Two NEMO-like Ubiquitin-Binding Domains in CEP55 Differently Regulate Cytokinesis.

CEP55 regulates the final critical step of cell division termed cytokinetic abscission. We report herein that CEP55 contains two NEMO-like ubiquitin-binding domains (UBDs), NOA and ZF, which regulate its function in a different manner. In vitro studies of isolated domains showed that NOA adopts a dimeric coiled-coil structure, whereas ZF is based on a UBZ scaffold. Strikingly, CEP55 knocked-down HeLa cells reconstituted with the full-length CEP55 ubiquitin-binding defective mutants, containing structure-guided mutations either in NOACEP55 or ZFCEP55 domains, display severe abscission defects. In addition, the ZFCEP55 can be functionally replaced by some ZF-based UBDs belonging to the UBZ family, indicating that the essential function of ZFCEP55 is to act as ubiquitin receptor. Our work reveals an unexpected role of CEP55 in non-degradative ubiquitin signaling during cytokinetic abscission and provides a molecular basis as to how CEP55 mutations can lead to neurological disorders such as the MARCH syndrome.

Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti.

The NF-κB pathway has critical roles in cancer, immunity and inflammatory responses. Understanding the mechanism(s) by which mutations in genes involved in the pathway cause disease has provided valuable insight into its regulation, yet many aspects remain unexplained. Several lines of evidence have led to the hypothesis that the regulatory/sensor protein NEMO acts as a biological binary switch. This hypothesis depends on the formation of a higher-order structure, which has yet to be identified using traditional molecular techniques. Here we use super-resolution microscopy to reveal the existence of higher-order NEMO lattice structures dependent on the presence of polyubiquitin chains before NF-κB activation. Such structures may permit proximity-based trans-autophosphorylation, leading to cooperative activation of the signalling cascade. We further show that NF-κB activation results in modification of these structures. Finally, we demonstrate that these structures are abrogated in cells derived from incontinentia pigmenti patients.

Human HOIP and LUBAC deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia.

Inherited, complete deficiency of human HOIL-1, a component of the linear ubiquitination chain assembly complex (LUBAC), underlies autoinflammation, infections, and amylopectinosis. We report the clinical description and molecular analysis of a novel inherited disorder of the human LUBAC complex. A patient with multiorgan autoinflammation, combined immunodeficiency, subclinical amylopectinosis, and systemic lymphangiectasia, is homozygous for a mutation in HOIP, the gene encoding the catalytic component of LUBAC. The missense allele (L72P, in the PUB domain) is at least severely hypomorphic, as it impairs HOIP expression and destabilizes the whole LUBAC complex. Linear ubiquitination and NF-κB activation are impaired in the patient's fibroblasts stimulated by IL-1ß or TNF. In contrast, the patient's monocytes respond to IL-1ß more vigorously than control monocytes. However, the activation and differentiation of the patient's B cells are impaired in response to CD40 engagement. These cellular and clinical phenotypes largely overlap those of HOIL-1-deficient patients. Clinical differences between HOIL-1- and HOIP-mutated patients may result from differences between the mutations, the loci, or other factors. Our findings show that human HOIP is essential for the assembly and function of LUBAC and for various processes governing inflammation and immunity in both hematopoietic and nonhematopoietic cells.

Phenotypic complementation of genetic immunodeficiency by chronic herpesvirus infection.

