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Hsia H.-E.,Max Planck Institute for Experimental Medicine | Kumar R.,Max Planck Institute for Experimental Medicine | Luca R.,Catholic University of Leuven | Luca R.,Vlaams Instituut Voor Biotechnologie Center for the Biology of Disease | And 24 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2014

Protein ubiquitination is a core regulatory determinant of neural development. Previous studies have indicated that the Nedd4-family E3 ubiquitin ligases Nedd4-1 and Nedd4-2 may ubiquitinate phosphatase and tensin homolog (PTEN) and thereby regulate axonal growth in neurons. Using conditional knockout mice, we show here that Nedd4-1 and Nedd4-2 are indeed required for axonal growth in murine central nervous system neurons. However, in contrast to previously published data, we demonstrate that PTEN is not a substrate of Nedd4-1 andNedd4-2, and that aberrant PTENubiquitination is not involved in the impairedaxongrowth upondeletion ofNedd4-1 and Nedd4-2. Rather, PTEN limits Nedd4-1 protein levels by modulating the activity of mTORC1, a protein complex that controls protein synthesis and cell growth. Our data demonstrate that Nedd4-family E3 ligases promote axonal growth and branching in the developing mammalian brain, where PTEN is not a relevant substrate. Instead, PTEN controls neurite growth by regulating Nedd4-1 expression.

Savas J.N.,Scripps Research Institute | De Wit J.,Vlaams Instituut Voor Biotechnologie Center for the Biology of Disease | De Wit J.,Catholic University of Leuven | De Wit J.,University of California at San Diego | And 5 more authors.
Nature Protocols | Year: 2014

Ligand-receptor interactions represent essential biological triggers that regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol that couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared with previous approaches, our analysis increases sensitivity, shortens analysis duration and boosts comprehensiveness. In this protocol, receptor extracellular domains are fused with the Fc region of IgG to generate fusion proteins that are purified from transfected HEK293T cells. These 'ecto-Fcs' are coupled to protein A beads and serve as baits for binding assays with prey proteins extracted from rodent brain. After capture, the affinity-purified proteins are digested into peptides and comprehensively analyzed by LCLC-MS/MS with ion-trap mass spectrometers. In 4 working days, this protocol can generate shortlists of candidate ligand-receptor protein-protein interactions. Our 'ecto-Fc MS' approach outperforms antibody-based approaches and provides a reproducible and robust framework for identifying extracellular ligand-receptor interactions. © 2014 Nature America, Inc. All rights reserved.

Thathiah A.,Vlaams Instituut voor Biotechnologie Center for the Biology of Disease
Nature medicine | Year: 2013

β-arrestins are associated with numerous aspects of G protein-coupled receptor (GPCR) signaling and regulation and accordingly influence diverse physiological and pathophysiological processes. Here we report that β-arrestin 2 expression is elevated in two independent cohorts of individuals with Alzheimer's disease. Overexpression of β-arrestin 2 leads to an increase in amyloid-β (Aβ) peptide generation, whereas genetic silencing of Arrb2 (encoding β-arrestin 2) reduces generation of Aβ in cell cultures and in Arrb2(-/-) mice. Moreover, in a transgenic mouse model of Alzheimer's disease, genetic deletion of Arrb2 leads to a reduction in the production of Aβ(40) and Aβ(42). Two GPCRs implicated previously in Alzheimer's disease (GPR3 and the β(2)-adrenergic receptor) mediate their effects on Aβ generation through interaction with β-arrestin 2. β-arrestin 2 physically associates with the Aph-1a subunit of the γ-secretase complex and redistributes the complex toward detergent-resistant membranes, increasing the catalytic activity of the complex. Collectively, these studies identify β-arrestin 2 as a new therapeutic target for reducing amyloid pathology and GPCR dysfunction in Alzheimer's disease.

Benilova I.,Vlaams Instituut voor Biotechnologie Center for the Biology of Disease | Benilova I.,Catholic University of Leuven | De Strooper B.,Vlaams Instituut voor Biotechnologie Center for the Biology of Disease | De Strooper B.,Catholic University of Leuven
Science | Year: 2013

A neuroimmune receptor has high affinity for Alzheimer's β-amyloid aggregates and mediates a synaptotoxic cascade in neurons.

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