Stimulation of cells with TNFα can promote distinct cell death pathways, including RIPK1-independent apoptosis, necroptosis, and RIPK1-dependent apoptosis (RDA)-the latter of which we still know little about. Here we show that RDA involves the rapid formation of a distinct detergent-insoluble, highly ubiquitinated, and activated RIPK1 pool, termed "iuRIPK1." iuRIPK1 forms after RIPK1 activation in TNF-receptor-associated complex I, and before cytosolic complex II formation and caspase activation. To identify regulators of iuRIPK1 formation and RIPK1 activation in RDA, we conducted a targeted siRNA screen of 1,288 genes. We found that NEK1, whose loss-of-function mutations have been identified in 3% of ALS patients, binds to activated RIPK1 and restricts RDA by negatively regulating formation of iuRIPK1, while LRRK2, a kinase implicated in Parkinson's disease, promotes RIPK1 activation and association with complex I in RDA. Further, the E3 ligases APC11 and c-Cbl promote RDA, and c-Cbl is recruited to complex I in RDA, where it promotes prodeath K63-ubiquitination of RIPK1 to lead to iuRIPK1 formation. Finally, we show that two different modes of necroptosis induction by TNFα exist which are differentially regulated by iuRIPK1 formation. Overall, this work reveals a distinct mechanism of RIPK1 activation that mediates the signaling mechanism of RDA as well as a type of necroptosis.

Ubiquitination of TNFR1-signaling-complex (TNF-RSC) controls the activation of RIPK1, a kinase critically involved in mediating multiple TNFα activated deleterious events. However, the molecular mechanism that coordinates different types of ubiquitination modifications to regulate the activation of RIPK1 kinase remains unclear. Here, we show that ABIN-1/NAF-1, a ubiquitin-binding protein, is recruited rapidly into TNF-RSC in a manner dependent upon M1 ubiquitinating complex LUBAC to regulate the recruitment of A20 to control K63 deubiquitination of RIPK1. ABIN-1 deficiency reduces the recruitment of A20 and licenses cells to die through necroptosis by promoting K63 ubiquitination and activation of RIPK1 with TNFα stimulation under conditions that would otherwise exclusively activate apoptosis in wild-type cells. Inhibition of RIPK1 kinase and RIPK3 deficiency block the embryonic lethality of Abin-1−/− mice. We propose that ABIN-1 provides a critical link between M1 ubiquitination mediated by LUBAC complex and K63 deubiquitination by phospho-A20 to modulate the activation of RIPK1.

Intraflagellar transport (IFT) particles of Chlamydomonas reinhardtii contain two distinct protein complexes, A and B, composed of at least 6 and 15 protein subunits, respectively. As isolated from C. reinhardtii flagella, IFT complex B can be further reduced to a ϳ500-kDa core that contains IFT88, 2؋ IFT81, 2؋ IFT74/72, IFT52, IFT46, IFT27, IFT25, and IFT22. In this study, yeast-based two-hybrid analysis was combined with bacterial coexpression to show that three of the core B subunits, IFT88, IFT52, and IFT46, interact directly with each other and, together, are capable of forming a ternary complex. Chemical cross-linking results support the IFT52-IFT88 interaction and provide additional evidence of an association between IFT27 and IFT81. With previous studies showing that IFT81 and IFT74/72 interact to form a (IFT81) 2 (IFT74/72) 2 heterotetramer and that IFT27 and IFT25 form a heterodimer, the architecture of complex B is revealing itself. Last, electroporation of recombinant IFT46 was used to rescue flagellar assembly of a newly identified ift46 mutant and to monitor in vivo localization and movement of the IFT particles.Found on the surface of many eukaryotic cells, cilia and flagella (redundant terms) are organelles consisting of membranebounded microtubular projections that emanate from basal body templates. Either motile or nonmotile, cilia have been adapted for a variety of functions, including cellular motility, directional fluid movement, sensory reception, and cellular signaling (reviewed in Refs. 1-4). Ciliary-based sensory reception includesvisionandolfaction,whereasciliary-mediatedreceptordependent signaling includes sonic hedgehog, noncanonical Wnt, and platelet-derived growth factor pathways (reviewed in Refs. 5-7). Defects in the assembly and function of these organelles have been associated with ciliopathies, an expanding list of human diseases that include immotile cilia and BardetBiedl syndromes and cystic kidney disorders, such as polycystic kidney disease and nephronophthisis (reviewed in Refs. 8 -12). Many of these ciliopathies have been linked to intraflagellar transport (IFT), 3 a conserved process required for the assembly and maintenance of eukaryotic cilia (reviewed in Refs. 13-15).IFT is characterized by the robust bidirectional movement of large proteinaceous particles along the length of the axonemal microtubules (16, 17). Kinesin-2 is responsible for driving the anterograde or outward movement (17-21), whereas the retrograde return to the cell body is powered by cytoplasmic dynein1b/2 (22, 23). Formerly known as rafts, the long IFT trains contain multiple copies of two distinct protein complexes, A and B (20, 24, 25). As isolated from the flagella of the green alga, Chlamydomonas reinhardtii, complex A contains at least six distinct proteins (IFT144, IFT140, IFT139, IFT122, IFT121, and IFT43), whereas complex B contains at least 13 proteins (IFT172, IFT88, IFT81, IFT80, IFT74/72, CrDYF-1, IFT57, IFT52, IFT46, IFT27, IFT25, IFT22, and IFT20); the subunit names reflect the relati...

