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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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A critical domain within the transmembrane region of human MHC-I is required for MIR2-mediated downregulation. (A) Cells stably expressing the control vector or MIR2 were transiently transfected with the human or the mouse MHC-I molecule (HLA.B7 and H2 Dd, respectively). The number of cell expressing the transfected MHC-I at the surface was measured by flow cytometry. (B) Chimeric proteins between human (bold) and mouse (underlined) MHC-I were engineered, and their susceptibility to MIR2-mediated downregulation was analyzed as above.
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fig7: A critical domain within the transmembrane region of human MHC-I is required for MIR2-mediated downregulation. (A) Cells stably expressing the control vector or MIR2 were transiently transfected with the human or the mouse MHC-I molecule (HLA.B7 and H2 Dd, respectively). The number of cell expressing the transfected MHC-I at the surface was measured by flow cytometry. (B) Chimeric proteins between human (bold) and mouse (underlined) MHC-I were engineered, and their susceptibility to MIR2-mediated downregulation was analyzed as above.

Mentions: The results presented thus far have focused upon the role of the cytosolic tail of the target protein in MIR-mediated regulation, and this role that can now be understood in terms of the provision of substrate lysines for ubiquitination by E2s recruited by MIR2's PHD domain. However, interaction with E2s is only one function of an E3. Another key role an E3 must play is target recognition, and in most E3s this function is subsumed by a different domain than that involved in E2 recruitment. We have found recently that the TM domains of MIR2 contribute critically to target selectivity (unpublished data). To better understand which regions of the target might contribute to MIR recognition, we took advantage of the fact that MIR2 can downregulate human but not murine MHC-I chains, even when the latter are expressed in a human cell (Fig. 7 A; Stevenson et al., 2000). Since both human and mouse MHC-I proteins contain cytosolic lysines, this suggests that the failure of MIR2 to regulate mouse MHC-I might reside at the level of target recognition. To further understand this issue, we constructed chimeras between mouse MHC-I (H2 Dd) and human MHC-I (HLA.B7) and tested their ability to be regulated by MIR2. BJAB cells stably expressing MIR2 or vector alone were transiently transfected with an expression vector for wt HLA B7 (which is not expressed endogenously by BJAB cells). All transfections proceeded with comparable efficiency (unpublished data). As shown in Fig. 7 A, a 15-fold decrease in the number of cells expressing HLA B7 was observed in cells expressing MIR2 compared with cells expressing the control vector. As expected, mouse MHC-I chains did not display this MIR2-mediated downregulation. Similar experiments were performed using the chimeras depicted in Fig. 7 B. As shown in Fig. 7 B, chimera 3, containing the TM and intracellular region of mouse MHC, cannot be downregulated by MIR2. On the other hand, chimera 2, which differs from chimera 3 principally in containing the TM and juxtamembrane region of human MHC, is efficiently downregulated by MIR2. This suggests that the TM and juxtamembrane regions of the target MHC chains contribute critically to recognition by MIR proteins.


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)

A critical domain within the transmembrane region of human MHC-I is required for MIR2-mediated downregulation. (A) Cells stably expressing the control vector or MIR2 were transiently transfected with the human or the mouse MHC-I molecule (HLA.B7 and H2 Dd, respectively). The number of cell expressing the transfected MHC-I at the surface was measured by flow cytometry. (B) Chimeric proteins between human (bold) and mouse (underlined) MHC-I were engineered, and their susceptibility to MIR2-mediated downregulation was analyzed as above.
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Related In: Results  -  Collection

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fig7: A critical domain within the transmembrane region of human MHC-I is required for MIR2-mediated downregulation. (A) Cells stably expressing the control vector or MIR2 were transiently transfected with the human or the mouse MHC-I molecule (HLA.B7 and H2 Dd, respectively). The number of cell expressing the transfected MHC-I at the surface was measured by flow cytometry. (B) Chimeric proteins between human (bold) and mouse (underlined) MHC-I were engineered, and their susceptibility to MIR2-mediated downregulation was analyzed as above.
Mentions: The results presented thus far have focused upon the role of the cytosolic tail of the target protein in MIR-mediated regulation, and this role that can now be understood in terms of the provision of substrate lysines for ubiquitination by E2s recruited by MIR2's PHD domain. However, interaction with E2s is only one function of an E3. Another key role an E3 must play is target recognition, and in most E3s this function is subsumed by a different domain than that involved in E2 recruitment. We have found recently that the TM domains of MIR2 contribute critically to target selectivity (unpublished data). To better understand which regions of the target might contribute to MIR recognition, we took advantage of the fact that MIR2 can downregulate human but not murine MHC-I chains, even when the latter are expressed in a human cell (Fig. 7 A; Stevenson et al., 2000). Since both human and mouse MHC-I proteins contain cytosolic lysines, this suggests that the failure of MIR2 to regulate mouse MHC-I might reside at the level of target recognition. To further understand this issue, we constructed chimeras between mouse MHC-I (H2 Dd) and human MHC-I (HLA.B7) and tested their ability to be regulated by MIR2. BJAB cells stably expressing MIR2 or vector alone were transiently transfected with an expression vector for wt HLA B7 (which is not expressed endogenously by BJAB cells). All transfections proceeded with comparable efficiency (unpublished data). As shown in Fig. 7 A, a 15-fold decrease in the number of cells expressing HLA B7 was observed in cells expressing MIR2 compared with cells expressing the control vector. As expected, mouse MHC-I chains did not display this MIR2-mediated downregulation. Similar experiments were performed using the chimeras depicted in Fig. 7 B. As shown in Fig. 7 B, chimera 3, containing the TM and intracellular region of mouse MHC, cannot be downregulated by MIR2. On the other hand, chimera 2, which differs from chimera 3 principally in containing the TM and juxtamembrane region of human MHC, is efficiently downregulated by MIR2. This suggests that the TM and juxtamembrane regions of the target MHC chains contribute critically to recognition by MIR proteins.

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