293T cells LC-MSMS

ABSTRACT: The leukocyte immunoglobulin-like receptor A3 (LILRA3) is a soluble protein primarily expressed by peripheral blood monocytes and is abundant in sera of healthy donors. Extracellular LILRA3 is anti-inflammatory and displays neuro-regenerative functions in vitro. However, its intracellular expression, distribution and function(s) remain unknown. Using a combination of high-resolution confocal and super-resolution microscopy, we identified intracellular expression of native LILRA3 in the nucleus of peripheral blood monocytes and in vitro derived macrophages. This unexpected nuclear localisation of LILRA3 was confirmed in LILRA3-GFP transfected HEK293T cells. Western blot of proteins fractionated from primary macrophages and the transfected HEK293T cells confirmed nuclear localisation of the native and expressed LILRA3 proteins. Interestingly, most of the LILRA3 in the nucleus was in a monomeric form like the biologically active secreted protein, while those in the other cellular compartments were mixed monomeric, dimeric, and oligomeric forms. The predominant presence of monomeric LILRA3 in the nucleus was independently corroborated in transfected live HEK293T cells using number and molecular brightness (N&B) analysis method. Immunoprecipitation and Mass spectrometric peptide sequencing studies revealed that nuclear LILRA3 co-immunoprecipitated with several nuclear proteins involved in host protein synthesis machinery via direct interaction to a key multifunctional RNA binding protein, the Ewing sarcoma breakpoint region 1 protein (EWS).

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Nicodemus Tedla

LAB HEAD: Nicodemus Tedla

PROVIDER: PXD024602 | Pride | 2021-04-08

REPOSITORIES: Pride

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Publications

Nuclear Leukocyte Immunoglobulin-like Receptor A3 Is Monomeric and Is Involved in Multiple Layers of Regulated Gene Expression and Translation.

An Hongyan H Richardson Alexander A Rajasekariah Poornima P Zhong Ling L Fernando Bentotage Samitha M BSM Macmillan Alexander A Klotzsch Enrico E Bryant Katherine K Kaakoush Nadeem O NO Tedla Nicodemus N

Journal of proteome research 20210401 6

The leukocyte immunoglobulin-like receptor A3 (LILRA3) is a soluble protein primarily expressed by peripheral blood monocytes and is abundant in sera of healthy donors. Extracellular LILRA3 is anti-inflammatory and displays neuro-regenerative functions in vitro. However, its intracellular expression, distribution, and function(s) remain unknown. Using a combination of high-resolution confocal and super-resolution microscopy, we identified intracellular expression of native LILRA3 in the nucleus ...[more]

PMID: 33793249

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Project description:Background: Atherosclerosis leads high mortality, highlighting an urgent need for new therapeutic strategies. Protein kinases orchestrate multiple cellular events in atherosclerosis and may provide new therapeutic targets for atherosclerosis. Here, we identified a protein kinase, WEE1 G2 checkpoint kinase (WEE1), promoting inflammatory response in atherosclerosis. Methods: The ApoE-/- mice without macrophage-specific WEE1 deletion (ApoE-/-WEE1f/f) and ApoE-/- mice with macrophage-specific WEE1 deletion (ApoE-/-WEE1MCKO) were generated using bone marrow transplantation. These mice and WEE1 inhibitor MK1775 were used in a high-fat diet (HFD)-induced atherosclerotic model. Human atherosclerotic tissues, mouse primary peritoneal macrophages (MPMs), 293T cells, and recombinant proteins were utilized to investigate the pathogenic role and underlying mechanisms of WEE1. Results: We identified up-regulated p-WEE1 in macrophages in atherosclerotic lesions of HFD-fed ApoE-/- mice. Transcriptome sequencing analysis indicated that WEE1 promotes oxLDL-induced inflammation in macrophages. We then demonstrated that macrophage-specific deletion or pharmacological inhibition of WEE1 attenuates atherosclerosis by reducing inflammation both in vivo and in vitro. The overexpression of wild-type WEE1 or activating-mutant WEE1, but not inactivating-mutant WEE1, exacerbates inflammation in macrophages. Mechanistically, transcriptome sequencing analysis and co-immunoprecipitation followed by quantitative proteomics analysis identified p65 as a binding protein of WEE1. We confirmed that WEE1 directly interacts with the RHD domain of p65 via kinase domain and phosphorylates p65 at S536, thereby facilitating subsequent NF-κB activation and inflammatory response in macrophages. Conclusions: Our findings demonstrated that macrophage WEE1 promotes inflammatory atherosclerosis by directly binding to p65 and phosphorylate it at S536. This study provides WEE1 as a new p65 regulator in inflammation and a potential therapeutic target for atherosclerosis.

Dataset Information

LILRA3-GFP and GFP only Transfected HEK293T cells LC-MSMS

Publications

Nuclear Leukocyte Immunoglobulin-like Receptor A3 Is Monomeric and Is Involved in Multiple Layers of Regulated Gene Expression and Translation.

Similar Datasets

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