Regulatory functions of ubiquitin, deubiquitination, ubiquitin binding domains and unconventional ubiquitin chains

In this section projects are summarized which study non-degradative functions of ubiquitin. For example, unconventional ubiquitin chains and ubiquitination are involved in signal transduction, DNA repair, the endocytic pathway and development.

Function of the UBZ-containing ATPase Wrnip1 in DNA replication and damage response

Prof. Dr. Ivan Dikic

Goethe University

Institut für Biochemie II

Postal address:

Theodor-Stern-Kai 7

60590 Frankfurt

t: +49 69 6301 5652

f: +49-69-6301-5577

Links:

Ubiquitin (Ub) and Ubiquitin-like modifiers (Ubls) are among the most diverse moieties covalently attached to proteins to maintain homeostasis under normal conditions and during stress such as DNA damage. We have previously shown that multiple proteins involved in the DNA damage response (DDR) - including the Y-family polymerases kappa and eta, Werner helicase (Wrn)-interacting protein 1 (Wrnip1), FAN-1 nuclease and Rad18 ubiquitin ligase - contain Ub-binding Zn finger (UBZ) domains that regulate their sub-nuclear localization inside focal structures. Here we propose to study the role of Wrnip1 in DNA replication, telomere maintenance and genome stability. We have found that Wrnip1 foci co-localize with heterochromatic regions including centromeres and telomeres and that Wrnip1 interacts with the telomeric protein TRF2 but not TRF1. We have also found that acute Wrnip1 depletion by RNA interference causes chromosomal instability involving both centromeres and telomeres. To dissect the functional role of Wrnip1, we will combine biochemical and cellular methods to study: a) the composition of Wrnip1-associated complexes; b) the dynamics of Wrnip1 foci in living cells under replication stress; c) the relevance of Wrnip1 for telomere maintenance, DNA replication and genome stability; d) the effects of the deletion of WRNIP1 and associated genes such as RAD18 and WRN in defined tissues and organs in genetically-engineered mice models. We expect that these studies will shed light onto the physiological role and mechanism of action of Wrnip1 during DDR.

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Elucidating the function of the deubiquitinating enzyme AMSH3 in cellular trafficking in Arabidopsis thaliana

Dr. Erika Isono

Technische Universität München

Center for Life and Food Sciences, Plant Systems Biology

Postal address:

Emil-Ramann-Str. 4

85354 Freising

t: +49 8161 71 2875

f: +49 8161-71-2886

Links:

Ubiquitination as well as deubiquitination regulate a broad range of cellular processes. AMSH proteins are evolutionarily conserved deubiquitinating enzymes. Human AMSH proteins interact with multiple proteins involved in cellular trafficking and are probably controlling the abundance and stability of plasma membrane localized receptors. Our analysis of the Arabidopsis AtAMSH3 has shown that it is essential for vacuole biogenesis, sorting of soluble cargo to the vacuole and endocytosis. Although AMSH proteins seem to play a critical role in intracellular transport in all organisms investigated so far, only a few interacting proteins have been identified. Combining immunoprecipitation and mass spectrometry analysis, we identified proteins that are either potential interactors or potential substrates of AtAMSH3. The goal of this proposal is (1) to elucidate the role of AtAMSH3 in cellular trafficking in the context of its interacting proteins, (2) to define the region in AtAMSH3 responsible for its K48 specificity, and (3) to investigate the chain type specific substrates of AtAMSH3. In carrying out the proposed project, we will contribute to the goals of the SPP (1) by identifying novel UbF-modified proteins, (2) by defining the regulatory functions of these modifications, and (3) by identifying the cellular machineries for UbF deconjugation.

