Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Investigating the potential role of MARCH proteins in regulating the Notch signalling pathway (#280)

Cyrus Tan 1 2 , Dina Stockwell 1 2 , Nicos A Nicola 1 2 , Nadia J Kershaw 1 2 , Jeff J Babon 1 2
  1. Walter and Eliza Hall Institute, Parkville, VIC, Australia
  2. Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia

The membrane-associated RING-CH (MARCH) are a family of membrane-bound RING-type E3 ubiquitin ligases that regulate the surface levels of a variety of cell surface proteins via ubiquitin-mediated pathways. While most of the MARCH proteins, especially MARCH1 and MARCH8, have been shown to regulate immunological cell surface receptors involved in antigen presentation and lymphocyte activation, the full substrate repertoire of the MARCH family has yet to be fully uncovered. Furthermore, the physiological roles of MARCH proteins have only been well described for MARCH1 and MARCH8, but not for the other MARCH family members. The Notch signalling pathway regulates critical biological processes during development, including tissue patterning and stem cell maintenance. Additionally, Notch signalling is also associated in controlling T and B cell fate decisions. Indeed, aberrant Notch signalling has been shown to drive a number of lymphoid malignancies, such as T-ALL and diffuse B cell lymphoma. The intracellular portion of Notch receptors are ubiquitinated by a host of E3 ubiquitin ligases in order to initiate signalling outcomes downstream of ligand engagement.

Here, we investigate the role of MARCH proteins as potential regulators of Notch receptors on the cell surface. Surface-capture proteomics identified Notch2 to be down-regulated from the surface of macrophage precursor M1 cells over-expressing MARCH2, 4, and 9 compared to cells that do not. In cell-based flow cytometry assays, MARCH2, 4 and 9 were observed to attenuate Notch1 and Notch2 surface levels when over-expressed in M1 cells. We used CRISPR-Cas9 technology to knock out endogenous MARCH2 and MARCH9 in M1 cells and observed a subtle difference in the surface levels of Notch1 and Notch2 compared to unedited M1 cells under steady-state conditions.

Overall, these preliminary findings suggest that specific MARCH proteins are able to regulate the surface levels of Notch1 and Notch2, thus implicating this family of E3 ligases as potential regulators of Notch signalling.

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