The role of MARCH1 in the ubiquitination and regulation of surface immunoreceptors

We are trying to find ways in which cells in our body hide from our immune system, which may allow them to develop into cancers.

To prevent a cell becoming cancerous and allow normal cell growth, all cells have receptors at their surface. These receptors need to be tightly controlled. The addition of small tags called ubiquitin onto these receptors causes their removal.

Herpesviruses have devised ingenious ways of adding ubiquitin tags to important cell surface receptors, leading to the receptors’ removal. Viruses are cunning and stole this idea from our own cells. This led to the discovery of a new family of genes –called the MARCH family. We found that MARCHs play an important role in regulating receptors of our immune system.

Our preliminary evidence suggests that MARCH proteins control immune cell activity to ensure that cells are neither over- nor under-active. We will use modern molecular techniques to find out how MARCH proteins control cell surface receptors.

This will be important for learning about (i) how immune cells work (ii) whether MARCH proteins will be good drug targets to prevent immune-mediated disease and (iii) whether they allow cells to escape the immune system and develop into cancers.

The regulation of cell surface receptors is essential for the maintenance of cell homeostasis. At the plasma membrane, ubiquitination is an important post-translational mechanism for controlling expression of many critical proteins including immunoreceptors, and targets receptors for degradation by proteasomal or lysosomal pathways. The critical ubiquitin enzyme is the E3 ligase which binds the substrate and therefore confers specificity to the reaction.

Our studies on viral immunoevasion of MHC class I identified the K3 family of viral membrane-associated ubiquitin E3 ligases which downregulate surface MHC class I and other critical immunoreceptors. These viral E3 ligases have been appropriated from the host genome and ten mammalian orthologues of the K3 viral family identified - the MARCH genes. Although their physiological function remains unclear, MARCH proteins are ubiquitin E3 ligases and downregulate cell surface receptors. A MARCH7 deficient mouse shows uncontrolled T cell proliferation and develops autoimmunity.

MARCH1 is an ubiquitin E3 ligase whose expression is tightly restricted to B cells. MARCH1 deficient mice are predicted to be unable to downregulate some B cell receptors and express increased surface levels of MARCH1 substrates. The aims of this project are (i) the generation of genetically modified MARCH1 mice, to allow (ii) the identification of MARCH1 substrate(s) and (iii) characterisation of the phenotype and physiological role of MARCH1 in the immune system. These targets will be identified initially using a candidate gene approach to compare expression of B cell receptors from MARCH1 deficient and wildtype cells by flow cytometry. For a more objective analysis we will collaborate with Dr Kathryn Lilley in the Cambridge Centre for Proteomics and profile B cell plasma membrane proteins to compare receptor expression in MARCH1 deficient and wildtype B cells using ITRAQ labelling and mass spectrometry.

Following identification of MARCH1 targets, the impact of impaired receptor regulation on B cell function will be determined in collaboration with Dr Ken Smith, a B cell immunologist in CIMR. The biochemical activity and mechanism of action of MARCH1 will be characterised and the impact of MARCH1 mutants tested in vivo and in vitro.

The work proposed in this project will identify the targets and physiological role of MARCH1, a ubiquitin E3 ligase we believe plays an important role in B cell regulation. It will provide insight into the role of ubiquitin in regulating B cell receptors and the importance of the MARCH family in immune cell function.