Mapping the Dysferlin Structure and Interactome

Brian Chait, D.Phil and Michael Rout, PhD

Rockefeller University

Brian Chait, D.Phil., is the Camille and Henry Dreyfus Professor at the Rockefeller University in New York and head of the Laboratory for Mass Spectrometry and Gaseous Ion Chemistry. He specializes in the development and use of mass spectrometry as a tool for investigating a variety of biological and biochemical phenomena. Michael Rout, Ph.D., is also a Professor at the Rockefeller University and head of the Laboratory of Cellular and Structural Biology. He uses biochemical, biophysical, and structural approaches to characterize macromolecular assemblies, with an emphasis on the nuclear pore complex, a key part of the pathway that relays information between the nucleus and cytoplasm. His goal is to further develop proteomic technologies that will enable the community to assemble detailed, dynamic representations of the interactions in the cell.

Research Projects

Objective: 
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.