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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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:HEK293 cells were cultured in two 10-cm dishes and transfected with BAG3-FLAG as well as Myc vector, CaMKIIβ(D-S)-Myc, and CaMKIIδ(D-S)-MYC, respectively. After the cells were transfected for 24 h, they were treated with the indicated regent. The cells were lysed with NP-40 alternative cell lysis buffer containing protease (Thermo Fisher Scientific, 87785) and phosphatase inhibitor mixtures (Thermo Fisher Scientific, 78427) on ice for 30 min and centrifuged at 14 000 g for 15 min at 4°C. For the supernatant, BAG3-FLAG was immunopurified with anti-FLAG Magnetic Beads (MCE, HY-K0207). Immunoprecipitated proteins were separated by SDS-PAGE and identified by staining with 0.25 % Coomassie brilliant blue R250. The BAG3-FLAG protein bands were cut out and digested with trypsin at 37°C overnight. The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient increased from 6-23% solvent B (0.1% formic acid in 98% acetonitrile) over 16 min, 23-35% in 8 min and climbing to 80% in 3 min, and then holding at 80% for the last 3 min. The flow rate remained constant at 400 nl/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for a full scan, and the intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting at 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan and 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The resulting MS/MS data were processed using Proteome Discoverer 2.4. Tandem mass spectra were searched against the Uniport protein database. Trypsin was specified as a cleavage enzyme allowing up to 2 missing cleavages. The mass error was set to 10 ppm and 0.02 Da for precursor and for fragment ions, respectively. Phospho-(S/T/Y) were chosen as variable modifications. Peptide confidence was set at high, and peptide ion score was set at > 20.

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	F147070.dat	Other
	Nicodemus-8-3-19-A3-eGFP-IgG-1ul.raw	Raw