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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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In vivo probe-binding studies and analysis of subcellular DUB localization.(A) Schemes of the EYFP fusion proteins: UCH-like Zinc Finger Motif (red), proteolytic UCH domain (blue, with the putative active-site cysteine depicted in yellow), ubiquitin-binding UBA-domain (light blue), ubiquitin-like domain (green), putative nuclear localization sequence (pink), EYFP fusion protein (yellow box). (B) In vivo binding of ubiquitin and ISG15 probes. Lysates obtained from 293T cells transfected with USP14EYFP or USP14C114S-EYFP were reacted with UbVME or ISG15VS and compared to untreated aliquots in an anti-YFP immunoblot. USP14EYFP but not USP14C114S-EYFP reacts with the probes. (C) Subcellular localization of DUBs analyzed via the distribution of C-terminal EYFP fusions. 24 hours post-transfection, 293T cells were fixed with paraformaldehyde and analyzed by confocal fluorescence microscopy. USP5EYFP can be found throughout the cell (upper left). USP13EYFP is expressed mainly within the nucleus, in a speckled pattern (upper right panel). USP14EYFP is detected predominantly in the cytoplasm, with lower levels in the nucleus (lower left panel). USP3EYFP and USP36EYFP (lower right panel) are detected in the nucleus and nucleolus, respectively.
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pone-0000679-g006: In vivo probe-binding studies and analysis of subcellular DUB localization.(A) Schemes of the EYFP fusion proteins: UCH-like Zinc Finger Motif (red), proteolytic UCH domain (blue, with the putative active-site cysteine depicted in yellow), ubiquitin-binding UBA-domain (light blue), ubiquitin-like domain (green), putative nuclear localization sequence (pink), EYFP fusion protein (yellow box). (B) In vivo binding of ubiquitin and ISG15 probes. Lysates obtained from 293T cells transfected with USP14EYFP or USP14C114S-EYFP were reacted with UbVME or ISG15VS and compared to untreated aliquots in an anti-YFP immunoblot. USP14EYFP but not USP14C114S-EYFP reacts with the probes. (C) Subcellular localization of DUBs analyzed via the distribution of C-terminal EYFP fusions. 24 hours post-transfection, 293T cells were fixed with paraformaldehyde and analyzed by confocal fluorescence microscopy. USP5EYFP can be found throughout the cell (upper left). USP13EYFP is expressed mainly within the nucleus, in a speckled pattern (upper right panel). USP14EYFP is detected predominantly in the cytoplasm, with lower levels in the nucleus (lower left panel). USP3EYFP and USP36EYFP (lower right panel) are detected in the nucleus and nucleolus, respectively.

Mentions: The wealth of USPs found in the human proteome likely reflects substrate specificity, but potentially also complementation in terms of expression profiles and subcellular distribution. We therefore sought to analyze the intracellular distribution pattern of a subset of our crossreactive DUBs, using confocal microscopy. The analysis of a genome-wide set of C-terminal GFP fusion proteins for yeast had shown remarkably few with altered function or subcellular distribution (<5%), validating the choice of such C-terminal modifications [32]. Using anti-G/YFP antibodies, we also utilized this tag to assay for activity of DUBs in cell lysate. We cloned and transiently expressed five USP-EYFP constructs in 293T cells: USP5, USP13, USP14, USP3, and USP36 (Figure 6A). Lysate of USP14EYFP transfected cells was incubated with the ubiquitin and the ISG15 probe, and assayed by anti-YFP immunoblot analysis (Figure 6B). Whereas the USP14EYFP construct reacted with both probes, the respective C114S mutant did not, in agreement with the results of our IVT screen. With respect to subcellular distribution, we were particularly interested in the expression pattern of USP5 and USP13, given that these two isoforms displayed different specificity in UbVME and ISG15VS labeling experiments. USP5EYFP was found throughout the cell (Figure 6C, upper left panel), similar to USP18 [33]. In contrast, its close relative USP13EYFP was expressed mainly in the nucleus in a speckled pattern (Figure 6C, upper right panel). Consistent with the in vivo interaction between USP14 and the proteasome [5], we observed USP14EYFP predominantly in the cytoplasm, though we also noticed fluorescence in the nucleus (Figure 6C, lower left panel). To demonstrate that the presence of the C-terminal YFP fusion does not interfere with the endogenous distribution of these enzymes, we analyzed USP3EYFP (Figure 6C, lower right panel) and USP36EYFP (Figure 6C, lower right panel). USP3 is predicted to be a nuclear protein [34] and USP36 was identified as a nucleolar protein [35]. Both proteins were detected in the expected subcellular compartment. Combined, these results indicate that ISG15-specific proteases are expressed throughout the cell – a feature also proposed for DUBs. This observation supports the notion that unlike SUMOylation, which is believed to mostly occur in the nucleus [36], ISG15-modification affects many cellular compartments.


Screen for ISG15-crossreactive deubiquitinases.

