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Characterisation of NBEAL2 interactome in HEK293T cells

ABSTRACT: Variants in NBEAL2 are causal of Grey Platelet Syndrome (GPS), a rare bleeding disorder characterized by absence of alpha- and specific- granules in platelets and neutrophils, respectively. The role of the scaffolding multidomain NBEAL2 protein in cell biology and granule homeostasis is unknown. We have performed proteomics to identify NBEAL2’s binding partners followed by different layers of validation including biochemical, cellular and functional analysis in vitro and in vivo.

INSTRUMENT(S): LTQ FT

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293t Cell, Cell Culture, Embryonic Stem Cell

SUBMITTER: James Wright

LAB HEAD: Jyoti Choudhary

PROVIDER: PXD006091 | Pride | 2018-02-14

REPOSITORIES: Pride

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Nbeal2 interacts with Dock7, Sec16a, and Vac14.

Mayer Louisa L Jasztal Maria M Pardo Mercedes M Aguera de Haro Salvadora S Collins Janine J Bariana Tadbir K TK Smethurst Peter A PA Grassi Luigi L Petersen Romina R Nurden Paquita P Favier Rémi R Yu Lu L Meacham Stuart S Astle William J WJ Choudhary Jyoti J Yue Wyatt W WW Ouwehand Willem H WH Guerrero Jose A JA

Blood 20171129 9

Mutations in <i>NBEAL2</i>, the gene encoding the scaffolding protein Nbeal2, are causal of gray platelet syndrome (GPS), a rare recessive bleeding disorder characterized by platelets lacking α-granules and progressive marrow fibrosis. We present here the interactome of Nbeal2 with additional validation by reverse immunoprecipitation of Dock7, Sec16a, and Vac14 as interactors of Nbeal2. We show that GPS-causing mutations in its BEACH domain have profound and possible effects on the interaction w ...[more]

PMID: 29187380

Similar Datasets

NBEAL2 deficiency in humans leads to low CTLA-4 expression in activated conventional T cells

Project description:Loss of NBEAL2 function leads to grey platelet syndrome (GPS), a bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets. A proportion of patients with GPS develop autoimmunity through an unknown mechanism, which might be related to the proteins NBEAL2 interacts with, specifically in immune cells. Here we show a comprehensive interactome of NBEAL2 in primary T cells, based on mass spectrometry identification of altogether 74 protein association partners. These include LRBA, a member of the same BEACH domain family as NBEAL2, recessive mutations of which cause autoimmunity and lymphocytic infiltration through defective CTLA-4 trafficking. Investigating the potential association between NBEAL2 and CTLA-4 signalling that is suggested by the mass spectrometry results, we confirm by co-immunoprecipitation that CTLA-4 and NBEAL2 interact with each-other. Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T cells, while their regulatory T cells appear unaffected. Knocking- down NBEAL2 in healthy primary T cells recapitulates the low CTLA-4 expression observed in T cells of GPS patients. Our results thus show that NBEAL2 is involved in the regulation of CTLA-4 expression in conventional T cells and provide a rationale for considering CTLA-4- immunoglobulin therapy in patients with GPS and autoimmune disease.

2023-05-12 | GSE196606 | GEO

EGAS00001000091-sc-20110715 - samples

Project description:Grey Platelet Syndrome (GPS)

| EGAD00001000029 | EGA

NBEAL2 interactome: comparison between healthy donors and grey platelet syndrome (NBEAL2 deficient) patients activated T cells samples.

2023-05-12 | PXD042180 | JPOST Repository

Grey Platelet Syndrome GPS

Project description:As part of the Bloodomics collaboration we have several categories of pedigrees with diseases/syndromes relevant to cardiovascular diseases (CVD). One such pedigree, Grey Platelet Syndrome (GPS) is a rare congenital bleeding disorder caused by a reduction or absence of alpha granules in platelets. Exome sequencing has been performed as part of a discovery program to ascertain potential causative variants of the clinical phenotype.

| EGAS00001000091 | EGA

Characterisation of Myst2 and Niam interactomes in mouse embryonic stem cells

Project description:Here we undertake the characterisation of Myst2 protein interactions in mouse embryonic stem cells by affinity purification and mass spectrometry using a Myst2-FTAP tagged cell line. This protein interaction study confirms that in mouse embryonic stem cells Myst2 is part of H3 and H4 histone acetylation complexes similar to those described in somatic cells. We identify a novel Myst2- associated protein, the tumour suppressor protein Niam (Nuclear Interactor of Arf and Mdm2), and show that it also interacts with subunits of Myst2 H4 HAT complexes. Human NIAM is involved in chromosome segregation, p53 regulation and cell proliferation in somatic cells, but its role in embryonic stem cells is unknown. We describe the first Niam embryonic stem cell interactome, which includes proteins with roles in DNA replication and repair, transcription, splicing and ribosome biogenesis. Many of Myst2 and Niam binding partners are required for correct embryonic development, implicating Myst2 and NIAM in the cooperative regulation of this process and suggesting a novel role for Niam in embryonic biology. The data provides a useful resource for exploring Myst2 and Niam essential cellular functions and should contribute to deeper understanding of organism development and survival as well as the development of cancer.

2017-08-22 | PXD005987 | Pride

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome

Project description:Gray platelet syndrome (GPS) is a rare inherited bleeding disorder characterized by absence of platelet ɑ-granules and pathogenic variants in NBEAL2. To discern the spectrum of pathological features, we obtained genotype and phenotype data from 47 GPS patients and performed RNA sequencing and protein mass spectrometry on blood cells and plasma in a subset of these patients. We identified 37 novel GPS-causing variants in NBEAL2. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4-lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Finally, we show that the plasma proteome of GPS patients has increased levels of proteins associated with inflammation and immune response, 27% of which are synthesized outside of the hematopoietic system, predominantly in the liver.

