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.

Keywords: UFM1; activity-based probes; chemical biology; native chemical ligation; post-translational modifications.

Figure 1

Schematic illustration of the UFM1 toolbox featuring activity‐based probes to study preference and selectivity of both proteases and ligases by covalently capturing active enzymes.

Scheme 1

Native chemical ligation strategy towards native UFM1 and UFM1 activity‐based probes.

Figure 2

Validation of synthetic UFM1. A) SDS‐PAGE gel showing synthetic and expressed UFM1. B) CD measurements of expressed UFM1 and synthetic UFM1 made by linear synthesis or native chemical ligation. Synth. FL=UFM1 generated by linear synthesis; Synth. NCL=UFM1 generated by NCL (6). C) UBA5 reacts with both expressed and synthetic UFM1. The asterisk (*) indicates UFM1‐thioester‐UBA5.

Figure 3

Reactivity of E1 (UBA5) towards UFM1. A) Scheme depicting the reactivity of UBA5 towards UFM1‐Dha permitting the formation of either the thioether (a) or the thioester adduct (b). B) SDS‐PAGE gel of reaction of synthetic UFM1 (6) or Rho‐UFM1‐Dha (8) and recombinant UBA5.

Figure 4

Assessment of UFM1‐PA reactivity and specificity against a panel of cysteine protease subfamilies. A) Immunoblot showing time‐dependent reactivity of Flag‐Ufsp1 (murine) towards Rho‐UFM1‐PA (10), B) and time‐dependent labeling of endogenous Ufsp2 (human). C,F) DUBs Flag‐UCHL‐1 and GFP‐OTUB2 do not react with UFM1‐PA. D,E) Flag‐SENP6 and Flag‐SENP1 react with S2‐PA (=SUMO2‐PA) but are unreactive towards UFM1‐PA. For fluorescence scans and actin blots see Figure S8.

Figure 5

Reactivity of Hela cells electroporated with Rho‐UFM1‐PA in the absence or presence of ectopically overexpressed murine Flag‐Ufsp1. A) Immunoblots of Flag‐Ufsp1 and Flag‐Ufsp1(C53A) and endogenous Ufsp2 in untransfected cells following electroporation. B) Confocal images of untransfected HeLa cells or C) in the presence of Flag‐Ufsp1 or Flag‐Ufsp1 (C53A) after probe electroporation. Cell boundaries and nuclei are indicated by a dashed line, and insets correspond to zoom‐ins. Scale bars=10 μm. Quantification of colocalization (Mander's overlap coefficient) is shown in Figure S11.