The filamin A actin binding domain structure and function: implications for a gain-of-function mechanism for the otopalatodigital syndrome: a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand

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The filamin family act as scaffolding proteins associating with actin filmanents, acting through a highly conserved actin binding domain (ABD). The ABD of the filamins is homologous to that found in other F-actin binding proteins such as dystrophin. Mutations in the filamin A gene cause a wide range of disease symptoms in humans reflecting the diversity of the roles that filamin A has in cell structure and signalling pathways. The diseases fall into two separate phenotypic groups. Periventricular nodular heterotopia (PVNH) generally results from the complete loss of filamin A protein, and affects the central nervous system. The clinically separate otopalatodigital disorders (OPD) spectrum disorders are skeletal disorders and were hypothesised to be gain of function phenotype diseases. At the beginning of this work, there was very little structural data available for the human filamins, and none for the crucial highly conserved actin binding domain. This lack of structural data limited the interpretation of the biochemical and genetic data and constrained our understanding of the disease associated mutations that cluster in this domain. These studies aimed to provide insights into the structure and mechanism of actin binding domains, and thus provide a better understanding of the diseases caused when this domain is mutated. A secondary structural analysis and crystal structures of the wildtype and OPD2 associated mutant ABDs were obtained. The overall fold of the three proteins was equivalent as determined by circular dichroism spectroscopy and x-ray crystallography. The ABD from filamin A E254K showed 3.7 fold increased F-actin affinity, accompanied by a reduced thermostability (of 5.6 °C). Western blotting of OPD2, frontometaphyseal dysplasia (FMD) and PVNH patient fibroblast lysates showed similar levels of filamin A compared to the control cells. In addition the OPD and PVNH patient fibroblasts were able to adhere to fibronectin and migrate with an equivalent rate to control cells. Together these results have allowed correlations to be developed between structure, protein stability, actin affinity, cellular phenotype and the overall clinical phenotype. Showing that, at least in one example, OPD2 may be due to an increased actin affinity providing further evidence for a gain of function mechanism of OPD2.
Filamin A gene, Actin binding domain, Microfilament proteins, Otopalatodigital syndrome spectrum disorders, Periventricular nodular heterotopia, Biochemistry