2020-07-28 | PXD017227 | Pride

Neutrophil specific granule and NETosis defect in Gray Platelet Syndrome

Project description:Gray Platelet Syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss of function variants in NBEAL2, a member of the family of BEACH genes, are causal of GPS. The gene is involved in fusion, fission and trafficking of vesicles and granules. Whether NBEAL2 controls the ontogeny of granules of myeloid cells remains disputed. We found that neutrophils obtained from the peripheral blood from GPS patients have a normal distribution of azurophilic granules, but show a deficiency of specific granules, as confirmed by immuno-electron microscopy and mass spectrometry proteomics analyses. In cultures from peripheral CD34+ hematopoietic stem cells (HSCs) into mature neutrophils, the time dynamics showed concordance of NBEAL2 and specific granule protein expression at transcriptional and protein level, which were discordant in neutrophils obtained GPS-HSCs. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as an important regulator of granule release (similar to platelets) which is suggested to occur upon egress into the blood stream. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis and killing of bacteria and fungi were intact. Since GPS patients do not excessively suffer from infections, the consequence of the reduced specific granule content and lack of NET formation for innate immunity remains to be explored.

2021-01-28 | PXD017784 | Pride

AhR interactome in early embryonic development

Project description:The transcriptional program of early embryonic development is tightly regulated by a set of well-defined transcription factors that suppress premature expression of differentiation genes and sustain the pluripotent identity. It is generally accepted that this program can be perturbed by environmental factors such as chemical pollutants, however the precise molecular mechanisms remain unknown. The Aryl Hydrocarbon Receptor (AHR) is a widely expressed nuclear receptor that senses environmental stimuli and modulates target gene expression. Here, we show that ectopic activation of AHR during early differentiation disrupts the differentiation program via the chromatin remodeling complex NuRD. The activated AHR/NuRD complex altered the expression of differentiation-specific genes that control the first two developmental decisions without affecting the pluripotency program. These findings identify a novel mechanism that allows environmental stimuli to disrupt embryonic development through AHR signaling.

2018-02-26 | PXD006089 | Pride

Long acting progestin-only contraceptives impair endometrial vasculature by inhibiting uterine vascular smooth muscle cell survival

Project description:Molecular mechanisms responsible for abnormal endometrial vasculature in women receiving long-acting progestin-only contraceptives (LAPCs) are unknown. We hypothesize that LAPCs impairs vascular smooth muscle cell (VSMC) and pericyte proliferation and/or migration producing thin-walled hyperdilated fragile microvessels prone to bleeding. Proliferating cell nuclear antigen (PCNA) and alpha smooth muscle actin (aSMA) double-immunohistochemistry assessed VSMC differentiation and proliferation in endometria from women pre- and postDepoProvera (Depo) treatment and from oophorectomized guinea pigs (OVX-GPs) treated with vehicle, estradiol (E2), medroxyprogesterone acetate (MPA) or E2+MPA pellets. After treating cultured VSMCs with MPA or etonogestrel (ETO), whole genome profiling, proliferation and migration assays were performed. Endometrial vasculature of Depo-administered women displayed reduced M-DM-.SMA immunoreactivity and fewer PCNA (+) nuclei among aSMA (+) cells (P<0.008). Microarray analysis of VSMCs identified several MPA and ETO-altered transcripts regulated by STAT1 signaling (p<2.22x10-6), including chemokine (C-C motif) ligand 2 (CCL2). Both MPA and ETO reduced VSMC proliferation and migration (p<0.001), recombinant CCL2 reversed this progestin inhibition, and, in turn, a STAT1 inhibitor abolished these CCL2 effects. Similarly, the endometria of MPA treated OVX-GPs displayed decreased aSMA staining and fewer PCNA (+) nuclei in VSMC (p<0.005). In conclusion, LAPCs promote abnormal endometrial vessel formation by inhibiting VSMC proliferation and migration. Total RNA (n=3) obtained from cultured TDCs incubated 6h with estradiol or estradio + medroxyprogesterone acetate with or without 1 ng/ml IL-1M-NM-2 for 6h.

2015-02-11 | E-GEOD-65835 | biostudies-arrayexpress

Gene expression analysis of murine wt and Nbeal2 -/- bone marrow derived mast cells (BMMCs) w/o IL-33 treatment

Project description:The Neurobeachin-like 2 (Nbeal2) protein is a BEACH (beige and chediak-higashi) family member. Loss of function mutations of the human NBEAL2 gene result in development of the gray platelet syndrome (GPS) a bleeding disorder characterized by macrothrombocytopenia, splenomegaly, and paucity of Î±-granules in megakaryocytes, platelets, mast cells (MCs), neutrophils and NK cells. We found that Nbeal2 deficiency in MCs leads to an enhanced resistance to growth factor deprivation, dysregulated distribution of c-Kit and of the multidrug resistant protein-1 (ABCB1), as well as to a pro-inflammatory MC-phenotype. These characteristics are caused by a defective protein degradation machinery resulting in accumulation of RSK and ribosomal proteins (RPS). These data demonstrate that Nbeal2 prevents oncogenic transformation by controlling protein homeostasis (proteostasis) in MCs.

2024-02-06 | GSE225692 | GEO

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