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The role of Nedd4 family E3 ubiquitin ligases in the regulation of neuronal cell surface receptors

Prof. Dr. Nils Brose

Max-Planck-Institut für experimentelle Medizin (MPIEM)

Abteilung Molekulare Neurobiologie

Postal address:

Hermann-Rein-Straße 3D

37075 Göttingen

t: +49 551 3899725

f: +49 551-3899715

Links:

Dr. Hiroshi Kawabe

Max-Planck-Institut für experimentelle Medizin (MPIEM)

Abteilung Molekulare Neurobiologie

Postal address:

Hermann-Rein-Straße 3D

37075 Göttingen

t: +49 551 3899720

f: +49 551-3899715

Links:

Signal transduction from trans-membrane cell surface receptors to nuclear transcription factors is regulated at multiple levels by protein ubiquitination, which can lead to downregulation of receptor surface expression, degradation of components of second messenger pathways, modification of the nuclear transport of transcription factors, or alterations in the epigenetic control of transcription by histone modification. In nonneuronal cells, monoubiquitination or K63-linked polyubiquitination are critical regulatory mechanisms that control the endocytosis and consequent downregulation of trans-membrane cell surface receptors, and thereby affect multiple cell biological processes - from cell differentiation to apoptosis. However, little is known about the role of monoubiquitination and K63-linked polyubiquitination of receptors on the nerve cell surface, although neuronal development and function are tightly controlled by extracellular signals. In the project proposed here, we will study the role of two HECT-type E3 ubiquitin ligases of the Nedd4 family, Nedd4-1 and Nedd4-2, in nerve cells - with a focus on their function in controlling cell surface receptors. Nedd4-1 and Nedd4-2 mediate monoubiquitination or K63-mediated polyubiquitination of substrates, are among the most abundant E3 ligases in neurons, and are particularly strongly expressed during early nerve cell development and differentiation, i.e. at developmental stages that are tightly controlled by extracellular signaling molecules such as growth factors. Moreover, Nedd4-1 and Nedd4-2 have been implicated in the control of multiple receptor proteins, although the corresponding evidence has often remained fragmentary. In preparation of our study, we generated conditional Nedd4-1 and Nedd4-2 KO mouse lines. We will employ these mouse models along with biochemical and cell biological approaches to identify transmembrane substrate proteins of Nedd4-1 and Nedd4-2 in neurons and to examine the consequences of such Nedd4-1/Nedd4-2-mediated ubiquitination processes for neuron and brain development and function.

 

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Functions of regulatory ubiquitin and ubiquitin-like modifications for NF-kappaB signaling in T cells

PD Dr. Daniel Krappmann

Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH)

Research Unit Cellular Signal Integration, Institute of Toxicology

Postal address:

Ingolstädter Landstraße 1

85764 Neuherberg

t: +49 89 3187 3461

f: +49 89 3187 3449

Links:

Assembly and disassembly of ubiquitin chains to cellular target proteins is involved in the regulation of many steps in T cell activation. We could previously show that K63-linked ubiquitination of Malt1 promotes the recruitment of IkB kinases (IKKs) to the high molecular weight Carma1-Bcl10-Malt1 (CBM) complex, which is a key regulator of T cells activation. We have discovered that TRAF6-catalyzed ubiquitination and A20-mediated deubiquitination of Malt1 balances IKK/NF-kB signaling in T cells. In addition, association of Malt1 with the COP9 signalosome – a cellular deneddylating complex – is required for optimal T cell activation and Malt1 is also prone to NEDD8 modifications. Within this project, we will investigate the mechanisms and cellular consequences of Malt1 neddylation. Specifically, we will address the question whether NEDD8 and ubiquitin modifications in Malt1 serve opposing functions that together help to coordinate T cell responses. Further, we will perform a siRNA screen to identify novel deubiquitinating enzymes (DUBs) that are involved in T cell activation. The aim is to obtain a comprehensive map of DUBs regulating T cell activation and to characterize their point of action. In addition, we will elucidate positive or negative functions of specific candidate DUBs for T cell biology.

