N4BP1 functions as a dimerization-dependent linear ubiquitin reader which regulates TNF signalling.

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) is a critical suppressor of proinflammatory cytokine production that acts as a regulator of innate immune signalling and inflammation. However, our current understanding about the molecular properties that enable N4BP1 to exert its suppressive potential remain limited. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerization-dependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure preferential recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under proapoptotic conditions, caspase-8 mediates proteolytic processing of N4BP1, resulting in rapid degradation of N4BP1 by the 26 S proteasome, and acceleration of apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a novel type of ubiquitin reader that selectively recognises linear ubiquitin which enables the timely and coordinated regulation of TNFR1-mediated inflammation and cell death.

Mesenchymal stem cells from biology to therapy.

Mesenchymal stem cells are as fascinating as they are enigmatic. They appear capable of performing a wide array of functions that cross skeletal biology, immunology and haematology. As therapeutics, mesenchymal stem cells or even just their secreted products may be used to regenerate tissue lost through injury or disease and suppress damaging immune reactions. However, these cells lack unique markers and are hard to identify and isolate as pure cell populations. They are often grown in laboratories using basic and undefined culture conditions. We cannot even agree on their name. While mesenchymal stem cells may lack the developmental understanding and defined differentiation hierarchies of their more illustrious stem cell cousins, they offer a compelling scientific challenge. In depth understanding of mesenchymal stem cell biology will enable us to exploit fully one of the most clinically valuable cell sources.

Epidermal growth factor can signal via ß-catenin to control proliferation of mesenchymal stem cells independently of canonical Wnt signalling.

Bone marrow mesenchymal stem/stromal cells (MSCs) maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal lineages. Signalling mechanisms that enable precise control of MSC function remain unclear. Here we report that by initially examining differences in signalling pathway expression profiles of individual MSC clones, we identified a previously unrecognised signalling mechanism regulated by epidermal growth factor (EGF) in primary human MSCs. We demonstrate that EGF is able to activate ß-catenin, a key component of the canonical Wnt signalling pathway. EGF is able to induce nuclear translocation of ß-catenin in human MSCs but does not drive expression of Wnt target genes or T cell factor (TCF) activity in MSC reporter cell lines. Using an efficient Design of Experiments (DoE) statistical analysis, with different combinations and concentrations of EGF and Wnt ligands, we were able to confirm that EGF does not influence the Wnt/ß-catenin pathway in MSCs. We show that the effects of EGF on MSCs are temporally regulated to initiate early "classical" EGF signalling mechanisms (e.g via mitogen activated protein kinase) with delayed activation of ß-catenin. By RNA-sequencing, we identified gene sets that were exclusively regulated by the EGF/ß-catenin pathway, which were distinct from classical EGF-regulated genes. However, subsets of classical EGF gene targets were significantly influenced by EGF/ß-catenin activation. These signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by classical EGF signalling.