Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology.
ABSTRACT Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.
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ABSTRACT: The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.The EMBO Journal 12/2014; 34(3). DOI:10.15252/embj.201489847 · 10.75 Impact Factor
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ABSTRACT: Global cardiac myofilament proteins phosphorylation levels, and their site-specific stoichiometry, are physiologically and clinically relevant for heart function. Unlike myofilament phosphorylation, other post-translational modifications (PTMs) such as O-GlcNAcylation, are just beginning to gain attention due to their potential physiological and clinical implications. This review will focus on what is currently known about cardiac Troponin I (cTnI) phosphorylation, and on the potential physiological and clinical impact of targeted proteomics including new findings on cTnI sites and stoichiometry. We will then discuss the increasing recognition of other myofilament PTMs functional relevance and the potential of targeted mass spectrometry approaches, particularly multiple reaction monitoring (MRM), for accelerating their systematic characterization. In addition, we will broadly discuss the development and application of MRM to quantitatively assess site-specific PTMs. Finally, we will give an overview of expert's consensus on MRM methods design/validation and best practices to develop MRM assays intended to reach clinical application. The unique ability of MRM and similar methods to identify and quantify cardiac myofilament PTMs is likely to become central in answering important biological questions in the field of cardiac integrative physiology.This article is protected by copyright. All rights reservedPROTEOMICS - CLINICAL APPLICATIONS 08/2014; 8(7-8). DOI:10.1002/prca.201400034 · 2.68 Impact Factor
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ABSTRACT: Posttranslational modification of cell-cycle regulators with ubiquitin chains is essential for eukaryotic cell division. Such chains can be connected through seven lysine residues or the amino terminus of ubiquitin, thereby allowing the assembly of eight homogenous and multiple mixed or branched conjugates. Although functions of homogenous chain types have been described, physiological roles of branched structures are unknown. Here, we report that the anaphase-promoting complex (APC/C) efficiently synthesizes branched conjugates that contain multiple blocks of K11-linked chains. Compared to homogenous chains, the branched conjugates assembled by the APC/C strongly enhance substrate recognition by the proteasome, thereby driving degradation of cell-cycle regulators during early mitosis. Our work, therefore, identifies an enzyme and substrates for modification with branched ubiquitin chains and points to an important role of these conjugates in providing an improved signal for proteasomal degradation.Cell 05/2014; 157(4):910-21. DOI:10.1016/j.cell.2014.03.037 · 33.12 Impact Factor