Dataset Information

Identification of novel SHARPIN binders

ABSTRACT: Sharpin, a multifunctional adaptor protein with oncogenic properties, regulates several signalling pathways. For example, Sharpin enhances signal-induced NF-κB signalling as part of the linear ubiquitin assembly complex (LUBAC) and inhibits integrins, the T cell receptor, caspase1 and PTEN. However, despite recent insights into Sharpin and LUBAC function, a systematic approach to identify signalling pathways regulated by Sharpin has not been reported. Here, we present the first ‘Sharpin interactome’, which identifies a large amount of novel potential Sharpin interactors. These data suggest that Sharpin and LUBAC might regulate a larger number of biological processes than previously identified, such as endosomal trafficking, RNA processing, metabolism and cytoskeleton regulation. Importantly, using the Sharpin interactome we have identified a novel role for Sharpin in lamellipodium formation. We demonstrate that Sharpin interacts with the Arp2/3 complex, a protein complex that catalyses actin filament branching, and that Sharpin and Arp2/3 colocalize in lamellipodia. We identified the Arp2/3-binding site in Sharpin and demonstrate using a specific Arp2/3-binding deficient mutant that the Sharpin-Arp2/3 interaction promotes lamellipodia formation in a LUBAC-independent fashion.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Guillaume Jacquemet

LAB HEAD: Jeroen Pouwels

PROVIDER: PXD004734 | Pride | 2017-08-11

REPOSITORIES: Pride

ACCESS DATA

Publications

The Sharpin interactome reveals a role for Sharpin in lamellipodium formation via the Arp2/3 complex.

Khan Meraj H MH Salomaa Siiri I SI Jacquemet Guillaume G Butt Umar U Miihkinen Mitro M Deguchi Takahiro T Kremneva Elena E Lappalainen Pekka P Humphries Martin J MJ Pouwels Jeroen J

Journal of cell science 20170803 18

Sharpin, a multifunctional adaptor protein, regulates several signalling pathways. For example, Sharpin enhances signal-induced NF-κB signalling as part of the linear ubiquitin assembly complex (LUBAC) and inhibits integrins, the T cell receptor, caspase 1 and PTEN. However, despite recent insights into Sharpin and LUBAC function, a systematic approach to identify the signalling pathways regulated by Sharpin has not been reported. Here, we present the first 'Sharpin interactome', which identifie ...[more]

PMID: 28775156

Similar Datasets

Project description:Development and homeostasis of blood vessels critically depend on the regulation of endothelial cell-cell junctions. Perturbations in cell-cell junction organization and function results in developmental defects and vascular pathologies including chronic inflammation, edema and atherosclerosis. Although many aspects of blood vessel formation and homeostasis depend on cell-cell junctions, the molecular mechanisms that regulate their dynamic rearrangement are not fully understood. The VEcad-catenin complex, which constitute the molecular basis of the adherens junctions (AJ), is connected to the actin cytoskeleton and its function is regulated by cytoskeletal contraction and actin-driven plasma membrane protrusions. Junction-associated intermitted lamellipodia (JAIL) are small actin-driven protrusions at cell-cell junctions controlled by the actin related protein 2/3 (Arp2/3)-complex that contribute to the regulation of cell-cell junctions. JAIL drive VEcad dynamics within the cell-cell junction thereby being critical for monolayer integrity, cell migration and angiogenesis. The molecular mechanisms regulating JAIL during vessel development are not completely understood. Coronin 1B (Coro1B) is an actin binding protein that controls actin networks at classical lamellipodia via both Arp2/3 complex and cofilin-mediated pathways. The role of Coro1B in endothelial cell (ECs) is not fully understood. In this study we demonstrate that Coro1B is a novel component and regulator of cell-cell junctions in ECs. Immunofluorescence studies show that Coro1B colocalizes with VEcad at cell-cell junctions in monolayers of ECs. Live-cell imaging reveal that Coro1B is recruited to, and operated at, actin-driven membrane protrusions at the cell-cell junctions. Coro1B recruitment to cell-cell junctions is regulated by cytoskeleton tension. By analyzing the Coro1B interactome, we identify integrin linked kinase (ILK) as new Coro1B-associated protein. Coro1B colocalizes with α-parvin, an interactor of ILK, at the leading edge of lamellipodia protrusions. Finally, functional experiments reveal that depletion of Coro1B causes defects in actin cytoskeleton and cell-cell junctions. In matrigel vessel network assays, depletion of Coro1B results in reduced network complexity, vessel number and vessel length. Together, our findings point towards a critical role for Coro1B in the dynamic remodeling of endothelial cell-cell junction and the assembly of vessel network.

Project description:The cytosolic protein Sharpin is as a component of the linear ubiquitin chain assembly complex (LUBAC), which regulates NF-κB signaling in response to specific ligands. Its inactivating mutation in Cpdm (chronic proliferative dermatitis mutation) mice causes multi-organ inflammation, yet this phenotype is not transferable into wildtype mice by hematopoietic stem cell transfer. Recent evidence demonstrated that Cpdm mice additionally display low bone mass, but the cellular and molecular causes of this phenotype remained to be established. Here we have applied non-decalcified histology together with cellular and dynamic histomorphometry to perform a thorough skeletal phenotyping of Cpdm mice. We show that Cpdm mice display trabecular and cortical osteopenia, solely explained by impaired bone formation, whereas osteoclastogenesis is unaffected. We additionally found that Cpdm mice display a severe disturbance of articular cartilage integrity in the absence of joint inflammation, supporting the concept that Sharpin-deficiency affects mesenchymal cell differentiation. Consistently, Cpdm mesenchymal cells displayed reduced osteogenic capacitiy ex vivo, yet this defect was not associated with impaired NF-κB signaling. A molecular comparison of wildtype and Cpdm bone marrow cell populations further revealed that Cpdm mesenchymal cells produce higher levels of Cxcl5 and lower levels of IL1ra. Collectively, our data demonstrate that skeletal defects of Cpdm mice are not caused by chronic inflammation, but that Sharpin is as a critical regulator of mesenchymal cell differentiation and gene expression. They additionally provide an alternative molecular explanation for the inflammatory phenotype of Cpdm mice and the absence of disease transfer by hematopoetic stem cell transplantation. Unsorted bone marrow cells from wildtype and Cpdm mice were cultured for 10 days in the presence of ascorbic acid and ß-glycerophosphate to induce osteogenic differentiation

Dataset Information

Identification of novel SHARPIN binders

Publications

The Sharpin interactome reveals a role for Sharpin in lamellipodium formation via the Arp2/3 complex.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure