Awarded to Professor Glenn Morris, Oswestry Hospital, May 2007.
Abstract
In SMA, reduction of SMN protein levels results in a specific loss of function leading to motor neuron degeneration. Understanding SMN's direct and indirect interactions with other proteins is the key to understanding its various functions and mass spectrometry (MS) is recognized as a major approach to identifying novel interactions without bias or preconception. This proposal arises because the RJAH research group has unusually good access to up-to-date MS and proteomics facilities. A member of our research group, Dr. Heidi Fuller, will run the Proteomics facility for Keele University based at RJAH and will contribute substantially to the present project. The Group also has produced a wide range of antibodies against SMN and gemins, available in unlimited quantity and these will be used for the purification of protein complexes for MS analysis by immunoaffinity capture onto magnetic beads. The most important factor for success is to characterize the capture mAb carefully for high specificity and efficiency in IP experiments.
The questions we propose to answer are:
1) Does the composition of SMN complexes vary between (a) different subcellular regions (cytoplasm, nucleoplasm, nucleoli, gems), (b) different cells and tissues (HeLa, NT2, PC12 nucleus and dendrites, skin fibroblasts, myoblasts and adult muscle)? Do any compositional variations cause functional variations? Does the composition of the SMN "interactome" change significantly when nucleic acids are removed by nuclease treatments? Are there any variations in posttranslational modification (such as phosphorylation) related to specific functions?
2) Using recently-developed techniques for quantitative MS, what quantitative changes in protein expression occur when SMN is reduced in SMA type I fibroblasts? How many of these changes are reversed by drugs suggested for SMA treatment, such as valproate, phenylbutyrate and indoprofen? How many additional, possibly undesirable, changes are produced by drug treatments? Is there an alternative biomarker to SMN itself that can be assayed more easily under drug testing conditions?
These studies are expected to contribute to our understanding of SMN complex function by identifying potential signalling pathways and downstream effectors and to provide further information of the mechanism of action of HDAC inhibitors. They may improve assay methods for effectiveness of drug treatments. They also focus on the anticipated differences between SMN function in nucleocytoplasmic snRNP transport and processing and its function in axonal/dendritic cytoplasm of neuronal cells. | 
