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Summary Cell cycle progression requires the E3 ubiquitin ligase anaphase-promoting complex (APC/C), which uses the substrate adaptors CDC20 and CDH1 to target proteins for proteasomal degradation. The APC CDH1 substrate cyclin A is critical for the G1/S transition and, paradoxically, accumulates even when APC CDH1 is active. We show that the deubiquitinase USP37 binds CDH1 and removes degradative polyubiquitin from cyclin A. USP37 was induced by E2F transcription factors in G1, peaked at G1/S, and was degraded in late mitosis. Phosphorylation of USP37 by CDK2 stimulated its full activity. USP37 overexpression caused premature cyclin A accumulation in G1 and accelerated S phase entry, whereas USP37 knockdown delayed these events. USP37 was inactive in mitosis because it was no longer phosphorylated by CDK2. Indeed, it switched from an antagonist to a substrate of APC CDH1 and was modified with degradative K11-linked polyubiquitin.

Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger 4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.

Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin. We engineered a K11 linkage-specific antibody and use it to demonstrate that K11 chains are highly upregulated in mitotic human cells precisely when substrates of the ubiquitin ligase anaphase-promoting complex (APC/C) are degraded. These chains increased with proteasomal inhibition, suggesting they act as degradation signals in vivo. Inhibition of the APC/C strongly impeded the formation of K11-linked chains, suggesting that a single ubiquitin ligase is the major source of mitotic K11-linked chains. Our results underscore the importance of K11-linked ubiquitin chains as critical regulators of mitotic protein degradation.

El pdf del artículo es al manuscrito de autor (PubMed).-- et al., The proteasome recognizes its substrates via a diverse set of ubiquitin receptors, including subunits Rpn10/S5a and Rpn13. In addition, shuttling factors, such as Rad23, recruit substrates to the proteasome by delivering ubiquitinated proteins. Despite the increasing understanding of the factors involved in this process, the regulation of substrate delivery remains largely unexplored. Here we report that Rpn10 is monoubiquitinated in vivo and that this modification has profound effects on proteasome function. Monoubiquitination regulates the capacity of Rpn10 to interact with substrates by inhibiting Rpn10's ubiquitin-interacting motif (UIM). We show that Rsp5, a member of NEDD4 ubiquitin-protein ligase family, and Ubp2, a deubiquitinating enzyme, control the levels of Rpn10 monoubiquitination in vivo. Notably, monoubiquitination of Rpn10 is decreased under stress conditions, suggesting a mechanism of control of receptor availability mediated by the Rsp5-Ubp2 system. Our results reveal an unanticipated link between monoubiquitination signal and regulation of proteasome function., This work was performed in the Institute of Molecular Biology of Barcelona (IBMB), supported by Spanish Government (MICINN) grant BFU2006-02928. MS analysis was performed at Harvard Medical School (HMS). We acknowledge National Institutes of Health (NIH) grants GM065592 (D.F.) and GM67945 (S.G).

Summary Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

Polyubiquitin is a diverse signal both in terms of chain length and linkage type. Lysine 48-linked ubiquitin is essential for marking targets for proteasomal degradation, but the significance and relative abundance of different linkages remain ambiguous. Here we dissect the relationship of two proteasome-associated polyubiquitin-binding proteins, Rpn10 and Dsk2, and demonstrate how Rpn10 filters Dsk2 interactions, maintaining proper function of the ubiquitin-proteasome system. Using quantitative mass spectrometry of ubiquitin, we found that in S. cerevisiae under normal growth conditions the majority of conjugated ubiquitin was linked via lysine 48 and lysine 63. In contrast, upon DSK2 induction, conjugates accumulated primarily in the form of lysine 48 linkages correlating with impaired proteolysis and cytotoxicity. By restricting Dsk2 access to the proteasome, extraproteasomal Rpn10 was essential for alleviating the cellular stress associated with Dsk2. This work highlights the importance of polyubiquitin shuttles such as Rpn10 and Dsk2 in controlling the ubiquitin landscape.

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain.

Ubiquitination of the EGF receptor (EGFR) is believed to play a critical role in regulating both its localization and its stability. To elucidate the role of EGFR ubiquitination, tandem mass spectrometry was used to identify six distinct lysine residues within the kinase domain of the EGFR, which can be conjugated to ubiquitin following growth factor stimulation. Substitution of these lysine residues with arginines resulted in a dramatic decrease in overall ubiquitination but preserved normal tyrosine phosphorylation of EGFR. Ubiquitination-deficient EGFR mutants displayed a severe defect in their turnover rates but were internalized at rates comparable to those of wild-type receptors. Finally, quantitative mass spectrometry demonstrated that more than 50% of all EGFR bound ubiquitin was in the form of polyubiquitin chains, primarily linked through Lys63. Taken together, these data provide direct evidence for the role of EGFR ubiquitination in receptor targeting to the lysosome and implicate Lys63-linked polyubiquitin chains in this sorting process.