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minimal medium with 2% dextrose and all required nutrients unless indicated; SG, synthetic minimal medium with 2% galactose and all required nutrients unless indicated; YPD, yeast peptone medium with 2% dextrose; ziNCD, zinc-induced necrotic cell deathAutophagy is essential for prolonging yeast survival during nutrient deprivation; however, this report shows that some autophagy proteins may also be accelerating population death in those conditions. While leucine starvation caused YCA1-mediated apoptosis characterized by increased annexin V staining, nitrogen deprivation triggered necrotic death characterized by increased propidium iodide uptake. Although a Datg8 strain died faster than its parental strain during nitrogen starvation, this mutant died slower than its parent during leucine starvation. Conversely, a Datg11 strain died slower than its parent during nitrogen starvation, but faster during leucine starvation. Curiously, although GFP-Atg8 complemented the Datg8 mutation, this protein made ATG8 cells more sensitive to nitrogen starvation, and less sensitive to leucine starvation. These results were difficult to explain if autophagy only extended life but could be an indication that a second form of autophagy could concurrently facilitate either apoptotic or necrotic cell death.

ABIN-1 (encoded by the gene Tnip1) is a ubiquitin-binding protein that can interact with ubiquitin-editing enzyme A20 (encoded by the gene TNFAIP3) to restrain the activation of necroptosis and NF-κB activation. Genetic variants in the genes Tnip1 and TNFAIP3 are both strongly associated with susceptibility to autoimmune chronic inflammatory diseases such as psoriasis vulgaris and systemic lupus erythematosus (SLE) in humans. Here we investigated the mechanism by which ABIN-1 regulated innate immune responses. We show that ABIN-1 heterozygosity sensitizes cells to antiviral response by mediating NF-κB-dependent and RIPK1-independent expression of pattern recognition molecules, including TLR3, RIG-I, and MDA5, in MEFs. Furthermore, we demonstrate that increased interaction of ABIN-1 and A20 with prolonged poly(I:C) stimulation of WT cells leads to A20-dependent reduction of ABIN-1 protein. Finally, we show that ABIN-1 heterozygosity sensitizes innate immune response of Abin-1 +/− mice in vivo by promoting the production of proinflammatory cytokines, which can be blocked upon inhibition of RIPK1 kinase. Inhibition of RIPK1 kinase activity in vivo partially reduces the expression of MDA5, RIG-I, and caspase-11 in Abin-1 +/− mice but not in WT mice. Thus, we conclude that ABIN-1 is a suppressor of innate immune response and the interaction of ABIN-1 with A20 controls innate immunity response through the NF-κB pathway and in both RIPK1 kinase activity-independent and dependent manner.