Variation in the presentation of hereditary immunodeficiencies may be explained by genetic or environmental factors. Patients with mutations in HOIL1 (RBCK1) present with amylopectinosis-associated myopathy with or without hyper-inflammation and immunodeficiency. We report that barrier-raised HOIL-1-deficient mice exhibit amylopectin-like deposits in the myocardium but show minimal signs of hyper-inflammation. However, they show immunodeficiency upon acute infection with Listeria monocytogenes, Toxoplasma gondii or Citrobacter rodentium. Increased susceptibility to Listeria was due to HOIL-1 function in hematopoietic cells and macrophages in production of protective cytokines. In contrast, HOIL-1-deficient mice showed enhanced control of chronic Mycobacterium tuberculosis or murine γ-herpesvirus 68 (MHV68), and these infections conferred a hyper-inflammatory phenotype. Surprisingly, chronic infection with MHV68 complemented the immunodeficiency of HOIL-1, IL-6, Caspase-1 and Caspase-1;Caspase-11-deficient mice following Listeria infection. Thus chronic herpesvirus infection generates signs of auto-inflammation and complements genetic immunodeficiency in mutant mice, highlighting the importance of accounting for the virome in genotype-phenotype studies.

TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO-IKK supramolecular structures.

Nuclear factor κB (NF-κB) essential modulator (NEMO), a regulatory component of the IκB kinase (IKK) complex, controls NF-κB activation through its interaction with ubiquitin chains. We show here that stimulation with interleukin-1 (IL-1) and TNF induces a rapid and transient recruitment of NEMO into punctate structures that are anchored at the cell periphery. These structures are enriched in activated IKK kinases and ubiquitinated NEMO molecules, which suggests that they serve as organizing centers for the activation of NF-κB. These NEMO-containing structures colocalize with activated TNF receptors but not with activated IL-1 receptors. We investigated the involvement of nondegradative ubiquitination in the formation of these structures, using cells deficient in K63 ubiquitin chains or linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination. Our results indicate that, unlike TNF, IL-1 requires K63-linked and linear ubiquitin chains to recruit NEMO into higher-order complexes. Thus, different mechanisms are involved in the recruitment of NEMO into supramolecular complexes, which appear to be essential for NF-κB activation.

The 4 Notch receptors play distinct and antagonistic roles in the proliferation and hepatocytic differentiation of liver progenitors.

The Notch signaling pathway is involved in liver development and regeneration. Here, we investigate the role of the 4 mammalian Notch paralogs in the regulation of hepatoblast proliferation and hepatocytic differentiation. Our model is based on bipotential mouse embryonic liver (BMEL) progenitors that can differentiate into hepatocytes or cholangiocytes in vitro and in vivo. BMEL cells were subjected to Notch antagonists or agonists. Blocking Notch activation with a γ-secretase inhibitor, at 50 µM for 48 h, reduced cell growth by 50%. S-phase entry was impaired, but no apoptosis was induced. A systematic paralog-specific strategy was set using lentiviral transduction with constitutively active forms of each Notch receptor along with inhibition of endogenous Notch signaling. This assay demonstrates that proliferation of BMEL cells requires Notch2 and Notch4 activity, resulting in significant down-regulation of p27(Kip1) and p57(Kip2) cyclin-dependent kinase inhibitors. Conversely, Notch3-expressing cells proliferate less and express 3-fold higher levels of p57(Kip2). The Notch3 cells present a hepatocyte-like morphology, enhanced multinucleation, and a ploidy shift. Moreover, Notch3 activity is conducive to hepatocytic differentiation in vitro, while its paralogs impede this fate. Our study provides the first evidence of a functional diversity among the mammalian Notch homologues in the proliferation and hepatocytic-lineage commitment of liver progenitors.

A glycosphingolipid binding domain controls trafficking and activity of the mammalian notch ligand delta-like 1.

The activity of Notch ligands is tightly regulated by trafficking events occurring both before and after ligand-receptor interaction. In particular endocytosis and recycling have been shown to be required for full signaling activity of the ligands before they encounter the Notch receptor. However little is known about the precise endocytic processes that contribute to ligand internalization. Here we demonstrate that endocytosis contributes to Dll1 signaling activity by preserving the ligand from shedding and degradation. We further show that the glycosphingolipid-binding motif originally identified in Drosophila Notch ligands is conserved in mammals and is necessary for Dll1 internalization. Mutation of its conserved tryptophan residue results in a Dll1 molecule which is rapidly inactivated by shedding and degradation, does not recycle to the cell surface and does not activate Notch signaling. Finally, silencing in the signal-sending cells of glucosylceramide synthase, the enzyme implicated in the initial phase of glycosphingolipid synthesis, down-regulates Notch activation. Our data indicate that glycosphingolipids, by interacting with Dll1, may act as functional co-factors to promote its biological activity.

