Crosstalk Between NDP52 and LUBAC in Innate Immune Responses, Cell Death, and Xenophagy

Abstract

Nuclear dot protein 52 kDa (NDP52, also known as CALCOCO2) functions as a selective autophagy receptor. The linear ubiquitin chain assembly complex (LUBAC) specifically generates the N-terminal Met1-linked linear ubiquitin chain, and regulates innate immune responses, such as nuclear factor-κB (NF-κB), interferon (IFN) antiviral, and apoptotic pathways. Although NDP52 and LUBAC cooperatively regulate bacterial invasion-induced xenophagy, their functional crosstalk remains enigmatic. Here we show that NDP52 suppresses canonical NF-κB signaling through the broad specificity of ubiquitin-binding at the C-terminal UBZ domain. Upon TNF-α-stimulation, NDP52 associates with LUBAC through the HOIP subunit, but does not disturb its ubiquitin ligase activity, and has a modest suppressive effect on NF-κB activation by functioning as a component of TNF-α receptor signaling complex I. NDP52 also regulates the TNF-α-induced apoptotic pathway, but not doxorubicin-induced intrinsic apoptosis. A chemical inhibitor of LUBAC (HOIPIN-8) cancelled the increased activation of the NF-κB and IFN antiviral pathways, and enhanced apoptosis in NDP52-knockout and -knockdown HeLa cells. Upon Salmonella-infection, colocalization of Salmonella, LC3, and linear ubiquitin was detected in parental HeLa cells to induce xenophagy. Treatment with HOIPIN-8 disturbed the colocalization and facilitated Salmonella expansion. In contrast, HOIPIN-8 showed little effect on the colocalization of LC3 and Salmonella in NDP52-knockout cells, suggesting that NDP52 is a weak regulator in LUBAC-mediated xenophagy. These results indicate that the crosstalk between NDP52 and LUBAC regulates innate immune responses, apoptosis, and xenophagy.

Innate immune response of host cells infected with Salmonella

“…In the extrinsic pathway, ligands bind to death receptors on the cell surface and recruit the adaptor Fasassociated protein with death domain (FADD) and initiator caspases, which interact and change the conformation of the initiator caspase (such as caspase-8), leading to its activation, triggering the death execution pathway. At present, there are three main death receptor signaling pathways of apoptosis: Fas signaling pathway, TNFR1 signaling pathway, and TRAIL signaling pathway [35]. Salmonella effector proteins mediate apoptosis through these signaling pathways.…”

Structures, functions, and inhibitors of LUBAC and its related diseases

“…Together, these advantages make HOIPIN‐8 the most potent and specific inhibitor for LUBAC to date (Table 1). Moreover, HOIPINs are detected to effectively induce cell death in activated B‐cell‐like diffuse large B‐cell lymphoma and attenuate psoriasis caught by imiquimod in model mice 98–100 …”

“…Moreover, HOIPINs are detected to effectively induce cell death in activated B-cell-like diffuse large Bcell lymphoma and attenuate psoriasis caught by imiquimod in model mice. [98][99][100] Collectively, emerging evidence has proved that LUBAC can be thus considered as a candidate target for the treatment of tumors or related immune diseases, so the discovery and development of its inhibitors are highly desirable and will play a vital role in the future treatment of diseases.…”

Novel functions of LUBAC-mediated M1 poly-ubiquitination in TNFR1-mediated necroptosis

“…Of note, HOIPIN-8 effectively inhibits NF-κB activation and IFN antiviral pathways in several human and murine cell lines, including HEK293T, HeLa, A549, Jurkat, MEF, murine bone marrow-derived macrophages (BMDM) [285,286,381], as well as in HT-29 (this study). In addition, HOIPIN-8 treatment has been shown to block xenophagy of invading Salmonella in HeLa cells, by inhibiting M1 Ub decoration of Salmonella and recruitment of LC3-positive membranes, which initiate bacterial clearance [381].…”

“…Interestingly, LUBAC is conserved among mammals, where it regulates innate and acquired immune responses, and an ortholog of HOIP, named linear ubiquitin E3 ligase (LUBEL), has been recently identified to control stress and inflammatory responses in drosophila [379,380], suggesting that the role of M1-linked ubiquitination in maintaining cellular homeostasis is evolutionary conserved [288]. Of note, HOIPIN-8 effectively inhibits NF-κB activation and IFN antiviral pathways in several human and murine cell lines, including HEK293T, HeLa, A549, Jurkat, MEF, murine bone marrow-derived macrophages (BMDM) [285,286,381], as well as in HT-29 (this study). In addition, HOIPIN-8 treatment has been shown to block xenophagy of invading Salmonella in HeLa cells, by inhibiting M1 Ub decoration of Salmonella and recruitment of LC3-positive membranes, which initiate bacterial clearance [381].…”