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  • thymidylate synthase Haj Yahya et al applied native


    Haj-Yahya et al. [52] applied native chemical ligation for the construction of diubiquitin probes. They attached a cysteine residue to the lysine side chain of the proximal ubiquitin and ligated the distal module using the C-terminal thioester of ubiquitin. Elimination of the sulfur thymidylate synthase resulted in the formation of a dehydroalanine warhead (Figure 4B-5). Lys48, Lys63-linked and linear diubiquitin probes were constructed and used for labeling of six purified DUBs. Notably, this probe is cleaved by DUBs, which can be expected on the basis of the positioning of the vinyl amide moiety. Mulder et al. recently developed a dedicated ligation handle designed to construct diubiquitin probes via native chemical ligation. Subsequent thiol elimination yields the warhead in the final step of synthesis (Figure 4B-6) []. These diubiquitin probes have the appropriate linker length and warhead correctly positioned with respect to the native diubiquitin isopeptide linkage. All seven diubiquitin isopeptide linkages were created and shown to label USP7. The Lys11 and Lys48 probes were fluorescently tagged and used to label DUBs in EL-4 cell lysate, which revealed distinct labeling patterns between these two linkages and monoubiquitin probe.
    Directions in ubiquitin probe research Ubiquitin-based probes have proven to be valuable tools for structural studies that aim to understand substrate recognition by DUBs on a molecular level using X-ray crystallography. This was predominantly achieved using pan-DUB probes but it will now become key thymidylate synthase to design probes that target one DUB or a family of DUBs selectively. Recent developments in this direction involve tuning of the affinity for ubiquitin by creating ubiquitin mutants that act as rather selective DUB inhibitors [54••, 55••]. The ubiquitin linkage specificity of DUBs remains an intriguing field of research and many studies have focused on the involvement of the S1 and S1′ ubiquitin binding pockets. However, for certain DUBs it has been shown that there is a significant contribution of the S1 and S2 pockets []. A specific probe that targets these sites remains to be reported (Figure 4A bottom panel).
    Conflict of interests
    References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:
    Acknowledgements This work was supported by grants from the Netherlands Foundation for Scientific Research and the European Research Council to H.O.
    Introduction The mammalian genome consists of approximately 3000 megabases of DNA packaged into chromatin within the cell nucleus. The basic subunits of chromatin are nucleosomes, which comprise the DNA double helix wrapped around octamers of histone proteins including H2A, H2B, H3, and H4, at 147 base pair intervals (Kornberg and Lorch, 1999, Luger et al., 2012). Nucleosomes are assembled into higher-order structures. Chromatin condensation state, defined as the higher-order compact organization of nucleosomes, can be highly dynamic and switch between closed heterochromatin and open euchromatin (Woodcock and Ghosh, 2010). This allows for tight regulation of the key DNA-centric cellular processes, including transcription, DNA replication, and genomic integrity maintenance. Beyond the genetic information encoded by the DNA sequence, there exists a secondary layer of epigenetic information defined by covalent modifications of DNA and histone proteins (Bernstein et al., 2007). Diverse post-translational modifications of histones have been discovered, including methylation, phosphorylation, acetylation, ubiquitination, and SUMOylation. These modifications primarily target the so-called tails of histone proteins, found at the N-terminus of all four histones and the C-terminus of histone H2A. The tails are historically defined by their susceptibility to proteases and extend from the core fold of the nucleosome, making them accessible for protein interactions and post-translational modifications. The modifications can have a direct effect on the structural stability of nucleosomes and chromatin compaction, and also modulate the specificities of various protein-DNA interactions. The patterns of histone modifications that regulate diverse cellular processes and interact in both cooperative and antagonistic manner are known as the “histone code” (Kouzarides, 2007, Strahl and Allis, 2000). The enzymes and other proteins that modulate histone modifications are often dysregulated in diseases including cancer, which demonstrates their essential role in supporting normal cellular functions and highlights them as potential targets for development of therapies (Baylin and Jones, 2011).