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A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition.

Coscoy L, Sanchez DJ, Ganem D - J. Cell Biol. (2001)

Bottom Line: Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells.This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins.Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Microbiology, University of California Medical Center, San Francisco, CA 94143, USA.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

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The MIR2 PHD domain can mediate ATP/E2-dependent ubiquitination in vitro. In vitro ubiquitination reactions were performed as described in Materials and methods. In the complete reaction mix, 10 μg of GS-bound GST, GST-MIR2 wt PHD domain, and GST-MIR2 mutant PHD domain were added to a cocktail of 10 mM ATP, 2 mg Ub, 40 ng of E1, and 1 μg of the E2 UbcH5a. In the remaining lanes, individual components of the full reaction mix were omitted as indicated above each lane. After the reaction, GS-bound material was recovered by centrifugation, eluted with SDS sample buffer, and examined by Western blotting with anti-Ub antibody.
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fig6: The MIR2 PHD domain can mediate ATP/E2-dependent ubiquitination in vitro. In vitro ubiquitination reactions were performed as described in Materials and methods. In the complete reaction mix, 10 μg of GS-bound GST, GST-MIR2 wt PHD domain, and GST-MIR2 mutant PHD domain were added to a cocktail of 10 mM ATP, 2 mg Ub, 40 ng of E1, and 1 μg of the E2 UbcH5a. In the remaining lanes, individual components of the full reaction mix were omitted as indicated above each lane. After the reaction, GS-bound material was recovered by centrifugation, eluted with SDS sample buffer, and examined by Western blotting with anti-Ub antibody.

Mentions: Accordingly, we tested the ability of wt and PHD mutant forms of MIR2 to functionally interact with a series of known eukaryotic E2 Ub-conjugating enzymes. In one standard assay of such interaction, in vitro incubation of glutathione S-transferase (GST)–E3 fusions bearing the RING finger region with active E1 and E2 enzymes leads to transfer of Ub to the GST region of the fusion protein (Joazeiro et al., 1999). To see if MIR2 could function in such an assay, we fused the NH2-terminal cytosolic region of MIR2 (bearing its PHD domain) to GST and expressed the resulting fusion protein in Escherichia coli. In parallel, a MIR2–GST fusion bearing mutations in key cysteine residues of the PHD/Zn finger was expressed. Both proteins were partially purified by binding to glutathione-Sepharose (GS), and equal amounts of each was added to a reaction mix containing ATP, Ub, purified E1, and the human E2 UbcH5a. After incubation, the resulting GST-containing protein products were purified by GS binding, separated by SDS-PAGE, transferred to filters, and probed with anti-Ub antibody. As shown in Fig. 6, in the presence of the wt MIR2–GST fusion a prominent band of high molecular weight products reactive with anti-Ub was observed; these products were not detected in reactions programmed with GST alone or with the MIR2–GST fusion protein bearing point mutations in the PHD domain. As expected, these ubiquitinated reaction products were dependent on the presence of ATP and E2 enzyme (Fig. 6).


A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition.

Coscoy L, Sanchez DJ, Ganem D - J. Cell Biol. (2001)

The MIR2 PHD domain can mediate ATP/E2-dependent ubiquitination in vitro. In vitro ubiquitination reactions were performed as described in Materials and methods. In the complete reaction mix, 10 μg of GS-bound GST, GST-MIR2 wt PHD domain, and GST-MIR2 mutant PHD domain were added to a cocktail of 10 mM ATP, 2 mg Ub, 40 ng of E1, and 1 μg of the E2 UbcH5a. In the remaining lanes, individual components of the full reaction mix were omitted as indicated above each lane. After the reaction, GS-bound material was recovered by centrifugation, eluted with SDS sample buffer, and examined by Western blotting with anti-Ub antibody.
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Related In: Results  -  Collection

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fig6: The MIR2 PHD domain can mediate ATP/E2-dependent ubiquitination in vitro. In vitro ubiquitination reactions were performed as described in Materials and methods. In the complete reaction mix, 10 μg of GS-bound GST, GST-MIR2 wt PHD domain, and GST-MIR2 mutant PHD domain were added to a cocktail of 10 mM ATP, 2 mg Ub, 40 ng of E1, and 1 μg of the E2 UbcH5a. In the remaining lanes, individual components of the full reaction mix were omitted as indicated above each lane. After the reaction, GS-bound material was recovered by centrifugation, eluted with SDS sample buffer, and examined by Western blotting with anti-Ub antibody.
Mentions: Accordingly, we tested the ability of wt and PHD mutant forms of MIR2 to functionally interact with a series of known eukaryotic E2 Ub-conjugating enzymes. In one standard assay of such interaction, in vitro incubation of glutathione S-transferase (GST)–E3 fusions bearing the RING finger region with active E1 and E2 enzymes leads to transfer of Ub to the GST region of the fusion protein (Joazeiro et al., 1999). To see if MIR2 could function in such an assay, we fused the NH2-terminal cytosolic region of MIR2 (bearing its PHD domain) to GST and expressed the resulting fusion protein in Escherichia coli. In parallel, a MIR2–GST fusion bearing mutations in key cysteine residues of the PHD/Zn finger was expressed. Both proteins were partially purified by binding to glutathione-Sepharose (GS), and equal amounts of each was added to a reaction mix containing ATP, Ub, purified E1, and the human E2 UbcH5a. After incubation, the resulting GST-containing protein products were purified by GS binding, separated by SDS-PAGE, transferred to filters, and probed with anti-Ub antibody. As shown in Fig. 6, in the presence of the wt MIR2–GST fusion a prominent band of high molecular weight products reactive with anti-Ub was observed; these products were not detected in reactions programmed with GST alone or with the MIR2–GST fusion protein bearing point mutations in the PHD domain. As expected, these ubiquitinated reaction products were dependent on the presence of ATP and E2 enzyme (Fig. 6).

Bottom Line: Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells.This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins.Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Microbiology, University of California Medical Center, San Francisco, CA 94143, USA.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

Show MeSH