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Regulated exocytosis in natural killer (NK) cells: a functional proteomics analysis

Reference	BB/C500201/1
Principal Investigator / Supervisor	Dr Eric Hewitt
Co-Investigators / Co-Supervisors
Institution	University of Leeds
Department	Inst of Molecular & Cellular Biology
Funding type	Research
Value (£)	223,640
Status	Completed
Type	Research Grant
Start date	20/01/2005
End date	19/01/2008
Duration	36 months

Abstract

This proposal will use a functional proteomics approach to identify secretory lysosome membrane proteins that are required for exocytosis of this organelle in natural killer (NK) cells. NK cells and cytotoxic T lymphocytes (CTLs) are specialised lymphocytes that play important roles in immunity to viruses and tumours. Although their recognition of infected and cancerous cells differs, both NK and CTLs kill target cells by the regulated exocytosis of pre-stored cytotoxic effector molecules from secretory lysosomes. Yet, despite the importance of regulated exocytosis of secretory lysosomes by NK cells and CTLs, little is known about how this organelle releases its contents. Recent advances in proteomic technologies offer an excellent opportunity to identify the secretory lysosome exocytic machinery. In this study secretory lysosomes will be isolated from NK cells by subcellular fractionation on centrifugal gradients and the membrane proteins identified using mass spectrometry. Those proteins that either share sequence identity with proteins known to regulate membrane traffic in other systems or whose sequences are novel will be subject to further characterisation. The localisation of these proteins to secretory lysosomes will be confirmed using immunofluorescence microscopy. Finally, the role of secretory lysosome membrane proteins in regulated exocytosis will be examined. Protein expression will be depleted using RNA interference and the effect upon the release of cytotoxic effector molecules measured.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Proteomics and Cell Function (PCF) [2003-2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

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