Patricia Chastagner

Monoubiquitination and endocytosis direct γ-secretase cleavage of activated Notch receptor

Activation of mammalian Notch receptor by its ligands induces TNFα-converting enzyme–dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent γ-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for γ-secretase processing of a constitutively active Notch derivative, ΔE, which mimics the TNFα-converting enzyme–processing product. PS interacts with this modified form of ΔE, ΔEu. 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 γ-secretase substrates.

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.

AIP4/Itch Regulates Notch Receptor Degradation in the Absence of Ligand

Background The regulation of Notch signaling heavily relies on ubiquitination events. Drosophila Su(dx), a member of the HECT family of ubiquitin-ligases, has been described as a negative regulator of Notch signaling, acting on the post-endocytic sorting of Notch. The mammalian ortholog of Su(dx), Itch/AIP4, has been shown to have multiple substrates, including Notch, but the precise events regulated by Itch/AIP4 in the Notch pathway have not been identified yet. Methodology/Principal Findings Using Itch-/- fibroblasts expressing the Notch1 receptor, we show that Itch is not necessary for Notch activation, but rather for controlling the degradation of Notch in the absence of ligand. Itch is indeed required after the early steps of Notch endocytosis to target it to the lysosomes where it is degraded. Furthermore Itch/AIP4 catalyzes Notch polyubiquitination through unusual K29-linked chains. We also demonstrate that although Notch is associated with Itch/AIP4 in cells, their interaction is not detectable in vitro and thus requires either a post-translational modification, or a bridging factor that remains to be identified. Conclusions/Significance Taken together our results identify a specific step of Notch regulation in the absence of any activation and underline differences between mammalian and Drosophila Notch pathways.

The Translation Initiation Factor 3f (eIF3f) Exhibits a Deubiquitinase Activity Regulating Notch Activation

The translation initiation factor complex eIF3f has an intrinsic deubiquitinase activity and regulates the Notch signaling pathway.

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

Background: Notch activity depends notably on the quantity of Notch receptor at the cell surface. Results: The ubiquitin-specific protease USP12 directly targets Notch and directs it to lysosomal degradation. Conclusion: USP12 is a novel, conserved negative regulator of Notch signaling. Significance: Notch signaling regulation by various deubiquitinating enzymes acting at different steps is of crucial importance. 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.

α-arrestin 1 (ARRDC1) and β-arrestins cooperate to mediate Notch degradation in mammals

Summary 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 &bgr;-arrestins are necessary for Itch–Notch interaction and for Itch-driven ubiquitylation and degradation of Notch. Interestingly, &bgr;-arrestins do not directly bind Itch but heterodimerize with a member of another subfamily of arrestins called ARRDC1 or &agr;-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 &bgr;-arrestin−/− or Itch−/− cells. Our data show for the first time that ARRDC1 and &bgr;-arrestins heterodimerize and cooperate in the same complex to promote non-activated Notch receptor degradation, thus acting as negative regulators of Notch signaling.

Human IL-Rβ chains form IL-2 binding homodimers

Two types of functional interleukin-2 receptor (IL-2Ralpha/IL-2Rbeta/gammac and IL-2Rbeta/gammac) have already been characterized in humans. Here we describe a new form consisting of IL-2Rbeta/beta homodimers that assemble spontaneously in the absence of gammac. Co-transfection of COS-7 cells with constructs expressing IL-2Rbeta chains tagged with either HA or MYC sequences results in the formation of IL-2Rbeta:HA/IL-2Rbeta:MYC complexes detectable by coimmunoprecipitation. The formation of these IL-2Rbeta:HA/IL-2Rbeta:MYC dimers is also observed in the absence of IL-2. Moreover, in COS cells expressing chimeras of IL-2Rbeta fused to fluorescence reporters such as IL-2Rbeta:ECFP and IL-2Rbeta:EYFP, we also observed specific FRET at the surface of living cells, as expected for dimer formation. Transiently transfected COS-7 cells expressing IL-2Rbeta bind 125I-labeled IL-2 (homodimers, Kd = 1nM) as cells expressing both IL-2Rbeta and gammac chains (heterodimers, Kd = 1 nM). IL-2Rbeta/IL-2Rbeta could represent either a decoy receptor or a new form of IL-2R involved in signaling when gammac expression is low.

Clonotypic dominance and variable gene elements of pathogenic anti-DNA autoantibodies from a single patient with lupus

This study explores the usage and diversity of the variable gene elements expressed by human lupus antibodies to DNA bearing the 0–81 idiotype, a marker of pathogenic anti‐DNA autoantibodies. Rather than studying DNA‐specific clonotypes from different patients, a panel of idiotype positive anti‐DNA autoantibody‐secreting clones from a single individual were analysed. By cloning and nucleotide‐sequeneing the heavy‐chain variable gene segments, evidence was found for dominance of clonotypic patterns. Also noted was a high rate of diversification among the variable (VH), diversity (Dh) and junctional (JH) gene segments utilized, with a pattern of mutations indicative of antigenic selection. These features suggest that the clones secreting the lupus pathogenic autoantibodies have been selected over multiple generations through an affinity‐maturation process that is reminiscent of antigen‐driven immune responses.

Cloning of a gene encoding a lupus-associated human autoantibody VK region using the polymerase chain reaction and degenerate primers.

The variable light-chain-encoding gene of a human autoantibody secreted by a B-cell hybridoma derived from a patient with systemic lupus erythematosus was amplified using the polymerase chain reaction and degenerate primers. After cloning, the nucleotide sequence of the EcoRI-HindIII region was determined. It is highly homologous to a previously described gene expressed by a human lymphoid cell line.

Induction of mouse p55 interleukin-2 receptor gene expression by IL-2 and IL-4 and characterization of its transcription initiation sites

Two murine T cell lines (C30.1 and Line 1) were used to study the expression of the p55 interleukin-2 receptor gene. C30.1 is an IL-2-dependent T cell line that can be stimulated for a short period of time by IL-4. Line 1 cells are propagated in IL-4 but they also proliferate in response to IL-2. In both cell lines stimulation by IL-2 leads to a strong induction of p55 IL-2 receptor mRNA while stimulation by IL-4 leads only to a very moderate increase in expression of this mRNA. The induction of p55 IL-2 receptor mRNA by IL-4 is comparable to that of beta-actin mRNA. These data confirm that IL-2 upregulates p55 IL-2 receptor gene expression while IL-4, which also activates T cells, does not lead to specific induction of this gene. We have also determined the transcription initiation sites utilized by the p55 IL-2 receptor gene in C30.1 and Line 1 cells. Seven sites were identified, one of which predominates. Resting cells, or cells stimulated with IL-2 or IL-4, display the same pattern of transcription site utilization.