Α-arrestin 1 (ARRDC1) and ß-arrestins cooperate to mediate Notch degradation in mammals.

Notch signaling is a conserved signaling pathway implicated in embryogenesis and adult tissue maintenance. Notch signaling strength is strictly regulated, notably by maintaining a controlled pool of functional receptor at the cell surface. Mammalian non-activated Notch receptor is internalized, ubiquitylated by the Itch E3 ubiquitin ligase and degraded in the lysosomes. Here, we show that ß-arrestins are necessary for Itch-Notch interaction and for Itch-driven ubiquitylation and degradation of Notch. Interestingly, ß-arrestins do not directly bind Itch but heterodimerize with a member of another subfamily of arrestins called ARRDC1 or α-arrestin 1, which harbors PPxY motifs that allow direct interaction with Itch. Cells transfected with ARRDC1 mutated in PPxY motifs show reduced Itch-mediated Notch ubiquitylation and impaired lysosomal degradation of Notch, as observed in ß-arrestin(-/-) or Itch(-/-) cells. Our data show for the first time that ARRDC1 and ß-arrestins heterodimerize and cooperate in the same complex to promote non-activated Notch receptor degradation, thus acting as negative regulators of Notch signaling.

Immunodeficiency, autoinflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency.

We report the clinical description and molecular dissection of a new fatal human inherited disorder characterized by chronic autoinflammation, invasive bacterial infections and muscular amylopectinosis. Patients from two kindreds carried biallelic loss-of-expression and loss-of-function mutations in HOIL1 (RBCK1), a component of the linear ubiquitination chain assembly complex (LUBAC). These mutations resulted in impairment of LUBAC stability. NF-κB activation in response to interleukin 1ß (IL-1ß) was compromised in the patients' fibroblasts. By contrast, the patients' mononuclear leukocytes, particularly monocytes, were hyper-responsive to IL-1ß. The consequences of human HOIL-1 and LUBAC deficiencies for IL-1ß responses thus differed between cell types, consistent with the unique association of autoinflammation and immunodeficiency in these patients. These data suggest that LUBAC regulates NF-κB-dependent IL-1ß responses differently in different cell types.

The ubiquitin-specific protease 12 (USP12) is a negative regulator of notch signaling acting on notch receptor trafficking toward degradation.

Notch signaling is critical for development and adult tissue physiology, controlling cell fate in a context-dependent manner. Upon ligand binding, the transmembrane Notch receptor undergoes two ordered proteolytic cleavages releasing Notch intracellular domain, which regulates the transcription of Notch target genes. The strength of Notch signaling is of crucial importance and depends notably on the quantity of Notch receptor at the cell surface. Using an shRNA library screen monitoring Notch trafficking and degradation in the absence of ligand, we identified mammalian USP12 and its Drosophila melanogaster homolog as novel negative regulators of Notch signaling. USP12 silencing specifically interrupts Notch trafficking to the lysosomes and, as a consequence, leads to an increased amount of receptor at the cell surface and to a higher Notch activity. At the biochemical level, USP12 with its activator UAF1 deubiquitinate the nonactivated form of Notch in cell culture and in vitro. These results characterize a new level of conserved regulation of Notch signaling by the ubiquitin system.

Plk1-dependent phosphorylation of optineurin provides a negative feedback mechanism for mitotic progression.

