Biochemical analysis of plectin isoforms and their molecular subdomains

Plectin is a widely expressed protein of very large size that has all the attributes of a multifunctional crosslinking and organizing element of the cytoskeleton. It displays a multi-domain structure, versatile binding activities, and subcellular localizations that enable it to strengthen cells against mechanical stress forces. Moreover, as revealed very recently, hereditary gene defects in plectin cause epidermolysis bullosa simplex (EBS-MD, a severe human skin blistering disease with muscular dystrophy; there is evidence that defects in plectin expression may also play a role in neurodegeneration. Recent unpublished studies indicate the existence and tissue-specific expression of various isoforms of plectin generated by differential splicing of the gene. The major objective of the proposed project is the characterization of plectin isoforms on the protein level, and the precision mapping and biochemical analysis of molecular binding domains involved in the interactions of plectin with different types of intermediate filaments (vimentin, cytokeratins, desmin), microtubules, actin filaments, and hemidesmosomes (integrin beta 4). In addition, it is planned to study the role of various carboxy-terminal phosphorylation sites in the regulation of plectin functions, including a unique site for M-phase specific p34cdc2 kinase. The experimental approach proposed is based primarily on the expression of defined mutant proteins (corresponding to identified contact sites/domains) in bacteria and structure/function analyses of such mutants, using a variety of proteinchemical and ultrastructural techniques. Protein isoforms of plectin will be purified from different cell lines and tissues, and analysed in a similar way. The biochemical analyses will be complemented with cell transfection studies using cDNA expression constructs encoding plectin mutant proteins. In addition, peptides corresponding to defined molecular contact sites will be synthesized and used for competitive in vitro binding assays, in vivo microinjection experiments, and antibody production. The proposed experimental approaches should enable a comprehensive analysis of the structure and function of plectin isoforms and their subdomains. Such an analysis is expected to provide new insights into molecular mechanisms governing the formation and maintenance of cellular cytoarchitecture and its defects and malformations due to pathological conditions.