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VCP/p97-governed sorting of mono-ubiquitinated proteins in the endocytic pathway

Prof. Dr. Hemmo Meyer

Universität Duisburg-Essen

Zentrum für Medizinische Biotechnologie (ZMB), Fakultät für Biologie,

Postal address:

Universitätsstraße, Gebäude S03 V04 F44

45177 Essen

t: +49 201 183 4217

f: +49 201-183-4257

Links:

Each cell in the human body contains a specific set of proteins on the surface that mediate exchange and communication with the environment. Controlled turnover of indivi­dual components by endocytosis is particularly important for signaling proteins that regulate cell proliferation and are therefore critical in cancer. Ubiquitin modification is known to trigger endo­cytosis and subsequent sorting to endolysosomes for degradation. The ubiquitin-selective segregase Valosin-Containing Protein (VCP)/p97 has emerged as a central regulator of ubiquitin-dependent processes. Along with its cofactor Ufd1-Npl4 it is best studied in proteasomal degradation of poly-ubiquitinated substrates. However, it also plays important roles in signaling pathways. In this project, we would like to explore whether and how VCP/p97 is involved in ubiquitin-dependent sorting of mono-ubiquitinated cargo in the endocytic pathway. Our work will hopefully shed light on a novel layer of ubiquitin-dependent signaling at the plasma membrane that may govern cellular homoeostasis and tumorigenesis, and will help to clarify the cellular relevance of VCP/p97 in health and disease.

 

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Mechanistic insight into A/B toxin cell entry via ubiquitin-mediated endocytosis

Prof. Dr. Manfred J. Schmitt

Universität des Saarlandes, Gebäude A 1.5

Fachrichtung Biowissenschaften - FR 8.3, Molekular- & Zellbiologie

Postal address:

Postfach 151150

66041 Saarbrücken

t: +49 681 3024730

f: +49 681-3024710

Links:

Microbial, plant and viral A/B toxins such as cholera and Shiga toxin, ricin and the viral A/B toxin K28 enter eukaryotic target cells by receptor-mediated endocytosis and retrograde transport through the secretory pathway (via early endosomes, Golgi and ER) before being retrotranslocated into the cytosol to finally kill their corresponding host. While most of these processes and cellular components involved are still poorly understood, the present project intends to address and dissect the initial step of intoxification which is A/B toxin uptake via ubiquitin-mediated receptor endocytosis and subsequent sorting into the endocytic pathway. Since some A/B toxins can use different strategies to enter and penetrate eukaryotic cells, detailed analysis of ubiquitin-mediated endocytosis of a viral A/B toxin cargo (K28) in yeast will possibly reveal non-canonical endocytic entry routes and extend our current knowledge of endocytosis and intracellular trafficking of medically relevant A/B toxins.

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Proteomic characterization of ubiquitin-dependent processes in the secretory pathway

Dr. Ernst Jarosch

Max-Delbrück-Center for Molecular Medicine

Postal address:

Robert-Rössle-Straße 10

13122 Berlin

t: +49 30 9406 3305

f: +49 30 9406 3363

Prof. Dr. Thomas Sommer

Max-Delbrück-Center for Molecular Medicine

Postal address:

Robert-Rössle-Straße 10

13122 Berlin

t: +49 30 9406 3753

f: +49 30 9406 3363

Links:

This research proposal aims at characterizing non-proteolytic functions of the ubiquitin system that are involved in maintaining homeostasis in the secretory pathway. To this end we plan to purify membrane-associated Cdc48 containing protein complexes and identify their functions by genetic and biochemical methods. The AAA-ATPase Cdc48, in mammals termed p97 or VCP, is a key player in many ubiquitin-dependent processes like homeotypic membrane fusion, cell cycle control, proteasome-mediated protein degradation and DNA repair. To fulfill these activities, Cdc48/p97 teams up with a large set of diverse ancillary proteins in a temporally and spatially controlled manner. Notably, the vast majority of the presently known Cdc48/p97 co-factors contain ubiquitin binding domains or are elsewise linked to the ubiquitin system. However, in most cases we lack in depth knowledge on their molecular function. In the course of this work we plan to isolate novel Cdc48 interacting proteins, determine their function and establish a network of Cdc48 co-factors that integrates this versatile enzyme into particular ubiquitin-dependent processes in the secretory pathway.

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