Mapping the Dysferlin Structure and Interactome
The molecular basis for the pathology of dysferlinopathies - why loss of the normal Dysferlin protein leads to the disease phenotype - remains incompletely understood. It is known that Dysferlin appears to be recruited to cell membranes to organize repair there, because muscle tissue undergoes constant and extreme mechanical wear and tear. However, crucially, the mechanism of this repair and the identification of all the players involved remains to be elucidated, as does determining how they function and how they interact with each other and different regions of dysferlin to initiate and complete the repair process. These dysferlin-interacting proteins also represent potentially important therapeutic targets, provided that we can identify their sites of interaction on dysferlin (and for this, we need a structural map of dysferlin itself), and in turn identify their proximal interactors, collectively making up the vicinal interactome of dysferlin.
Our group has developed technologies that can purify and preserve, with high fidelity, various defined forms of the hierarchical arrangement of interactors surrounding any chosen protein, in defined complexes isolated natively from living cells. We will apply these technologies to mapping the structure of dysferlin and its complexes from human myotube cells – a tissue culture model for living muscle. We will use chemical crosslinking with mass spectrometric readout to provide distance restraints for accurately modeling the structure of dysferlin. We will also isolate a series of dysferlin complexes that range from proximally interacting “core complexes” to protein assemblies that include increasingly distal components to ultimately define the vicinal interactome. We will use a newly developed technology that we term ProxMap on this series of protein complexes to determine the proximity and interaction architecture of both proximal and distal players, thereby providing a comprehensive picture of how dysferlin is structured and how it and its partners form the key membrane repair complexes in human muscles.