Generation of the UFM1 Toolkit for Profiling UFM1‐Specific Proteases and Ligases

Abstract

Ubiquitin‐fold modifier 1 (UFM1) is a reversible post‐translational modifier that is covalently attached to target proteins through an enzymatic cascade and removed by designated proteases. Abnormalities in this process, referred to as Ufmylation, have been associated with a variety of human diseases. Given this, the UFM1‐specific enzymes represent potential therapeutic targets; however, understanding of their biological function has been hampered by the lack of chemical tools for activity profiling. To address this unmet need, a diversifiable platform for UFM1 activity‐based probes (ABPs) utilizing a native chemical ligation (NCL) strategy was developed, enabling the generation of a variety of tools to profile both UFM1 conjugating and deconjugating enzymes. The use of the probes is demonstrated in vitro and in vivo for monitoring UFM1 enzyme reactivity, opening new research avenues.

Hybrid Chains: A Collaboration of Ubiquitin and Ubiquitin-Like Modifiers Introducing Cross-Functionality to the Ubiquitin Code

“…So far, only (semi)-synthetic strategies for obtaining ubiquitinated Rub1, the yeast NEDD8 homolog (Singh et al, 2014) and SUMO-2-K63diUb hybrid chains (Bondalapati et al, 2017) have been reported. Only in the last decade, efforts to devise synthetic strategies for UbL proteins such as Nedd8 (Mulder et al, 2014), SUMO (Dobrota et al, 2012;Wucherpfennig et al, 2014;Mulder et al, 2018) and Ufm1 (Ogunkoya et al, 2012;Witting et al, 2018) have been undertaken. More recently, ISG15 synthesis has been accomplished as a modular synthesis of both domains and its subsequent ligation (Xin et al, 2019).…”

Profiling DUBs and Ubl-specific proteases with activity-based probes

“…Along this line, similar ABPs for Ubl-specific proteases have been developed by replacing the C-terminal residue in the respective Ubl for an electrophilic moiety, such as -Prg, -VME, or -VS and these include ABPs based on SUMO1,-2, À3, Nedd8, ISG15, and UFM1. Currently, many of these Ub and Ub-like ABPs are commercially available but are also readily obtained by chemical synthesis (Basters et al, 2017;Ekkebus et al, 2013;Mulder et al, 2018;Witting et al, 2018), or semisynthesis, such as the use of intein chemistry for Ub(l)ΔG-thioester formation, followed by reaction of this thioester with an amine nucleophile (Hemelaar et al, 2004).…”

Highly Specialized Ubiquitin-Like Modifications: Shedding Light into the UFM1 Enigma

“…Despite having a catalytic triad universally shared by cysteine proteases, UFSP enzymes lack sequence homology to deubiquitinating enzymes (DUBs) or even other proteases, thus representing a novel subfamily of cysteine proteases [ 50 , 51 ]. UFSP1, has a papain-like fold harboring an unusual active site configuration consisting of both a conserved Cys and His box domain as opposed to the classical Cys-His-Asp catalytical triad [ 50 , 51 ] Intriguingly, while murine UFSP1 processes UFM1 effectively, human UFSP1 seems to be catalytically inactive due to the shorter N-terminus and thus lack of the conserved cysteine active site [ 49 , 52 ]. In contrast, crystal structures reveal that murine UFSP2 contains two domains—a C-terminal catalytic domain with a similar architecture as that of murine UFSP1 in addition to a uniquely structured N-terminal domain key to recruitment by DDRGK1 and subsequent relocalization to the ER membrane [ 13 , 51 ].…”