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Proteolytic regulation of the 14.3.3. proteins in cell function

Principal Supervisor: Prof Ed Rainger - Institute of Cardiovascular Sciences

Co-supervisor: Dr Alex Conner

PhD project title: Proteolytic regulation of the 14.3.3. proteins in cell function

University of Registration: Birmingham

Project outline:

The behaviour of distinct cell lineages during homeostatic function of organs is regulated by intercellular communications that regulate coordinated and complementary responses within the tissue. Such signals can be of many different types including lipids, proteins and metabolites. Recently we published (in Nature Medicine1) the identity of a novel pathway that regulates the migration of immune cells during the homeostatic function of this cellular system. Migration is regulated by a novel mediator, PEPtide Inhibitor of Trans-Endothelial Migration (PEPITEM) which is proteolytic cleavage product of the 14.3.3. protein, and is released into the extracellular environment at sites where regulation of migration is required. PEPITEM is not a member of, nor does it have sequence homology to, any known family of cell-regulatory molecules, and thus represents a new signalling paradigm in cell biology. Interestingly, there are 7 members of the 14.3.3 family in vertebrates and to date all of their described functions are within the cytosol of cells. However, although only 14.3.3. possess the core PEPITEM motif, other 14.3.3 proteins vary at only 1 or 2 amino acids. Moreover, all of the 14.3.3 proteins possess predicted protease excision sequences flanking the PEPITEM-like motif. Do these represent a wider family of biologically active peptides? If so, are they functional inside the cell or do they have extracellular signalling functions, thereby representing a new regulatory paradigm for the diverse functions of 14.3.3 proteins systemically? You will have the chance to answer these questions by conducting the first studies in this field and thereby establishing the founding models for a new paradigm of 14.3.3 protein function.

As we have done for PEPITEM, we will explore the derivation of these peptides from the parent proteins. Protease cleavage sites and the putative identity of the proteases driving peptide excision will be modelled in silico using available software resources and a list of target proteases will be generated for inhibitor studies. In collaboration with Dr Alex Conner, we will make protease resistant mutants of the 14.3.3 proteins as well as transient transfection constructs of the peptides for over expression studies. In alliance with siRNA knockdown strategies we will be able to investigate how dynamic regulation of proteolytic cleavage and peptide generation affects downstream cellular function. The roles of 14.3.3 proteins are diverse, but migration and integrin function, apoptosis, phagocytosis and growth of cells are published functions in which 14.3.3 proteins are known to contribute. As necessary and relevant there is the possibility to diversify these studies into animal models to ascertain how changes in 14.3.3 function have impact at a systemic level.

References:

  1. Chimen M, McGettrick HM, Apta B, Kuravi SJ, Yates CM, Kennedy A, Odedra A, Alassiri M, Harrison M, Martin A, Barone F, Nayar S, Hitchcock JR, Cunningham AF, Raza K, Filer A, Copland DA, Dick AD, Robinson J, Kalia N, Walker LSK, Buckley CD, Nash GB, Narendran P, Rainger GE. Homeostatic regulation of T cell trafficking by a B cell–derived peptide is impaired in autoimmune and chronic inflammatory disease. Nature Medicine. 21; 467–475 [10.1038/nm.3842].

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

  • In silico modelling of protease excision sequences in 14.3.3 proteins
  • Site mutagenesis of 14.3.3 protein sequences to make protease resistant constructs
  • Transfection of mutant and native 14.3.3 proteins for overexpression and loss of function studies
  • siRNA knockdown of 14.3.3. genes
  • Flow cytometry
  • qRTPCR
  • Western blotting
  • Leukocyte adhesion assays (flow or static based as appropriate)
  • Apoptosis assays
  • Phagocytosis assays
  • In vivo analysis of leukocyte trafficking (Peritonitis models or intravital microscopy of the microcirculation

Contact: Professor Ed Rainger, University of Birmingham