Dystrophin

Introduction

Dystrophin is a 427 kDa protein with 4 major domains involved in the dystrophin-associated glycoprotein complex which lies between the sarcolemma and myofilaments in muscle fibers. By action of linking various support proteins to actin filaments through it’s , dystrophin is classified as a cohesive protein. It connects the elements of the sarcomere to the sarcolemma. The sarcolemma, or the cell membrane of striated muscle fibers, is linked to these actin filaments through the dystrophin-associated glycoprotein complex, or also known as the costamere. ^[1] Skeletal muscle tissue contains a surprisingly small amount of dystrophin, about 0.002% of total muscle protein. However, the absence of this protein amounts to disruption of the stability of the myofibril as well as the linkage to nearby myofibrils, vastly reduces the stiffness of muscle cells, and compromises the mechanical stability of costameres. ^[1] Disruption or loss of dystrophin through mutations leads to muscular dystrophy, a condition in which the patient experiences progressive weakness and loss of muscle mass. The most common of the nine types of muscular dystrophy is Duchenne’s, where the patient completely lacks the protein. Duchenne’s is a more severe form of Becker’s dystrophy, where the patient only has a decreased number (or a weakening) of dystrophin.

Genetics and Expression of the Protein

Dystrophin is a member of the β-spectrin/α-actinin protein family and is expressed from one of the largest genes in the human genome, DMD (Duchenne Muscular Dystrophy Gene) spanning 2.3 megabases at locus Xp21 . The tissue distribution of the protein’s expression is indicated by the three separate promoters for dystrophin expression present in the brain, muscle, and purkinji’s, although the protein is most abundant in striated muscle fibers found in skeletal muscles and cardiac muscle ^[2]. Additionally, internal promoters that lie within the transcript allow for genesis of shortened expressions of COOH-terminal isoforms; these isoforms contain binding sites for association with multiple dystrophin-associated proteins (DAPs) ^[3]. These truncated forms expressed by alternative promoters can be used in non-muscle tissues with unique amino-terminus sites. ^[2].

Structure and Function

Dystrophin is a cytoplasmic protein that connects the inner cytoskeleton elements of a muscle fiber to the extracellular matrix (the sarcolemma) by means of binding to various other muscle proteins through the plasma membrane, known as the dystrophin-associated complex. Dystrophin, along with other integral and peripheral proteins such as sarcoglycan and dystroglycan, act to promote stability of the muscle cell and allow for force transduction during muscle contraction. Dystrophin specifically binds to F-actin on its N-terminus and it’s carboxy terminus anchors the muscle cell to the extracellular dystrophin-associated glycoprotein (DAG) complex, effectively stabilizing and linking muscle cells to the extracellular matrix ^[4]. This overlying structure is known as the costamere or the dystrophin-associated protein complex; this complex links the sarcomere of the muscle to the cell membrane. The DAG complex consists of sarcospan, dystrobrevins, syntrophin, sarcoglycans, and dystroglycans in addition to dystrophin. These proteins exist in 3 categories based on their location: the extracellular protein is α-dystroglycan; the transmembrane proteins consist of β-dystroglycan, sarcoglycans, and sarcospan; and cytoplasmic proteins consist of dystrophin, dystrobrevin, and syntrophin ^[5]. The interaction of caveolin-3 with β-dystroglycan has been hypothesized to competitively regulate the recruitment of dystrophin to the plasma membrane. Several studies show that dystrophin may also play a role in the stability, stiffness and organization of the sarcolemma, as well as protecting it from membrane stress suffered during muscle contraction. These cellular roles spawned it’s perceived function as a key mechanical scaffold of muscle cells; this role includes bulwarking against micro-tears and damages brought on by various forces including normal muscle contraction, as well as preventing non-specific ion (including calcium) and cellular content leakages ^[3]. Additionally, ZZ modules in the cysteine-rich domain of dystrophin have been hypothesized to contain calmodulin-binding domains, allowing regulation of the interactions with the other DAG complex elements directly with the use of calcium ions; current hypothesis involve conjecture that the DAP complex is involved with cellular signaling. The complex is hypothesized to anchor cellular signaling agents to the overall site. ^[3] Disruption of the DAGC possibly leads to not only the leakage of cellular contents or leakage of ions, but more critically the activation of calcium-dependent proteases (as well as overall disruption of calcium homeostasis), generating the progressive cellular necrosis seen in the pathology of Duchenne’s Muscular Dystrophy.

Dystrophin comprises 4 major domains. The crystal structure of the dystrophin actin-binding domain (ABD) has been determined at 2.6 A resolution. The structure is an antiparallel dimer of two ABDs each comprising two calponin homology domains (CH1 and CH2) that are linked by a central alpha helix located at the amino terminal. The CH domains are both alpha-helical globular folds ^[2]. The calponin homology (CH) domain is a protein module of about 100 residues that was first identified at the N-terminus of calponin, an actin-binding protein playing a major regulatory role in muscle contraction. The second and largest domain is composed of 24 triple helical spectrin-like repeats thought to majorly contribute dystrophin’s overall shape that resembles a stretched out and flexible rod. The third domain is cysteine-rich and encodes two EF hand-like modules bounded by which is a module known to mediate regulatory protein complexes (scene shown is the structure of the dystrophin WW domain fragment in complex with a beta-dystroglycan peptide) and ZZ (a zinc-finger and cysteine rich domain near the C-terminus involved in stabilizing the interaction between dystrophin and β-dystroglycan) modules ^{[2] [6]}. The fourth domain, the carboxy terminus is unique to dystrophin and contains two regions forming α-helical coiled coils forming the binding site for dystrobrevin ^[2].

Disease Pathology Associated with Dystrophin

Duchenne’s muscular dystrophy is an x-linked recessive disease characterized by a general loss of dystrophins throughout the patient’s body. The lack of this important multifaceted protein diminished the levels of DAPC associated elements including dystroglycans, sarcoglycans, integrins and caveolin, resulting in cellular content leakage, destabilization of the myofibrils, ion leakage, calcium-dependent protease overactivation, and a major loss of signaling elements, as well as other functions of this complicated structure and constituents. It is hypothesized that these alterations allow calcium ions to enter the mitochondria and cause it to burst. This in turn leads to amplification of stress-induced cytosolic calcium signals and in an amplification of stress-induced reactive oxygen species production ^[7]. This complicate cascade of events results ultimately in the death of the muscle cell and a replacement with adipose or connective tissue. Patient’s diagnosed with DMD experience muscle loss around age 4 and are generally wheelchair-bound by age 12 and pass away from respiratory failure in their early twenties. Becker muscular dystrophy, a milder form of the disease, is characterized by a reduction in the amount or the actual size of the dystrophin proteins, rather than widespread loss. Large insertions or deletions with consequent downstream frameshift errors account for approximately 60% of cases of DMD, and 40% involve point mutations or small rearrangements ^[8]. Dystrophin or dystrophin-associated glycoprotein elements may also undergo spontaneous mutations that lead to muscular degrative pathologies ^[9]. DMD is currently uncurable, but several treatments do exist including gene therapy, where either viruses or plasmids are used to deliver dystrophin sequences to the patient, myoblast transplantation, therapy involving stem-cells which have been shown to proliferate longer than myoblasts, and proteasome inhibitors. Advancements in studying the function of dystrophin and its interaction with other molecular subunits and signaling roles will aid in the search for more effective treatments for Duchenne’s muscular dystrophy ^[8].