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The E3 Ubiquitin Ligase MARCH8 Degrades the Interferon Gamma Receptor
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A4706 - The E3 Ubiquitin Ligase MARCH8 Degrades the Interferon Gamma Receptor
Author Block: J. D. Londino1, L. S. Masa1, R. K. Mallampalli2; 1PACCM, University of Pittsburgh, Pittsburgh, PA, United States, 2PACCM, UPMC Montefiore, Pittsburgh, PA, United States.
RATIONALE:
Interferon gamma signaling plays an important role in the resolution of bacterial and viral infection. Ubiquitination and degradation of the IFNGR1 receptor decreases the availability of IFNGR1 for signaling. Previously, the Kaposi's sarcoma-associated herpesvirus virus encoded e3 ubiquitin ligases K3 and K5 were shown to degrade IFNGR1. We therefore speculated that their mammalian homologs, the Membrane associated RING-CH (MARCH) proteins, may endogenously regulate IFNGR1.
METHODS:
Experiments were performed in HEK and A549 cell lines. HEK cells were transfected with plasmids expressing MARCH e3 ubiquitin ligases and IFNGR1 stability was examined via immunoblotting. The stability of IFNGR1 was examined in the alveolar epithelial cell line A549 via CHX chase. MARCH 1 and MARCH8 e3 ligase mutants generated via site directed mutagenesis.
RESULTS:
We examined the stability of IFNGR1 receptor in HEK cells by overexpressing MARCH family e3 ligases. The highly similar e3 ubiquitin ligases MARCH1 and MARCH8 both appeared to decrease IFNGR1 stability. E3 ligase inactive RING domain mutants of MARCH1 and MARCH8 failed to decrease IFNGR1 stability. Due to the preferential expression of MARCH8 is in the epithelium, we knocked down and overexpressed of MARCH8 in epithelial cell lines and measured stability via a CHX chase assay. Increased MARCH8 expression destabilized IFNGR1 while knockdown of endogenous MARCH8 increased the stability the receptor suggesting a role for MARCH8 in the regulation of IFNGR1.
CONCLUSIONS:
Our experiments show that IFNGR1 stability is altered by the E3 ubiquitin ligase MARCH8. Alteration of interferon signaling via MARCH8 may have implications in the disease pathology of viral and bacterial infection as well as in cancer. Further examination of the mechanism of ubiquitination and degradation of IFNGR1 will provide new insight into IFN-γ signaling and may lead to novel therapeutic targets.
Author Block: J. D. Londino1, L. S. Masa1, R. K. Mallampalli2; 1PACCM, University of Pittsburgh, Pittsburgh, PA, United States, 2PACCM, UPMC Montefiore, Pittsburgh, PA, United States.
RATIONALE:
Interferon gamma signaling plays an important role in the resolution of bacterial and viral infection. Ubiquitination and degradation of the IFNGR1 receptor decreases the availability of IFNGR1 for signaling. Previously, the Kaposi's sarcoma-associated herpesvirus virus encoded e3 ubiquitin ligases K3 and K5 were shown to degrade IFNGR1. We therefore speculated that their mammalian homologs, the Membrane associated RING-CH (MARCH) proteins, may endogenously regulate IFNGR1.
METHODS:
Experiments were performed in HEK and A549 cell lines. HEK cells were transfected with plasmids expressing MARCH e3 ubiquitin ligases and IFNGR1 stability was examined via immunoblotting. The stability of IFNGR1 was examined in the alveolar epithelial cell line A549 via CHX chase. MARCH 1 and MARCH8 e3 ligase mutants generated via site directed mutagenesis.
RESULTS:
We examined the stability of IFNGR1 receptor in HEK cells by overexpressing MARCH family e3 ligases. The highly similar e3 ubiquitin ligases MARCH1 and MARCH8 both appeared to decrease IFNGR1 stability. E3 ligase inactive RING domain mutants of MARCH1 and MARCH8 failed to decrease IFNGR1 stability. Due to the preferential expression of MARCH8 is in the epithelium, we knocked down and overexpressed of MARCH8 in epithelial cell lines and measured stability via a CHX chase assay. Increased MARCH8 expression destabilized IFNGR1 while knockdown of endogenous MARCH8 increased the stability the receptor suggesting a role for MARCH8 in the regulation of IFNGR1.
CONCLUSIONS:
Our experiments show that IFNGR1 stability is altered by the E3 ubiquitin ligase MARCH8. Alteration of interferon signaling via MARCH8 may have implications in the disease pathology of viral and bacterial infection as well as in cancer. Further examination of the mechanism of ubiquitination and degradation of IFNGR1 will provide new insight into IFN-γ signaling and may lead to novel therapeutic targets.
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