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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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Related in: MedlinePlus

Structures of USP2 and USP14, modeled with ubiquitin and ISG15.Both USP2 and USP14 accommodate ubiquitin (green) in a shallow groove, measuring approximately 25–30 Å in diameter (view from top shown in the upper panels, view from front shown in the lower panels). Based on these complexes, the C-terminal domain of ISG15 (red) can be modeled into the groove to replace ubiquitin. The N-terminal domain of ISG15 is not involved in the hydrolysis reaction, and is located outside of the catalytic core domain of both proteases. The N-termini of USP2 and USP14 are lacking in these representations and extend to the right side of the structure models.
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pone-0000679-g007: Structures of USP2 and USP14, modeled with ubiquitin and ISG15.Both USP2 and USP14 accommodate ubiquitin (green) in a shallow groove, measuring approximately 25–30 Å in diameter (view from top shown in the upper panels, view from front shown in the lower panels). Based on these complexes, the C-terminal domain of ISG15 (red) can be modeled into the groove to replace ubiquitin. The N-terminal domain of ISG15 is not involved in the hydrolysis reaction, and is located outside of the catalytic core domain of both proteases. The N-termini of USP2 and USP14 are lacking in these representations and extend to the right side of the structure models.

Mentions: Our data show the existence of multiple ISG15-reactive DUBs, a finding that further strengthens the similarities between ubiquitin and ISG15. USP2 is a highly active protease [37] and it represents one of only few mammalian DUBs with a known target. USP2 exhibits oncogenic potential in prostate cancer by stabilizing its substrate Fatty Acid Synthase (FAS) [38], and FAS has indeed been identified as a target of ISG15 modification [39]. Furthermore, USP2 has been implicated in the regulation of the p53 pathway [40]. The recently solved structures of USP2 and USP14 [41], [42] show that both proteases accommodate the ubiquitin molecule in a shallow pan-like protrusion. Based on the orientation of the ubiquitin protein in both structures, ISG15 easily fits into the catalytic domain of USP2, USP14, and USP5 (data not shown) without apparent steric clashes (Figure 7) [23], [43].


Screen for ISG15-crossreactive deubiquitinases.

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

Structures of USP2 and USP14, modeled with ubiquitin and ISG15.Both USP2 and USP14 accommodate ubiquitin (green) in a shallow groove, measuring approximately 25–30 Å in diameter (view from top shown in the upper panels, view from front shown in the lower panels). Based on these complexes, the C-terminal domain of ISG15 (red) can be modeled into the groove to replace ubiquitin. The N-terminal domain of ISG15 is not involved in the hydrolysis reaction, and is located outside of the catalytic core domain of both proteases. The N-termini of USP2 and USP14 are lacking in these representations and extend to the right side of the structure models.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0000679-g007: Structures of USP2 and USP14, modeled with ubiquitin and ISG15.Both USP2 and USP14 accommodate ubiquitin (green) in a shallow groove, measuring approximately 25–30 Å in diameter (view from top shown in the upper panels, view from front shown in the lower panels). Based on these complexes, the C-terminal domain of ISG15 (red) can be modeled into the groove to replace ubiquitin. The N-terminal domain of ISG15 is not involved in the hydrolysis reaction, and is located outside of the catalytic core domain of both proteases. The N-termini of USP2 and USP14 are lacking in these representations and extend to the right side of the structure models.
Mentions: Our data show the existence of multiple ISG15-reactive DUBs, a finding that further strengthens the similarities between ubiquitin and ISG15. USP2 is a highly active protease [37] and it represents one of only few mammalian DUBs with a known target. USP2 exhibits oncogenic potential in prostate cancer by stabilizing its substrate Fatty Acid Synthase (FAS) [38], and FAS has indeed been identified as a target of ISG15 modification [39]. Furthermore, USP2 has been implicated in the regulation of the p53 pathway [40]. The recently solved structures of USP2 and USP14 [41], [42] show that both proteases accommodate the ubiquitin molecule in a shallow pan-like protrusion. Based on the orientation of the ubiquitin protein in both structures, ISG15 easily fits into the catalytic domain of USP2, USP14, and USP5 (data not shown) without apparent steric clashes (Figure 7) [23], [43].

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