Plk1 activation is required for progression through mitotic entry to cytokinesis. Here we show that at mitotic entry, Plk1 phosphorylates Optineurin (Optn) at serine 177 and that this dissociates Optn from the Golgi-localized GTPase Rab8, inducing its translocation into the nucleus. Mass spectrometry analysis revealed that Optn is associated with a myosin phosphatase complex (MP), which antagonizes the mitotic function of Plk1. Our data also indicate that Optn functionally connects this complex to Plk1 by promoting phosphorylation of the myosin phosphatase targeting subunit 1 (MYPT1). Accordingly, silencing Optn expression increases Plk1 activity and induces abscission failure and multinucleation, which were rescued upon expression of wild-type (WT) Optn, but not a phospho-deficient mutant (S177A) that cannot translocate into the nucleus during mitosis. Overall, these results highlight an important role of Optn in the spatial and temporal coordination of Plk1 activity.

Regrowing the adult brain: NF-κB controls functional circuit formation and tissue homeostasis in the dentate gyrus.

Cognitive decline during aging is correlated with a continuous loss of cells within the brain and especially within the hippocampus, which could be regenerated by adult neurogenesis. Here we show that genetic ablation of NF-κB resulted in severe defects in the neurogenic region (dentate gyrus) of the hippocampus. Despite increased stem cell proliferation, axogenesis, synaptogenesis and neuroprotection were hampered, leading to disruption of the mossy fiber pathway and to atrophy of the dentate gyrus during aging. Here, NF-κB controls the transcription of FOXO1 and PKA, regulating axogenesis. Structural defects culminated in behavioral impairments in pattern separation. Re-activation of NF-κB resulted in integration of newborn neurons, finally to regeneration of the dentate gyrus, accompanied by a complete recovery of structural and behavioral defects. These data identify NF-κB as a crucial regulator of dentate gyrus tissue homeostasis suggesting NF-κB to be a therapeutic target for treating cognitive and mood disorders.

NEMO is a key component of NF-κB- and IRF-3-dependent TLR3-mediated immunity to herpes simplex virus.

BACKGROUND: Children with germline mutations in Toll-like receptor 3 (TLR3), UNC93B1, TNF receptor-associated factor 3, and signal transducer and activator of transcription 1 are prone to herpes simplex virus-1 encephalitis, owing to impaired TLR3-triggered, UNC-93B-dependent, IFN-α/ß, and/or IFN-λ-mediated signal transducer and activator of transcription 1-dependent immunity. OBJECTIVE: We explore here the molecular basis of the pathogenesis of herpes simplex encephalitis in a child with a hypomorphic mutation in nuclear factor-κB (NF-κB) essential modulator, which encodes the regulatory subunit of the inhibitor of the Iκß kinase complex. METHODS: The TLR3 signaling pathway was investigated in the patient's fibroblasts by analyses of IFN-ß, IFN-λ, and IL-6 mRNA and protein levels, by quantitative PCR and ELISA, respectively, upon TLR3 stimulation (TLR3 agonists or TLR3-dependent viruses). NF-κB activation was assessed by electrophoretic mobility shift assay and interferon regulatory factor 3 dimerization on native gels after stimulation with a TLR3 agonist. RESULTS: The patient's fibroblasts displayed impaired responses to TLR3 stimulation in terms of IFN-ß, IFN-λ, and IL-6 production, owing to impaired activation of both NF-κB and IRF-3. Moreover, vesicular stomatitis virus, a potent IFN-inducer in human fibroblasts, and herpes simplex virus-1, induced only low levels of IFN-ß and IFN-λ in the patient's fibroblasts, resulting in enhanced viral replication and cell death, as reported for UNC-93B-deficient fibroblasts. CONCLUSION: Herpes simplex encephalitis may occur in patients carrying NF-κB essential modulator mutations, due to the impairment of NF-κB- and interferon regulatory factor 3-dependent-TLR3-mediated antiviral IFN production.

New mechanism of X-linked anhidrotic ectodermal dysplasia with immunodeficiency: impairment of ubiquitin binding despite normal folding of NEMO protein.

