Collett, Georgina Doreen May; (1999) Analysis of the distribution and molecular interactions of the novel protein muskelin. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Muskelin is a novel intracellular protein involved in cell responses to the adhesion modulating matrix component, Thrombospondin-1 (TSP-1). The muskelin polypeptide sequence contains six motifs with homology to the tandem kelch motifs, initially identified in the Drosophila Kelch protein, a component of ring canals. The focus of the thesis project has been to identify the molecular interactions of muskelin by use of a number of approaches including the yeast dihybrid system, copprecipitation, pharmacological agents and gel overlays. The two hybrid approach did not prove to be appropriate, because expression of muskelin had toxic effects on yeast cells. Treatment of cell extracts with a panel of pharmacological agents, to target components of cell signalling pathways, had no effect on the distribution of muskelin between detergent soluble (cytosol and membranes) and insoluble (cytoskeleton) fractions. Using a panel of cell lines including skeletal myoblasts, a set of proteins migrating with apparent molecular weights between 45-57 kDa were specifically detected upon muskelin overlays of SDS-polyacrylamide gels. These proteins did not correspond to the most abundant proteins within cell extract. Interaction of muskelin with these proteins was detectable throughout myoblast fusion into myotubes. Fascin, tubulin and actin were investigated as potential candidates for the proteins detected by muskelin gel overlay. A combination of muskelin and fascin overlays of cell extracts demonstrated that muskelin did not directly bind fascin. Muskelin did not interact with purified tubulin and actin in gel overlay assays and cosedimentation studies revealed that muskelin does not interact direct with microtubules or microfilaments. The most prominant interaction of muskelin in a gel overlay was with a 45 kDa protein of pI 6.0. This interaction was enhanced during myoblast fusion. Apparent postranslational modification of the 45 kDa protein was detected by muskelin overlay of extracts resolved on 2-Dimensional IEF/SDS-PAGE gels. A two step approach was developed to isolate the 45 kDa protein by using a strong anion exchange column to enrich for the 45 kDa protein, followed by 2D gel anaKsis. Identifying muskelin's binding partners will enhance understanding of its functional role.

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