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Screen for ISG15-crossreactive deubiquitinases.

Catic A, Fiebiger E, Korbel GA, Blom D, Galardy PJ, Ploegh HL - PLoS ONE (2007)

Bottom Line: USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome.By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin.Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 mice.

View Article: PubMed Central - PubMed

Affiliation: Program in Immunology, Harvard Medical School, Boston, Massachusetts, United States of America; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

ABSTRACT

Background: The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15.

Results: We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome.

Conclusions: By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 mice.

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Activity-based profiling of DUBs.IVT was used to generate DUBs for profiling activity toward ISG15VS, UbVME and SUMO1VME probes. After incubation with the probes, samples were analyzed by SDS-PAGE, as shown for USP18 (A), USP14 (B), USP5 (C), USP13 (D), USP2 (E), and CGI-77 (F). Binding was inhibited by preincubation of the proteases with NEM. The SUMO protease SENP2 interacts specifically with SUMO1VME (G). Molecular weight in kDa is indicated on the left.
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pone-0000679-g002: Activity-based profiling of DUBs.IVT was used to generate DUBs for profiling activity toward ISG15VS, UbVME and SUMO1VME probes. After incubation with the probes, samples were analyzed by SDS-PAGE, as shown for USP18 (A), USP14 (B), USP5 (C), USP13 (D), USP2 (E), and CGI-77 (F). Binding was inhibited by preincubation of the proteases with NEM. The SUMO protease SENP2 interacts specifically with SUMO1VME (G). Molecular weight in kDa is indicated on the left.

Mentions: Figure 1 shows a consensus phylogram based on the alignment of catalytic core sequences of DUBs, including the majority of known human USP homologs. In this tree, the ISG15-protease USP18 clusters close to USP5 (IsoT1) and its isoform USP13 (IsoT3). Previous work had identified USP5 as a protease with affinity for both ubiquitin [27] and ISG15, as shown by its reaction with an electrophilic ISG15 derivative, ISG15-vinyl sulfone (ISG15VS) [28]. To probe for additional ISG15-reactive proteases, we have cloned and expressed a total of 22 human DUB homologs from different clades of this phylogram (indicated with arrows), 17 of which reacted with a ubiquitin-based probe and/or an ISG15-based probe (see below). The screen was based on in vitro transcription and translation (IVT) of cloned cDNAs, which affords a rapid method to generate radiochemically pure proteins. This technique allows the generation of DUBs that cannot be readily expressed in bacterial systems, or that are sequestered in subcellular compartments or in multimolecular complexes when expressed in cell lines. To profile for DUB specificity, we used recombinantly expressed ubiquitin, SUMO1 and ISG15, and installed an electrophilic trap at their C-terminus to obtain the active-site probes ubiquitin-vinylmethyl ester (UbVME), SUMO1VME and ISG15VS, respectively [28]. DUBs generated by IVT were incubated with each of these three probes (Figure 2), followed by direct analysis of the reaction mixture by SDS-PAGE. We have determined by X-ray crystallography that probes of this type form a covalent adduct with the catalytic cysteine residue of active DUBs to yield a thioether-linked adduct between enzyme and probe [26]. When unmodified IVT products are run adjacent to samples incubated with these activity-based probes, the adduct is readily detected through a shift in apparent molecular mass. USP2, USP5, USP13 and USP14 reacted with ISG15VS (Figure 2B, C, D, E).


Screen for ISG15-crossreactive deubiquitinases.

Catic A, Fiebiger E, Korbel GA, Blom D, Galardy PJ, Ploegh HL - PLoS ONE (2007)

Activity-based profiling of DUBs.IVT was used to generate DUBs for profiling activity toward ISG15VS, UbVME and SUMO1VME probes. After incubation with the probes, samples were analyzed by SDS-PAGE, as shown for USP18 (A), USP14 (B), USP5 (C), USP13 (D), USP2 (E), and CGI-77 (F). Binding was inhibited by preincubation of the proteases with NEM. The SUMO protease SENP2 interacts specifically with SUMO1VME (G). Molecular weight in kDa is indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1919423&req=5

pone-0000679-g002: Activity-based profiling of DUBs.IVT was used to generate DUBs for profiling activity toward ISG15VS, UbVME and SUMO1VME probes. After incubation with the probes, samples were analyzed by SDS-PAGE, as shown for USP18 (A), USP14 (B), USP5 (C), USP13 (D), USP2 (E), and CGI-77 (F). Binding was inhibited by preincubation of the proteases with NEM. The SUMO protease SENP2 interacts specifically with SUMO1VME (G). Molecular weight in kDa is indicated on the left.
Mentions: Figure 1 shows a consensus phylogram based on the alignment of catalytic core sequences of DUBs, including the majority of known human USP homologs. In this tree, the ISG15-protease USP18 clusters close to USP5 (IsoT1) and its isoform USP13 (IsoT3). Previous work had identified USP5 as a protease with affinity for both ubiquitin [27] and ISG15, as shown by its reaction with an electrophilic ISG15 derivative, ISG15-vinyl sulfone (ISG15VS) [28]. To probe for additional ISG15-reactive proteases, we have cloned and expressed a total of 22 human DUB homologs from different clades of this phylogram (indicated with arrows), 17 of which reacted with a ubiquitin-based probe and/or an ISG15-based probe (see below). The screen was based on in vitro transcription and translation (IVT) of cloned cDNAs, which affords a rapid method to generate radiochemically pure proteins. This technique allows the generation of DUBs that cannot be readily expressed in bacterial systems, or that are sequestered in subcellular compartments or in multimolecular complexes when expressed in cell lines. To profile for DUB specificity, we used recombinantly expressed ubiquitin, SUMO1 and ISG15, and installed an electrophilic trap at their C-terminus to obtain the active-site probes ubiquitin-vinylmethyl ester (UbVME), SUMO1VME and ISG15VS, respectively [28]. DUBs generated by IVT were incubated with each of these three probes (Figure 2), followed by direct analysis of the reaction mixture by SDS-PAGE. We have determined by X-ray crystallography that probes of this type form a covalent adduct with the catalytic cysteine residue of active DUBs to yield a thioether-linked adduct between enzyme and probe [26]. When unmodified IVT products are run adjacent to samples incubated with these activity-based probes, the adduct is readily detected through a shift in apparent molecular mass. USP2, USP5, USP13 and USP14 reacted with ISG15VS (Figure 2B, C, D, E).

Bottom Line: USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome.By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin.Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 mice.

View Article: PubMed Central - PubMed

Affiliation: Program in Immunology, Harvard Medical School, Boston, Massachusetts, United States of America; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

ABSTRACT

Background: The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15.

Results: We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome.

Conclusions: By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 mice.

Show MeSH
Related in: MedlinePlus