Biomedical subjects

Alain Israël

Publications and source records attributed to Alain Israël.

At least 19 recordsLinked to original sources

PubMed2006

Itch/AIP4 mediates Deltex degradation through the formation of K29-linked polyubiquitin chains.

Deltex (DTX) and AIP4 are the human orthologues of the Drosophila deltex and Suppressor of deltex, which have been genetically described as being antagonistically involved in the Notch signalling pathway. Both genes encode E3 ubiquitin ligases of the RING (Really interesting new gene)-H2 and HECT (Homologous to E6AP carboxyl terminus) families, respectively. In an attempt to understand the molecular basis of their genetic interactions, we studied the relationship between DTX and AIP4 in the absence of activation of the Notch pathway. We show here that both molecules interact and partially colocalize to endocytic vesicles, and that AIP4 targets DTX for lysosomal degradation. Furthermore, AIP4-generated polyubiquitin chains are mainly conjugated through lysine 29 of ubiquitin in vivo, indicating a link between this type of chain and lysosomal degradation.

Animals↗

PubMed2006

NF-kappaB activation: Nondegradative ubiquitination implicates NEMO.

Non-degradative ubiquitination events have been recently demonstrated to have an important role in activating the IkappaB kinase (IKK) complex, the central component of the nuclear factor (NF)-kappaB signaling cascade. Two new papers demonstrate that NF-kappaB essential modulator (NEMO)-IKKgamma, the structural and regulatory component of the IKK complex, specifically recognizes Lys63 polyubiquitin chains attached to the receptor-interacting protein (RIP)1 kinase following tumor necrosis factor treatment, and that this recognition is a requirement for IKK activation.

Enzyme Activation↗

PubMed2006

X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production.

Germline mutations in five autosomal genes involved in interleukin (IL)-12-dependent, interferon (IFN)-gamma-mediated immunity cause Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular basis of X-linked recessive (XR)-MSMD remains unknown. We report here mutations in the leucine zipper (LZ) domain of the NF-kappaB essential modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The mutant proteins were produced in normal amounts in blood and fibroblastic cells. However, the patients' monocytes presented an intrinsic defect in T cell-dependent IL-12 production, resulting in defective IFN-gamma secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-kappaB/c-Rel-mediated CD40 signaling after the stimulation of monocytes and dendritic cells by CD40L-expressing T cells and fibroblasts, respectively. However, the CD40-dependent up-regulation of costimulatory molecules of dendritic cells and the proliferation and immunoglobulin class switch of B cells were normal. Moreover, the patients' blood and fibroblastic cells responded to other NF-kappaB activators, such as tumor necrosis factor-alpha, IL-1beta, and lipopolysaccharide. These two mutations in the NEMO LZ domain provide the first genetic etiology of XR-MSMD. They also demonstrate the importance of the T cell- and CD40L-triggered, CD40-, and NEMO/NF-kappaB/c-Rel-mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans.

Adolescent↗

PubMed2006

NF-kappaB regulates spatial memory formation and synaptic plasticity through protein kinase A/CREB signaling.

Synaptic activity-dependent de novo gene transcription is crucial for long-lasting neuronal plasticity and long-term memory. In a forebrain neuronal conditional NF-kappaB-deficient mouse model, we demonstrate here that the transcription factor NF-kappaB regulates spatial memory formation, synaptic transmission, and plasticity. Gene profiling experiments and analysis of regulatory regions identified the alpha catalytic subunit of protein kinase A (PKA), an essential memory regulator, as a new NF-kappaB target gene. Consequently, NF-kappaB inhibition led to a decrease in forskolin-induced CREB phosphorylation. Collectively, these results disclose a novel hierarchical transcriptional network involving NF-kappaB, PKA, and CREB that leads to concerted nuclear transduction of synaptic signals in neurons, accounting for the critical function of NF-kappaB in learning and memory.

Animals↗

PubMed2005

A point mutation in NEMO associated with anhidrotic ectodermal dysplasia with immunodeficiency pathology results in destabilization of the oligomer and reduces lipopolysaccharide- and tumor necrosis factor-mediated NF-kappa B activation.

