Specific and Covalent Targeting of Conjugating and Deconjugating Enzymes of Ubiquitin-Like Proteins

Abstract

Modification of proteins by ubiquitin (Ub)-like proteins (UBLs) plays an important role in many cellular processes, including cell cycle progression, nuclear transport, and autophagy. Protein modification occurs via UBL-conjugating and -deconjugating enzymes, which presumably exert a regulatory function by determining the conjugation status of the substrate proteins. To target and identify UBL-modifying enzymes, we produced Nedd8, ISG15, and SUMO-1 in Escherichia coli and equipped them with a C-terminal electrophilic trap (vinyl sulfone [VS]) via an intein-based method. These C-terminally modified UBL probes reacted with purified UBL-activating (E1), -conjugating (E2), and -deconjugating enzymes in a covalent fashion. Modified UBLs were radioiodinated and incubated with cell lysates prepared from mouse cell lines and tissues to allow visualization of polypeptides reactive with individual UBL probes. The cell type-and tissue-specific labeling patterns observed for the UBL probes reflect distinct expression profiles of active enzymes, indicating tissuespecific functions of UBLs. We identify Ub C-terminal hydrolase L1 (UCH-L1) and DEN1/NEDP1/SENP8, in addition to UCH-L3, as proteases with specificity for Nedd8. The Ub-specific protease isopeptidase T/USP5 is shown to react with ISG15-VS. Furthermore, we demonstrate that the desumoylation enzyme SuPr-1 can be modified by SUMO-1-VS, a modification that is dependent on the SuPr-1 active-site cysteine. The UBL probes described here will be valuable tools for the further characterization of the enzymatic pathways that govern modification by UBLs.Ubiquitin (Ub) is a conserved 76-amino-acid protein attached posttranslationally to substrate proteins. This conjugation occurs through an isopeptide bond between the C-terminal carboxylate of Ub and the ε-NH 2 of a lysine side chain in the target protein. Conjugation is achieved by the sequential action of an E1 activating enzyme, E2 conjugating enzymes, and E3 ligases (22). The removal of Ub from substrates is carried out by deubiquitinating enzymes. The long-known Ubspecific cysteine protease families of Ub C-terminal hydrolases (UCHs) and Ub-specific processing proteases (UBPs/USPs) were recently joined by a Ub-specific JAMM motif containing metalloprotease and cysteine proteases containing an OTU domain (2,8,13,63,67).Ub-like proteins (UBLs) are a set of small proteins that share with Ub the ability to be conjugated to a lysine residue in a substrate protein (26). Many UBLs are related in sequence to Ub, and a three-dimensional fold similar to that in Ub has been reported for Nedd8 and SUMO (3, 66). The UBLs ISG15 (also called UCRP) and FAT10 resemble two Ub moieties fused in tandem (19, 54). UBLs do not generally appear to be assembled into multimeric chains upon conjugation to substrates, with the possible exception of SUMO-2 and SUMO-3 (62). Like Ub, most UBLs are expressed as inactive precursors, with extensions at the C terminus, which prevent direct conjugation (26) ( Table 1). These precursors must be processed by sp...

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation

“…Following binding to phosphorylated ubiquitin, the Parkin catalytic Cys 431 , which is normally occluded by the RING0 domain, becomes exposed as demonstrated by Ub-vinyl sulfone accessibility (Fig. 6B) (14,24,39,40). This result strongly suggests that Parkin structural remodeling occurs after binding to phosphorylated ubiquitin.…”

Characterization and Structural Studies of the Plasmodium falciparum Ubiquitin and Nedd8 Hydrolase UCHL3

“…The probes used to identify PfUCHL3 were derived from the mouse Ub and Nedd8 sequences and have been characterized extensively (11). Briefly, these probes consist of the UBL equipped with an electrophilic warhead on the C-terminus, capable of forming a covalent bond with the active site cysteine of target enzymes.…”

Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity

“…DNA extractions and purification were performed using Qiagen system kits, and His-tagged proteins were isolated using the Ni-NTA resin from the same supplier. [α-32 P]UTP (6000 Ci/mmol) was purchased from New England Nuclear, nucleoside 5′-triphosphates (all nucleotides were ultrapure solutions) and Q Sepharose fast flow were purchased from Amersham Pharmacia Biotech, poly(rA) was purchased from Sigma, dT 15 was purchased from Integrated DNA Technologies, all RNA oligonucleotides were purchased from Dharmacon Research, NZCYM was purchased from Invitrogen, and phosphocellulose (P-11) and DE-81 filter paper were purchased from Whatman. All other reagents were of the highest grade available from Sigma or Fisher.…”

“…Proteases specific for SUMO conjugates include yeast Ulp1 and Ulp2 [9,10] and human SENP1 and SENP2 [11,12]. Other known ULPs include UBP43, which cleaves ISG15 conjugates [13], and DEN1 (SENP8), which deconjugates Nedd8 from protein substrates [14,15]. Two recently identified classes of deubiquitinating enzymes (DUBs) are the otubains, cysteine proteases belonging to the OTU (ovarian tumor) superfamily of proteins [16], and the JAMM family enzymes, which are quite different from most DUBs in that they are metalloproteases that cleave ubiquitin from substrates in a Zn 2+ -and ATP-dependent manner [17].…”