Nuclear factor-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase complex, is a critical component of the NF-κB pathway. Hypomorphic mutations in the X-linked human NEMO gene cause various forms of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID). All known X-linked EDA-ID-causing mutations impair NEMO protein expression, folding, or both. We describe here 2 EDA-ID-causing missense mutations that affect the same residue in the CC2-LZ domain (D311N and D311G) that do not impair NEMO production or folding. Structural studies based on pull-down experiments showed a defect in noncovalent interaction with K63-linked and linear polyubiquitin chains for these mutant proteins. Functional studies on the patients' cells showed an impairment of the classic NF-κB signaling pathways after activation of 2 NEMO ubiquitin-binding-dependent receptors, the TNF and IL-1ß receptors, and in the CD40-dependent NF-κB pathway. We report the first human NEMO mutations responsible for X-linked EDA-ID found to affect the polyubiquitin binding of NEMO rather than its expression and folding. These experiments demonstrate that the binding of human NEMO to polyubiquitin is essential for NF-κB activation. They also demonstrate that the normal expression and folding of NEMO do not exclude a pathogenic role for NEMO mutations in patients with EDA-ID.

The adaptor-associated kinase 1, AAK1, is a positive regulator of the Notch pathway.

The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. The extracellular part of the receptor is removed by a metalloprotease of the ADAM family and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent γ-secretase activity. γ-Secretase processing of Notch has been shown to depend upon monoubiquitination as well as clathrin-mediated endocytosis (CME). We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of the γ-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization. These results suggest that after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates.

IKK regulation and human genetics.

The IKK kinase complex is the core element of the NF-κB cascade. It is essentially made of two kinases (IKKα and IKKß) and a regulatory subunit, NEMO/IKKγ. Additional components may exist, transiently or permanently, but their characterization is still uncertain. In this review, we will focus on the NEMO molecule, and describe the results which have been obtained, and the hypotheses which have been proposed, to explain how NEMO controls the activation of the IKK complex. NEMO is one of the very few non-redundant components of the NF-κB cascade, and the localization of the gene that encodes it on the X chromosome suggests it is likely to be the target of mutations leading to pathologies: this is indeed the case, and we will also present the current status of our knowledge regarding NEMO-associated pathologies.

The translation initiation factor 3f (eIF3f) exhibits a deubiquitinase activity regulating Notch activation.

Activation of the mammalian Notch receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.

The IKK complex, a central regulator of NF-kappaB activation.

The IKK kinase complex is the core element of the NF-kappaB cascade. It is essentially made of two kinases (IKKalpha and IKKbeta) and a regulatory subunit, NEMO/IKKgamma. Additional components may exist, transiently or permanently, but their characterization is still unsure. In addition, it has been shown that two separate NF-kappaB pathways exist, depending on the activating signal and the cell type, the canonical (depending on IKKbeta and NEMO) and the noncanonical pathway (depending solely on IKKalpha). The main question, which is still only partially answered, is to understand how an NF-kappaB activating signal leads to the activation of the kinase subunits, allowing them to phosphorylate their targets and eventually induce nuclear translocation of the NF-kappaB dimers. I will review here the genetic, biochemical, and structural data accumulated during the last 10 yr regarding the function of the three IKK subunits.

IKK connects autophagy to major stress pathways.

Cells respond to stress by activating cytoplasmic mechanisms as well as transcriptional programs that can lead to adaptation or death. Autophagy represents an important cytoprotective response that is regulated by both transcriptional and transcription-independent pathways. NFkappaB is perhaps the transcription factor most frequently activated by stress and has been ascribed with either pro- or anti-autophagic functions, depending on the cellular context. Our results demonstrate that activation of the IKK (IkappaB kinase) complex, which is critical for the stress-elicited activation of NFkappaB, is sufficient to promote autophagy independent of NFkappaB, and that IKK is required for the optimal induction of autophagy by both physiological and pharmacological autophagic triggers.