A biochemical and molecular analysis of functional differences between dystrophin and utrophin

The DMD gene encodes the protein dystrophin, a 427kD cytoplasmic protein responsible for linking the actin cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex. Mutations in dystrophin that abolish its expression lead to Duchenne muscular dystrophy (DMD). Patients with DMD become wheelchair bound in their early teens and succumb to fatal cardiac and/or respiratory failure in their mid-twenties to early thirties. There is currently no effective or widely available treatment for DMD beyond ventilatory support and the use of corticosteroids. Many therapies for treating dystrophin deficiency aim at upregulating its autosomal homolog utrophin due to its structural similarity and ability to bind an almost identical repertoire of proteins that dystrophin binds. It was previously shown that utrophin cannot bind neuronal nitric oxide synthase (nNOS) even though dystrophin binds nNOS, establishing for the first time a functional difference between dystrophin and utrophin. Here, we show that transgenic overexpression of utrophin on the mdx mouse background (Fiona-mdx) is not sufficient to rescue the disorganized microtubule network of the mdx mouse. Thus, we have elucidated a second functional difference between dystrophin and utrophin. Additionally, Fiona-mdx mice lack full recovery of cage activity after mild exercise. Our results suggest that any deficiency in nNOS binding or microtubule lattice function caused by loss of dystrophin may not be restored by upregulation of utrophin. Previously, our lab reported that dystrophin directly binds to microtubules and organizes them beneath the sarcolemma. Using in vitro microtubule cosedimentation assays, we show that dystrophin binds to microtubules with strong affinity (KD=0.33µM). Through the use of various recombinant constructs tested via in vitro microtubule cosedimentation we show that spectrin-like repeats 20-22 of the dystrophin central rod are responsible for microtubule binding activity. However, we show that these repeats require flanking regions of dystrophin for proper binding activity, making microtubule binding context-dependent. Additionally, we show that recombinant utrophin does not bind microtubules in vitro, corroborating our in vivo findings of the disorganized subsarcolemmal microtubule lattice of the Fiona-mdx mouse. We also provide evidence showing that dystrophin functions as a molecular guidepost to organize microtubules into a rectilinear lattice.

Author dc:creator

• Belanto, Joseph John

• DMD
• Duchenne muscular dystrophy
• Dystrophin
• mdx
• Microtubules
• Utrophin
• Molecular, cellular, developmental biology and genetics

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/11299/170101

OAI identifier oai:identifier

oai:conservancy.umn.edu:11299/170101