The NEMO (NF-kappaB essential modulator) protein plays a crucial role in the canonical NF-kappaB pathway as the regulatory component of the IKK (IkappaB kinase) complex. The human disease anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has been recently linked to mutations in NEMO. We investigated the effect of an alanine to glycine substitution found in the NEMO polypeptide of an EDA-ID patient. This pathogenic mutation is located within the minimal oligomerization domain of the protein, which is required for the IKK activation in response to diverse stimuli. The mutation does not dramatically change the native-like state of the trimer, but temperature-induced unfolding studied by circular dichroism showed that it leads to an important loss in the oligomer stability. Furthermore, fluorescence studies showed that the tyrosine located in the adjacent zinc finger domain, which is possibly required for NEMO ubiquitination, exhibits an alteration in its spectral properties. This is probably due to a conformational change of this domain, providing evidence for a close interaction between the oligomerization domain and the zinc finger. In addition, functional complementation assays using NEMO-deficient pre-B and T lymphocytes showed that the pathogenic mutation reduced TNF-alpha and LPS-induced NF-kappaB activation by altering the assembly of the IKK complex. Altogether, our findings provide understanding as to how a single point mutation in NEMO leads to the observed EDA-ID phenotype in relation to the NEMO-dependent mechanism of IKK activation.

Amino Acid Sequence↗

PubMed2005

Generation and characterization of mutant cell lines defective in gamma-secretase processing of Notch and amyloid precursor protein.

Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. We have set up a genetic screen based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. We selected clones deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. We further analyzed two of these clones and identified previously undescribed mutations in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired.

Amino Acid Sequence↗

PubMed2004

The notch ligand Delta1 recruits Dlg1 at cell-cell contacts and regulates cell migration.

Delta1 acts as a membrane-bound ligand that interacts with the Notch receptor and plays a critical role in cell fate specification. By using peptide affinity chromatography followed by mass spectrometry, we have identified Dlg1 as a partner of the Delta1 C-terminal region. Dlg1 is a human homolog of the Drosophila Discs large tumor suppressor, a member of the membrane-associated guanylate kinase family of molecular scaffolds. We confirmed this interaction by co-immunoprecipitation experiments between endogenous Dlg1 and transduced Delta1 in a 3T3 cell line stably expressing Delta1. Moreover, we showed that deletion of a canonical C-terminal PDZ-binding motif (ATEV) in Delta1 abrogated this interaction. Delta4 also interacted with Dlg1, whereas Jagged1, another Notch ligand, did not. In HeLa cells, transfected Delta1 triggered the accumulation of endogenous Dlg1 at sites of cell-cell contact. Expression of Delta1 also reduced the motility of 3T3 cells. Finally, deletion of the ATEV motif totally abolished these effects but did not interfere with the ability of Delta1 to induce Notch signaling and T cell differentiation in co-culture experiments. These results point to a new, probably cell-autonomous function of Delta1, which is independent of its activity as a Notch ligand.

3T3 Cells↗

PubMed2004

Inhibition of NF-kappa B activation by peptides targeting NF-kappa B essential modulator (nemo) oligomerization.

NF-kappa B essential modulator/IKK-gamma (NEMO/IKK-gamma) plays a key role in the activation of the NF-kappa B pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (Agou, F., Traincard, F., Vinolo, E., Courtois, G., Yamaoka, S., Israel, A., and Veron, M. (2004) J. Biol. Chem. 279, 27861-27869). To search for drugs inhibiting NF-kappa B activation, we have rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. Peptide transduction was monitored by fluorescence-activated cell sorter, and their effect on lipopolysaccharide-induced NF-kappa B activation was quantified using an NF-kappa B-dependent beta-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. We show that the peptides corresponding to the LZ and CC2 subdomains inhibit NF-kappa B activation with an IC(50) in the mum range. Control peptides, including mutated CC2 and LZ peptides and a heterologous coiled-coil peptide, had no inhibitory effect. The designed peptides are able to induce cell death in human retinoblastoma Y79 cells exhibiting constitutive NF-kappa B activity. Our results provide the "proof of concept" for a new and promising strategy for the inhibition of NF-kappa B pathway activation through targeting the oligomerization state of the NEMO protein.

