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The role of PML Nuclear Domains in the Cellular Response to stress: a nuclear apoptosome?

Reference	BB/C509823/1
Principal Investigator / Supervisor	Professor Andrew Wyllie
Co-Investigators / Co-Supervisors	Dr Tina Rich
Institution	University of Cambridge
Department	Pathology
Funding type	Research
Value (£)	207,197
Status	Completed
Type	Research Grant
Start date	01/04/2005
End date	31/03/2008
Duration	36 months

Abstract

We wish to examine the proteome of a subnuclear organelle that integrates multiple cell fate stimuli. This organelle, the PML nuclear domain, is a matrix-attached structure, up to 0.8 microns in diameter. PML-NDs may be viewed as a shell that hosts the activities of fifty or more nucleoproteins. These proteins traffic to and from PML-NDs according to the demands placed on their activities by the cell. Some proteins are brought together in PML-NDs in order to catalyse their activities, whereas others are exported from PML-NDs to their site of action. Examples of the former include P53 and Crebs binding protein. The latter scenario would apply to DNA repair proteins such as chk2, that engage in phospho-transfer reactions with PML in order to transmit the genotoxic damage signal. To sample proteomes of relevance to apoptosis we have developed a methodology whereby we can inducibly over-express cMyc to render cells more susceptible to apoptosis. This is achieved using tamoxifen induction of a mutant oestrogen receptor cMyc fusion (Littlewood et al., 1995). The technical difficulties in extracting intact PML-NDs are considerable, not least because PML-NDs are matrix attached, largely insoluble and labile in terms of the release of their constituent proteins. To address these issues we have adapted a protocol designed to purify functionally active nuclei from cells, based on the cell-free replication assay (Stoeber et al., 98). These nuclei are then sonicated according to protocols developed in the Lamond laboratory for the purification of Cajal bodies (Lam et al., 2002). We will minimise the stripping of SUMO-1 from PML-NDs by the inclusion of inhibitors of SENP-1. Western blotting of fractions eluted by gel filtration media-Sephacryl-300 (res-10,000-1.5MDa) show that PMLpositive SUMO-1positive complexes elute in the void volume. Thus the PML containing complexes are larger than 1.5Mda. This is not altogether unexpected as the BASC complex, fractionated by a Superose 6 gel-filtration column, also eluted in the void volume (Want et al., 2000). We are repeating these analyses using Sephacryl-400), which has a size exclusion limit of about 8MD. We shall also use a cross-linking approach, using photoactivatable reagents that can be coupled to proteins that localise to PML-NDs. Two cleavable crosslinkers that are activated by UV light will be used. These are aryl azide N-((2-pyridyldithio)ethyl)-4-azidosalicylamide (PEAS)-Molecular probes) and SAED (Sulfosuccinimidyl- 2-[7-amino-4-methylcoumarin-3-acetamido]ethyl-1,3 prime dithiopropionate) from Pierce.. The chariot reagent will then be used to convey the modified protein into the nucleus. Normal protein targeting then resumes. The bait, which targets to PML-NDs, can be coupled to target proteins by the delivery of UV irradiation. The proteome profile of PML-ND cargo proteins will be resolved by LC-MS/MS in a collaboration established with Dr Kathryn Lilley from the Cambridge Centre for Proteomics.

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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