Molecular-Functional Comparison of the Dysferlin Membrane Repair Complex with the Otoferlin Synaptic Complex
Genetically-defective dysferlin causes LGMD2B muscular dystrophy and mutated otoferlin causes DFNB9 deafness. Both proteins normally function by forming complexes with other proteins that either repair muscle (dysferlin) or promote hearing through sound-signal transmission (otoferlin). We are investigating the molecular role of dysferlin in muscle, guided by known ferlin interactions in inner ear hair cells, in an examination of the generalized molecular properties of dysferlin across tissues. By using biochemical and molecular-biological binding methods, we are detecting the strongest protein-protein interactions and thus identifying key protein players likely to be part of the dysferlin (and otoferlin) protein complexes responsible for membrane repair and fusion, as well as for muscular apoptotic processes which can result in fat replacement. We are further assessing binding properties - such as interaction with double-stranded RNA - for particular regions of dysferlin, providing information on the structure and function of the protein. Understanding these protein complexes through a comparative approach guides therapeutic intervention.
Summary of Findings:
As a critical component of a membrane repair complex, dysferlin is hypothesized to direct sealing of membrane defects in muscles, with dysferlin mutation and consequent dysfunction giving rise to limb girdle muscular dystrophy 2B. We analyzed presumptive protein candidates which together would form a repair complex with dysferlin, each by direct binding to specific motifs of the dysferlin molecule. On the basis of conserved ferlin homology between otoferlin and dysferlin, we identified new dysferlin interacting proteins: FKBP8, an anti-apoptotic outer mitochondrial membrane protein and ALG-2 (PDCD6), a cell-death/apoptotic protein. Dysferlin and annexin A2 appear to compete for binding to ALG-2. Interaction between individual proteins was quantitatively studied by surface plasmon resonance analysis as a function of calcium concentration, a correlate of membrane repair. Dysferlin-interacting proteins were immunolocalized in skeletal muscle with the avidin–biotin–complex peroxidase method and co-localized with confocal Z-stack immunofluorescence microscopy. Our evidence supports the hypothesis that prior to injury, at low calcium, dysferlin C2 domains self-interact, giving rise to a folded, compact structure as indicated for otoferlin. With elevation of intracellular calcium in injury, dysferlin unfolds, exposing the canonical C2A domain to interact with repair proteins, and in particular, realign from its interactions with PDCD6 and annexin A2 at basal calcium levels to strongly interact with FKBP8, merging mitochondrial and sarcolemmal sites.