Amino Acid Sequence↗

PubMed2004

Visualization of lymphatic vessels through NF-kappaB activity.

The molecular biology of lymphatics is only rudimentary owing to the long-standing absence of specific markers, and scanty is the information regarding bladder lymphatic vessels. By using mice with a reporter gene for nuclear factor kappaB (NF-kappaB) activity (kappaB-lacZ) in combination with immunohistochemical staining with a specific lymphatic marker (LYVE-1), we show, for the first time, that NF-kappaB is constitutively active in lymphatic endothelium in the urinary bladder, uterus, intestine, heart, and airways. Tie2-lacZ mice confirmed that the structures observed in kappaB-lacZ mice were not blood vessels. In addition, acute instillation of lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-alpha) into the kappaB-lacZ mouse bladder revealed the capacity of this transgenic in reporting inducible NF-kappaB activity. Our findings demonstrate an overriding constitutive NF-kappaB activity in the lymphatic system. They also provide a working model for detecting lymphatic vessels and evoke testable hypotheses regarding the role of lymphatic vessels in health and disease.

Animals↗

PubMed2004

Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor.

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other gamma-secretase substrates.

Amino Acid Sequence↗

PubMed2004

Molecular analysis of the genetic defect in a large cohort of IP patients and identification of novel NEMO mutations interfering with NF-kappaB activation.

Incontinentia Pigmenti (IP) is an X-linked genodermatosis that is lethal for males and present in females with abnormal skin pigmentation and high variable clinical signs, including retinal detachment, anodontia, alopecia, nail dystrophy and nervous system defects. The NF-kappaB essential modulator (NEMO) gene, responsible for IP, encodes the regulatory subunit of the IkappaB kinase (IKK) complex required for nuclear factor kappaB (NF-kappaB) activation. We analyzed the NEMO gene in 122 IP patients and identified mutations in 83 (36 familiar and 47 sporadic cases). The recurrent NEMO exon 4-10 deletion that is the major cause of the disease was present in 73 females (59.8%). In addition 10 point alterations (8.2% of females) were identified: three frameshift, three nonsense, three missense and one in-frame deletion of a single amino acid. We measured the effects of these NEMO point-mutations on NF-kappaB signaling in nemo(-/-) deficient murine pre-B cells. A mutation in the N-terminal domain, required for IKK assembly, reduced but did not abolish NF-kappaB activation following lipopolysaccharide stimulation. Mutations that disrupt the C-terminal domain, required for the recruitment of upstream factors, showed lower or no NF-kappaB activation. A phenotype score based on clinical features of our IP patients was applied for summarizing disease severity. The score did not correlate with mutation type or domain affected indicating that other factors influence the severity of IP. Such a factor is likely to be X-inactivation. Indeed, 64% of our patients have extremely skewed X-inactivation pattern (>/=80 : 20). Overall IP pathogenesis thus depends on a combination of X-inactivation and protein domain that recruit upstream factors and activate NF-kappaB.

Animals↗

PubMed2004

The trimerization domain of NEMO is composed of the interacting C-terminal CC2 and LZ coiled-coil subdomains.

NEMO (NF-kappaB essential modulator) plays a key role in the canonical NF-kappaB pathway as the scaffold/regulatory component of the IkappaB kinase (IKK) complex. The self-association of NEMO involves the C-terminal halves of the polypeptide chains containing two putative coiled-coil motifs (a CC2 and a LZ leucine zipper), a proline-rich region, and a ZF zinc finger motif. Using purified truncation mutants, we showed that the minimal oligomerization domain of NEMO is the CC2-LZ segment and that both CC2 and LZ subdomains are necessary to restore the LPS-dependent activation of the NF-kappaB pathway in a NEMO-deficient cell line. We confirmed the association of the oligomerization domain in a trimer and investigated the specific role of CC2 and LZ subdomains in the building of the oligomer. Whereas a recombinant CC2-LZ polypeptide self-associated into a trimer with an association constant close to that of the wild-type protein, the isolated CC2 and LZ peptides, respectively, formed trimers and dimers with weaker association constants. Upon mixing, isolated CC2 and LZ peptides associated to form a stable hetero-hexamer as shown by gel filtration and fluorescence anisotropy experiments. We propose a structural model for the organization of the oligomerization domain of activated NEMO in which three C-terminal domains associate into a pseudo-hexamer forming a six-helix bundle. This model is discussed in relation to the mechanism of activation of the IKK complex by upstream activators.

