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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.MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity.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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MIR1 and MIR2 mediate ubiquitination of their targets. wt HeLa cells or HeLa cells expressing B7.2 were stably transduced by pMX-pie, pBP-MIR1/EGFP, or pBP-MIR2/EGFP. B7.2 molecules (right) or MHC-I molecules (left) were immunoprecipitated, and their ubiquitination status was determined by Western blot analysis using an anti-Ub antibody.
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fig4: MIR1 and MIR2 mediate ubiquitination of their targets. wt HeLa cells or HeLa cells expressing B7.2 were stably transduced by pMX-pie, pBP-MIR1/EGFP, or pBP-MIR2/EGFP. B7.2 molecules (right) or MHC-I molecules (left) were immunoprecipitated, and their ubiquitination status was determined by Western blot analysis using an anti-Ub antibody.

Mentions: Accordingly, we looked for evidence that B7.2 and MHC-I chains might undergo ubiquitination in the presence of MIR proteins. HeLa cells stably expressing B7.2 were transduced with pMX-pie, pB-MIR1/EGFP, or pB-MIR2/EGFP. After puromycin selection, chains were immune precipitated with anti-B7.2 antibody, and the resulting precipitates were fractionated by SDS-PAGE, transferred to membranes, and blotted with anti-Ub antibody. As shown in Fig. 4 (right), no ubiquitinated forms were observed in vector or MIR-1–expressing cells. However, in MIR2-expressing cells, ubiquitination of B7.2 chains was readily detectable, producing a characteristic heterogeneous array that is presumably due to either polyubiquitin addition, ubiquitination of multiple target lysines, or both. Similarly (Fig. 4, left), cells expressing either MIR1 or MIR2 display ubiquitination of their endogenous MHC-I chains, whereas such chains are not detected in control cells lacking expression of either MIR1 or MIR2.


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)

MIR1 and MIR2 mediate ubiquitination of their targets. wt HeLa cells or HeLa cells expressing B7.2 were stably transduced by pMX-pie, pBP-MIR1/EGFP, or pBP-MIR2/EGFP. B7.2 molecules (right) or MHC-I molecules (left) were immunoprecipitated, and their ubiquitination status was determined by Western blot analysis using an anti-Ub antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199318&req=5

fig4: MIR1 and MIR2 mediate ubiquitination of their targets. wt HeLa cells or HeLa cells expressing B7.2 were stably transduced by pMX-pie, pBP-MIR1/EGFP, or pBP-MIR2/EGFP. B7.2 molecules (right) or MHC-I molecules (left) were immunoprecipitated, and their ubiquitination status was determined by Western blot analysis using an anti-Ub antibody.
Mentions: Accordingly, we looked for evidence that B7.2 and MHC-I chains might undergo ubiquitination in the presence of MIR proteins. HeLa cells stably expressing B7.2 were transduced with pMX-pie, pB-MIR1/EGFP, or pB-MIR2/EGFP. After puromycin selection, chains were immune precipitated with anti-B7.2 antibody, and the resulting precipitates were fractionated by SDS-PAGE, transferred to membranes, and blotted with anti-Ub antibody. As shown in Fig. 4 (right), no ubiquitinated forms were observed in vector or MIR-1–expressing cells. However, in MIR2-expressing cells, ubiquitination of B7.2 chains was readily detectable, producing a characteristic heterogeneous array that is presumably due to either polyubiquitin addition, ubiquitination of multiple target lysines, or both. Similarly (Fig. 4, left), cells expressing either MIR1 or MIR2 display ubiquitination of their endogenous MHC-I chains, whereas such chains are not detected in control cells lacking expression of either MIR1 or MIR2.

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.MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity.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