![]() ![]() These results revealed an unexpected role for the Ca 2+-dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain.Ĭytotoxic T lymphocytes and natural killer cells recognise and destroy virus-infected and virus-transformed cancerous cells through the exquisitely regulated secretory granule exocytosis pathway. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin’s cytotoxic function. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. ![]() On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca 2+ binding loop. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca 2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. However, the regulation of perforin proteostasis remains unexplored. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. doi:10.The pore forming, Ca 2+-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. In: Schwab M (ed) Encyclopedia of Cancer, 3rd edn. Scott JD, Pawson T (2009) Cell signaling in space and time: where proteins come together and when they’re apart. Polo SE, Jackson SP (2011) Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Good MC, Zalatan JG, Lim WA (2011) Scaffold proteins: hubs for controlling the flow of cellular information. Giglia-Mari G, Zotter A, Vermeulen W (2011) DNA damage response. This process is experimental and the keywords may be updated as the learning algorithm improves.īrown MD, Sacks DB (2009) Protein scaffolds in MAP kinase signaling. These keywords were added by machine and not by the authors. The organization of signaling complexes on scaffold proteins is dynamic, and there is an order of assembly that may vary. Thus, there is a cellular need to organize pathways both in time and space, a role that is achieved by scaffold proteins. These molecules need to be organized into complexes that will affect timing, location, and even specificity and magnitude of a single response, such as to growth factors or protection against DNA damage, for example. Thus, pathway components need to be partially preorganized so that signal transduction is not limited by the diffusion kinetics of individual molecules. ![]() An individual cell has over one billion proteins, of which ten percent form part of signaling pathways and networks, and none of them is exclusive for a single pathway.
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