Amino Acid Motifs↗

PubMed2004

Sleep deprivation increases the activation of nuclear factor kappa B in lateral hypothalamic cells.

Sleep deprivation increases sleep propensity in rats and mice as well as the production of several sleep-regulatory substances. Nuclear factor kappa B (NF-kappa B) is a transcription factor implicated in the activation of many of these sleep-promoting substances. A unique population of neurons immunoreactive for the p65 subunit of NF-kappa B was previously localized within the caudal dorsolateral hypothalamus of rats. Therefore, we evaluated the effect of sleep deprivation on NF-kappa Bp65-immunoreactivity (IR) in cells of this region in rats as well as its nuclear translocation in a kappa B-lacZ transgenic mouse line. In rats after 6 h of sleep deprivation beginning at light onset, the number of neurons with NF-kappa Bp65-IR increased significantly in the caudal lateral hypothalamus, specifically the magnocellular lateral hypothalamus adjacent to the subthalamus. Sleep deprivation also significantly increased the number of cells expressing NF-kappa B-dependent beta-galactosidase in the magnocellular lateral hypothalamus, zona incerta dorsal, as well as the adjacent subthalamus in the transgenic mice. These results suggest that NF-kappa B expressing cells within the lateral hypothalamus may be important in the maintenance of the sleep-wake cycle.

Active Transport, Cell Nucleus↗

PubMed2004

Combined deficiency in IkappaBalpha and IkappaBepsilon reveals a critical window of NF-kappaB activity in natural killer cell differentiation.

Nuclear factor kappaB (NF-kappaB) transcription factors are key regulators of immune, inflammatory, and acute-phase responses and are also implicated in the control of cell proliferation and apoptosis. While perturbations in NF-kappaB activity impact strongly on B- and T-cell development, little is known about the role for NF-kappaB in natural killer (NK) cell differentiation. Inhibitors of NF-kappaB (IkappaBs) act to restrain NF-kappaB activation. We analyzed the cell-intrinsic effects of deficiencies in 2 IkappaB members (IkappaBalpha and IkappaBepsilon) on NK cell differentiation. Neither IkappaBalpha nor IkappaBepsilon deficiency had major effects on NK cell generation, while their combined absence led to NF-kappaB hyperactivation, resulting in reduced NK cell numbers, incomplete NK cell maturation, and defective interferon gamma (IFN-gamma) production. Complementary analysis of transgenic mice expressing an NF-kappaB-responsive reporter gene showed increased NF-kappaB activity at the stage of NK cell development corresponding to the partial block observed in IkappaBalpha x IkappaBepsilon-deficient mice. These results define a critical window in NK cell development in which NF-kappaB levels may be tightly controlled.

Animals↗

PubMed2004

T-cell-receptor- and B-cell-receptor-mediated activation of NF-kappaB in lymphocytes.

B and T cells sense antigens through specific receptors, which, when activated, induce signalling cascades leading to the activation of a series of transcription factor families, such as NF-kappaB. These transcription factors control differentiation, cytokine production and proliferation, and they protect against apoptosis. Much progress has been made during the past two years in the understanding of the molecular events leading to NF-kappaB activation, but, although most of the molecules in this signalling cascade have now been identified, the detailed molecular events remain obscure; in particular regarding the molecules that specifically connect the T-cell receptor (TCR)- and B-cell receptor (BCR)-proximal adaptors and kinases to the central core of the NF-kappaB cascade, the IkappaB kinase complex. As these events are likely to be specific for both extremities of the signalling cascade (the TCR or BCR on one end, and NF-kappaB target genes on the other) they will ultimately represent the best targets to specifically manipulate this response in lymphocytes.

Animals↗

PubMed2004

[Human pathologies associated with NF-kappaB defects].

