2021 Fiscal Year Annual Research Report
| Project/Area Number |
21J14073
|
|---|
| Research Institution | The University of Tokyo |
|---|
Principal Investigator |
趙 東儀 東京大学, 薬学系研究科, 特別研究員(DC2)
|
|---|
| Project Period (FY) |
2021-04-28 – 2023-03-31
|
| Keywords | Type I IFN / Apoptosis / MAVS / ASK1 |
|---|
| Outline of Annual Research Achievements |
A MAP3K ASK1 phosphorylates its downstream kinase MAP2Ks, which in turn activates MAPKs. We found that overexpression of ASK1 induced the band shift of MAVS in immunoblotting, suggesting that ASK1 promotes phosphorylation of MAVS. Overexpression of an active form of MAP2Ks also promoted the shifted band of MAVS. Furthermore, with the MAPK inhibitor treatment, we found that downstream MAPK of ASK1 mediated the band shift of MAVS.
With Mass-spec analysis, we identified putative phosphorylation sites of MAVS in response to ASK1 activation. To determine ASK1-mediated phosphorylation sites of MAVS, I established plasmids encoding MAVS SA mutants. One of the mutations blocked the MAVS shifted band. By the use of this specific antibody, we found that either ASK1 overexpression, VSV or Sendai virus infection increased the phosphorylation of MAVS.
To examine the role of the ASK1-mediated MAVS phosphorylation, MAVS-/- MEFs were reconstituted with MAVS harboring single mutation and were stimulated with VSV, Sendai virus or poly(I:C). I found that the MAVS mutant reduced the ability of MAVS to induce IFN production in the early phase of poly(I:C) transfection and SeV infection. Consistent with these results, SeV replication dramatically increased in MAVS mutant MEFs.
|
| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
I firstly confirmed that ASK1 promote the phosphorylation of MAVS and then examined ASK1-induced phosphorylation sites of MAVS.The most important improvement of my project started after generation of the specific phospho-MAVS antibody. Then, I further check whether ASK1 and viral infection induced phosphorylation of MAVS at specific serine site.
Meanwhile, for excluding the endougenous level of MAVS, I generated the MAVS expressed MAVS serine to alanine mutant. Then further check whether the phosphorylation of MAVS mediate type I IFN production and viral repication in response to viral infection.
|
| Strategy for Future Research Activity |
Furthermore, to detect the phosphorylation of this specific serine residue, I generated an antibody specific for phospho-MAVS. By the use of this specific antibody, we found that either ASK1 overexpression, VSV or Sendai virus infection increased the phosphorylation of MAVS. As a next step, I would like to examine whether the viral infection induces phosphorylation of MAVS through ASK1.
Meanwhile, I would like to examine whether the phosphorylation of MAVS promote MAVS aggregation. Furthermore, I would like to take advantage of live imaging to detect downstream kinase of ASK1 mediated IFN production or apoptosis induction.
|