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

In vivo probe-binding studies and analysis of subcellular DUB localization.(A) Schemes of the EYFP fusion proteins: UCH-like Zinc Finger Motif (red), proteolytic UCH domain (blue, with the putative active-site cysteine depicted in yellow), ubiquitin-binding UBA-domain (light blue), ubiquitin-like domain (green), putative nuclear localization sequence (pink), EYFP fusion protein (yellow box). (B) In vivo binding of ubiquitin and ISG15 probes. Lysates obtained from 293T cells transfected with USP14EYFP or USP14C114S-EYFP were reacted with UbVME or ISG15VS and compared to untreated aliquots in an anti-YFP immunoblot. USP14EYFP but not USP14C114S-EYFP reacts with the probes. (C) Subcellular localization of DUBs analyzed via the distribution of C-terminal EYFP fusions. 24 hours post-transfection, 293T cells were fixed with paraformaldehyde and analyzed by confocal fluorescence microscopy. USP5EYFP can be found throughout the cell (upper left). USP13EYFP is expressed mainly within the nucleus, in a speckled pattern (upper right panel). USP14EYFP is detected predominantly in the cytoplasm, with lower levels in the nucleus (lower left panel). USP3EYFP and USP36EYFP (lower right panel) are detected in the nucleus and nucleolus, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1919423&req=5

pone-0000679-g006: In vivo probe-binding studies and analysis of subcellular DUB localization.(A) Schemes of the EYFP fusion proteins: UCH-like Zinc Finger Motif (red), proteolytic UCH domain (blue, with the putative active-site cysteine depicted in yellow), ubiquitin-binding UBA-domain (light blue), ubiquitin-like domain (green), putative nuclear localization sequence (pink), EYFP fusion protein (yellow box). (B) In vivo binding of ubiquitin and ISG15 probes. Lysates obtained from 293T cells transfected with USP14EYFP or USP14C114S-EYFP were reacted with UbVME or ISG15VS and compared to untreated aliquots in an anti-YFP immunoblot. USP14EYFP but not USP14C114S-EYFP reacts with the probes. (C) Subcellular localization of DUBs analyzed via the distribution of C-terminal EYFP fusions. 24 hours post-transfection, 293T cells were fixed with paraformaldehyde and analyzed by confocal fluorescence microscopy. USP5EYFP can be found throughout the cell (upper left). USP13EYFP is expressed mainly within the nucleus, in a speckled pattern (upper right panel). USP14EYFP is detected predominantly in the cytoplasm, with lower levels in the nucleus (lower left panel). USP3EYFP and USP36EYFP (lower right panel) are detected in the nucleus and nucleolus, respectively.
Mentions: The wealth of USPs found in the human proteome likely reflects substrate specificity, but potentially also complementation in terms of expression profiles and subcellular distribution. We therefore sought to analyze the intracellular distribution pattern of a subset of our crossreactive DUBs, using confocal microscopy. The analysis of a genome-wide set of C-terminal GFP fusion proteins for yeast had shown remarkably few with altered function or subcellular distribution (<5%), validating the choice of such C-terminal modifications [32]. Using anti-G/YFP antibodies, we also utilized this tag to assay for activity of DUBs in cell lysate. We cloned and transiently expressed five USP-EYFP constructs in 293T cells: USP5, USP13, USP14, USP3, and USP36 (Figure 6A). Lysate of USP14EYFP transfected cells was incubated with the ubiquitin and the ISG15 probe, and assayed by anti-YFP immunoblot analysis (Figure 6B). Whereas the USP14EYFP construct reacted with both probes, the respective C114S mutant did not, in agreement with the results of our IVT screen. With respect to subcellular distribution, we were particularly interested in the expression pattern of USP5 and USP13, given that these two isoforms displayed different specificity in UbVME and ISG15VS labeling experiments. USP5EYFP was found throughout the cell (Figure 6C, upper left panel), similar to USP18 [33]. In contrast, its close relative USP13EYFP was expressed mainly in the nucleus in a speckled pattern (Figure 6C, upper right panel). Consistent with the in vivo interaction between USP14 and the proteasome [5], we observed USP14EYFP predominantly in the cytoplasm, though we also noticed fluorescence in the nucleus (Figure 6C, lower left panel). To demonstrate that the presence of the C-terminal YFP fusion does not interfere with the endogenous distribution of these enzymes, we analyzed USP3EYFP (Figure 6C, lower right panel) and USP36EYFP (Figure 6C, lower right panel). USP3 is predicted to be a nuclear protein [34] and USP36 was identified as a nucleolar protein [35]. Both proteins were detected in the expected subcellular compartment. Combined, these results indicate that ISG15-specific proteases are expressed throughout the cell – a feature also proposed for DUBs. This observation supports the notion that unlike SUMOylation, which is believed to mostly occur in the nucleus [36], ISG15-modification affects many cellular compartments.

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