NF-kappaB is the generic name of a family of transcription factors which play a critical role in the immune, inflammatory and anti-apoptotic responses. Homo- or heterodimers between the five members of the family are retained in the cytoplasm by inhibitory molecules of the IkappaB family, which mask their nuclear localization signal. Three of these inhibitory molecules have been described: IkappaBalpha, IkappaBbeta and IkappaBepsilon. Following cellular stimulation, IkappaB proteins become phosphorylated by the IkappaB kinase (IKK) complex, ubiquitinated and finally degraded by the proteasome. NF-kappaB is then released and translocated to the nucleus, where it activates its target genes by binding to specific sites in their regulatory regions. The IKK complex is constituted of at least three subunits: two kinases, IKKalpha and IKKbeta, and one regulatory subunit (NEMO/IKKgamma), and it constitutes an integrator of most if not all signals which activate NF-kappaB. Although the mechanisms leading to the degradation of the IkappaB proteins are relatively well understood, the precise molecular mechanisms which result in the activation of the high-molecular-weight kinase complex remain to be elucidated. The central role of the IKK complex is consistent with its involvement in a series of human pathologies. We describe here four pathologies: two are due to mutations in the gene encoding the NEMO molecule, a third one in the gene encoding the IkappaBalpha inhibitor, while the fourth one is due to mutations in a gene which had been described as a tumor suppressor. This gene encodes a protein which interacts with NEMO and exhibits deubiquitinase activity, therefore strengthening the recent hypothesis of the role of non-degradation-linked ubiquitination in NF-kappaB activation.

Apoptosis↗

PubMed2003

IkappaBalpha/IkappaBepsilon deficiency reveals that a critical NF-kappaB dosage is required for lymphocyte survival.

In most cells, the NF-kappaB transcription factor is sequestered in the cytoplasm by interaction with inhibitory proteins, the IkappaBs. Here, we show that combined IkappaBalpha/IkappaBepsilon deficiency in mice leads to neonatal death, elevated kappaB binding activity, overexpression of NF-kappaB target genes, and disruption of lymphocyte production. In IkappaBalpha/IkappaBepsilon-deficient fetuses, B220+IgM+ B cells and single-positive T cells die by apoptosis. In adults, IkappaBalpha-/-IkappaBepsilon-/- reconstituted chimeras exhibit a nearly complete absence of T and B cells that is not rescued by cotransfer with wild-type bone marrow. These findings demonstrate that IkappaBs tightly control NF-kappaB activity in vivo and that increased NF-kappaB activity intrinsically impairs lymphocyte survival. Because reduction or rise of NF-kappaB activity leads to similar dysfunction, they also reveal that only a narrow window of NF-kappaB activity is tolerated by lymphocytes.

Animals↗

PubMed2003

Forebrain-specific neuronal inhibition of nuclear factor-kappaB activity leads to loss of neuroprotection.

The transcription factor Rel/nuclear factor (NF)-kappaB is known for its fundamental role in regulating immune and inflammatory responses. In the brain, constitutive NF-kappaB activity has been detected exclusively in neurons, and a large diversity of stimuli have been reported to induce NF-kappaB activity. Yet the function of this transcription factor in the nervous system remains unclear, and its role in neuroprotection or neurodegeneration is open to debate. Recently it was suggested that kappaB-driven gene expression in neurons is controlled by Sp1-like factors. To clarify such controversy, we have characterized here a novel mouse model in which the entire NF-kappaB-dependent transcriptional response is abolished in the forebrain. Calcium-calmodulin-dependent kinase II alpha promoter-driven tetracycline transactivator was used for regulated expression of a transdominant negative mutant of inhibitor kappaBalpha (super-repressor) together with a green fluorescent protein tracer. Inhibition of expression of a kappaB-dependent lacZ transgene was shown in triple transgenic mice, which correlated with the loss of kappaB-specific DNA binding. In transgenic organotypic hippocampal slice cultures, expression of the super-repressor led to strong cell death after neurotoxic insults. These data demonstrate for the first time that neuron-restricted ablation of NF-kappaB-driven gene expression increases neurodegeneration. This might lead to the path for new treatments of neurodegenerative diseases.

Animals↗