Chemistry and biology of the ubiquitin signal

Ubiquitination is one of the most utilized posttranslational modifications in eukaryotes and is involved in a wide range of cellular processes, but is mostly known as a signal for proteasomal degradation. Recently, it has become clear that the ubiquitin signal is far more complex and is dictated by the ubiquitin component and the substrate. The remarkable diversity of the ubiquitin signaling process has triggered an incredible amount of effort to investigate the role of ubiquitination on biological processes. However, despite more than three decades of studies, several important questions remain unanswered. A major hurdle is the inability to obtain homogeneous ubiquitin bioconjugates in sufficient amounts from cells or by application of the enzymatic machinery. Recent breakthroughs in chemical and semisynthetic strategies, however, offer solutions to these challenges. In this Review, we survey the fundamental biological aspects of the ubiquitin signal and present the emerging non-enzymatic approaches for overcoming these obstacles.     

MS‐based strategies for identification of protein SUMOylation modification

Protein SUMOylation modification conjugated with small ubiquitin‐like modifiers (SUMOs) is one kind of PTMs, which exerts comprehensive roles in cellular functions, including gene expression regulation, DNA repair, intracellular transport, stress responses, and tumorigenesis. With the development of the peptide enrichment approaches and MS technology, more than 6000 SUMOylated proteins and about 40 000 SUMO acceptor sites have been identified. In this review, we summarize several popular approaches that have been developed for the identification of SUMOylated proteins in human cells, and further compare their technical advantages and disadvantages. And we also introduce identification approaches of target proteins which are co‐modified by both SUMOylation and ubiquitylation. We highlight the emerging trends in the SUMOylation field as well. Especially, the advent of the clustered regularly interspaced short palindromic repeats/ Cas9 technique will facilitate the development of MS for SUMOylation identification.     

Hydrogen bonds in human ubiquitin reflected in temperature coefficients of amide protons

Analysis of amide proton temperature coefficients (deltasigma(HN)/DeltaT) in human ubiquitin shows their usefulness in indicating hydrogen bonds. The availability of a very accurate solution structure of ubiquitin enables the precise determination of hydrogen bonds and increases the reliability of the analysis of chemical shift temperature gradients. Values of deltasigma(HN)/DeltaT more positive than -4.6 ppb/K are very good indicators of hydrogen bonds. Additionally, a weak temperature dependence of non-hydrogen-bonded amides was observed for amide protons that are significantly shifted upfield. We observed that temperature gradients of amide protons involved in short hydrogen bonds are related to donor-acceptor distances.     

An E1‐Catalyzed Chemoenzymatic Strategy to Isopeptide‐N‐Ethylated Deubiquitylase‐Resistant Ubiquitin Probes

Triazole‐based deubiquitylase (DUB)‐resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain‐specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB‐resistant Ub probes is reported based on isopeptide‐N‐ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one‐pot, ubiquitin‐activating enzyme (E1)‐catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi‐milligram scale. Proteomic studies using label‐free quantitative (LFQ) MS indicated that the isopeptide‐N‐ethylated Ub probes may complement the triazole‐based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies.