No abstract

Ubiquitin-specific proteases (USPs) are papain-like isopeptidases with variable inter- and intramolecular regulatory domains. To understand the effect of these domains on USP activity, we have analyzed the enzyme kinetics of 12 USPs in the presence and absence of modulators using synthetic reagents. This revealed variations of several orders of magnitude in both the catalytic turnover (k(cat)) and ubiquitin (Ub) binding (K(M)) between USPs. Further activity modulation by intramolecular domains affects both the k(cat) and K(M), whereas the intermolecular activators UAF1 and GMPS mainly increase the k(cat). Also, we provide the first comprehensive analysis comparing Ub chain preference. USPs can hydrolyze all linkages and show modest Ub-chain preferences, although some show a lack of activity toward linear di-Ub. This comprehensive kinetic analysis highlights the variability within the USP family.

Thiolysine‐mediated chemical ligation has generated fluorescence polarisation assay reagents based on isopeptide‐linked ubiquitin‐like protein conjugates. These have been used to monitor the activity of ubiquitin(‐like) proteases. Thus, it is now possible to generate assay reagents that contain substrate‐derived elements around the isopeptide linkage, with no practical limitation.

SUMMARY Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and the polyUb chains to a range of cellular protein targets. Best documented Ub signaling pathway is the one that delivers polyUb-proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (UbPT), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb-conjugates. Our work demonstrates the value of UbPT and we expect that its future uses will help define and investigate the ubiquitin interactome.

Höchst effizient ist die hier vorgestellte lineare Festphasenpeptidsynthese von Ubiquitin (Ub), die es auf einfache Weise ermöglicht, bestimmte Markierungen und Mutationen einzuführen (siehe Bild; blau: ein Pseudoprolindipeptid, rot: ein Dimethoxybenzyldipeptid) sowie spezifische C‐terminale Modifikationen vorzunehmen und alle Diubiquitinkonjugate in hoher Ausbeute und Reinheit aufzubauen.

SUMO is a post‐translational modifier critical for cell cycle progression and genome stability that plays a role in tumorigenesis, thus rendering SUMO‐specific enzymes potential pharmacological targets. However, the systematic generation of tools for the activity profiling of SUMO‐specific enzymes has proven challenging. We developed a diversifiable synthetic platform for SUMO‐based probes by using a direct linear synthesis method, which permits N‐ and C‐terminal labelling to incorporate dyes and reactive warheads, respectively. In this manner, activity‐based probes (ABPs) for SUMO‐1, SUMO‐2, and SUMO‐3‐specific proteases were generated and validated in cells using gel‐based assays and confocal microscopy. We further expanded our toolbox with the synthesis of a K11‐linked diSUMO‐2 probe to study the proteolytic cleavage of SUMO chains. Together, these ABPs demonstrate the versatility and specificity of our synthetic SUMO platform for in vitro and in vivo characterization of the SUMO protease family.

Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signaling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research.

Proteins and other macromolecules can be delivered into live cells by noninvasive techniques using cell‐penetrating peptides. These peptides are easily synthesised by solid‐phase peptide synthesis and can be conjugated onto cargo molecules to mediate cellular delivery. We designed a TAT‐based cell‐penetrating ubiquitin (Ub) reagent by conjugating a dimeric disulfide‐linked TAT peptide to the C terminus of a rhodamine‐labelled Ub (RhoUb) protein. This reagent efficiently enters the cell by endocytosis and escapes from endosomes into the cytoplasm. Once the conjugate is inside the cytoplasm, the delivery vehicle is proteolytically removed by endogenous deubiquitinases (DUBs), at which point the intrinsic ubiquitination machinery is able to incorporate the RhoUb into ubiquitin conjugates. Our approach enables the controlled delivery of labelled or mutant Ub derivatives into cells, increasing our options for studying the ubiquitin system.