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Insights into Notch3 activation and inhibition mediated by antibodies directed against its negative regulatory region.

ABSTRACT: Notch receptors are single-pass transmembrane proteins that regulate development and tissue homeostasis in all metazoan organisms. Prior to ligand-induced signaling, Notch receptors adopt a proteolytic resistant conformation maintained by a critical interdomain interface within a negative regulatory region (NRR), which sits immediately external to the plasma membrane. Signaling is initiated when ligand binding induces exposure of the proteolytic cleavage site, termed S2, within the NRR. Here, we use hydrogen exchange in conjunction with mass spectrometry to study the dynamics of the human Notch3 NRR in four distinct biochemical states: in its unmodified quiescent form, in a proteolytically "on" state induced by ethylenediaminetetraacetic acid, and in complex with either agonist or inhibitory antibodies. Induction of the on state by either ethylenediaminetetraacetic acid or the agonist monoclonal antibody leads to accelerated deuteration in the region of the S2 cleavage site, reflecting an increase in S2 dynamics. In contrast, complexation of the Notch3 NRR with an inhibitory antibody retards deuteration not only across its discontinuous binding epitope but also around the S2 site, stabilizing the NRR in its "off" state. Together with previous work investigating the dynamics of the Notch1 NRR, these studies show that key features of autoinhibition and activation are shared among different Notch receptors and provide additional insights into mechanisms of Notch activation and inhibition by modulatory antibodies.

SUBMITTER: Tiyanont K

PROVIDER: S-EPMC3751422 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Insights into Notch3 activation and inhibition mediated by antibodies directed against its negative regulatory region.

Tiyanont Kittichoat K Wales Thomas E TE Siebel Christian W CW Engen John R JR Blacklow Stephen C SC

Journal of molecular biology 20130606 17

Notch receptors are single-pass transmembrane proteins that regulate development and tissue homeostasis in all metazoan organisms. Prior to ligand-induced signaling, Notch receptors adopt a proteolytic resistant conformation maintained by a critical interdomain interface within a negative regulatory region (NRR), which sits immediately external to the plasma membrane. Signaling is initiated when ligand binding induces exposure of the proteolytic cleavage site, termed S2, within the NRR. Here, we ...[more]

PMID: 23747483

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Project description:The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer.

Dataset Information

Insights into Notch3 activation and inhibition mediated by antibodies directed against its negative regulatory region.

Publications

Insights into Notch3 activation and inhibition mediated by antibodies directed against its negative regulatory region.

Similar Datasets

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