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  • br Experimental Procedures br Author Contributions M B


    Experimental Procedures
    Author Contributions M.B., with input and help from T.T.H., conceived the project, designed and performed the experiments, analyzed the data, and wrote the manuscript. B.B.B. and F.D.M. synthesized the wild-type and (nonhydrolyzable, NH) Ub tetramers and contributed Figure 1G; K.O. and E.L.D. performed and analyzed the experiments with the TRF2F/F Valrocemide (Figures 4C–4E, S4B, and S4C); M.J.J. generated the Flp-in stable cells (used in Figure 5); S.B.C. cultured Flp-in cells and carried out the TNF-α experiment (Figure S4A) and aided in mass-spectrometry preparation. Protein identification by mass spectrometry was carried out by J.R.C. and B.M.U.
    Acknowledgments The authors wish to thank Kristin Burns-Huang for critically reading the manuscript, members of the Lima, Sfeir, Smith, Reinberg, and Huang labs for reagents, resources, and discussions, and Shaun K. Olsen for the S. pombe E1 and for assistance with structure-based modeling. We express our gratitude to Chris Lima for hosting M.B. during the Hurricane Sandy lab relocation at NYU. M.B. is a recipient of an NRSA postdoctoral fellowship (1F32GM100598), and research in the Huang lab is supported by grants from the NIH (GM084244) and from ACS (RSG-12-158-DMC). E.L.D. is supported by the Pew Scholars Award. B.B.B. and F.D.M. Valrocemide are employees of Boston Biochem.
    Introduction Besides ubiquitin, several ubiquitin-like proteins (Ubl) are known that have an important function in immunity. Ubls are conjugated and deconjugated through mechanisms similar to the ubiquitylation cascade, sometimes sharing components with the ubiquitin pathway. The Ubls SUMO and Nedd8 are also involved in the NF-κB pathway. Nedd8 modification of the E3 SCF ligase complex is required for the recruitment of E2 and ubiquitylation of IκBα. On the other hand, SUMO modification of IκBα counteracts ubiquitylation at the same lysine residue, adding another regulatory step in this complex pathway [13]. ISG15, one of the first identified Ubls, is upregulated by type I interferon stimulation during infection [14, 15, 16]. It consists of two Ub-like domains connected by a small linker and plays an important role in the defense against viral and bacterial infections, possibly by modification of viral and host proteins. However, the exact function of ISGylation remains largely unclear [17]. FAT10 is another Ubl implicated in immune defense and also consists of two Ub-like modules. It is expressed in mature dendritic cells and B cells, and expression can be induced by IFN-γ and IFN-α. Mono-FATylated proteins are targeted to the proteasome for degradation [18]. Thus far, no deconjugating enzyme has been identified. Interestingly, pathogens have evolved highly effective mechanisms to modulate host immune signaling pathways through Ub/Ubl remodeling to secure replication, infection and pathogenesis. For example, the Herpes Simplex Virus-1 (HSV-1) tegument protein UL36 possesses deubiquitylating activity [19], which is conserved in homologs found in murine cytomegalovirus and Epstein Barr Virus [20]. The SARS coronavirus PLpro processing protease acts on a broad range of ubiquitylated and ISG15ylated host proteins and is required for viral replication [21, 22]. Crimean-Congo hemorrhagic fever (CCHF) virus harbors an OTU-like DUB which acts as both a deubiquitylase and a deISGylase [23, 24, 25]. In addition, Yersinia virulence factor YopJ has deubiquitylating activity that results in deubiquitylation of IκBα and hence inhibition of NF-κB signaling [26, 27]. Moreover, Chlamydia trachomatis expresses two DUB-like proteases that possess deubiquitylating and deneddylating activity [28].
    Synthesis of ubiquitin and Ubl reagents The development of the first ubiquitin-based assay reagents relied on transpeptidation reactions. Often trypsin is used in these reactions, which removes the last two glycine residues of ubiquitin under native conditions [29]. During the cleavage reaction, an intermediate ester is formed with the active-site serine residue. This intermediate ester can undergo a transpeptidation reaction resulting in a new peptide bond with an amine nucleophile of choice, if a large amount of nucleophile is present. The fluorogenic substrate Ub-AMC has initially been synthesized using this method [30]. Another method uses intein chemistry for a more convenient preparation of reactive ubiquitin thioester intermediates [31]. These intermediates can be chemically converted to introduce a reactive group in the synthesis of ubiquitin-based active site-directed probes. Intein chemistry is generally preferred over transpeptidation methods as it is more generally applicable [32, 33, 34].