1. Dystrophin and the Extracellular Matrix
4/25/2017
Dystrophin and the Extracellular Matrix
Protein domain structure
Dystrophin glycoprotein complex
Contraction unit
Cytoskeletal linkages at sarcolemma
Muscle proteins and disease
Creatine kinase and CKI
In situ staining and Western blots
Eric Niederhoffer
SIU-SOM

2. Dystrophin Domain Structure
4/25/2017
Dystrophin Domain Structure
Domains of the dystrophin molecule. The N terminus contains the primary actin-binding site, whereas the C terminus contains the -dystroglycan, dystrobrevin, and syntrophin-binding sites. The N- and C-terminal domains are connected by 24 spectrin-like repeats, some of which have been shown to bind actin. The four "hinge" regions are denoted H1-H4.
Dystrophin gene is 2.5 Mbp
Dystrophin protein is 427 kDa (thought to be a dimer), phosphorylation sites in C-terminus
Recall that DNA has both a noncoding and template strand. During transcription the template strand is read 3’ to 5’ to produce mRNA that runs 5’ to 3’. The mRNA is translated to produce polypeptide chain in the N- to C-terminal direction.
Proposed signal transduction related phosphorylation sites are located in the C-terminal region.

3. Dystrophin Glycoprotein Complex
4/25/2017
Dystrophin Glycoprotein Complex
Schematic representation of the dystrophin-associated glycoprotein complex. The N-terminal, actin-binding domain of dystrophin in purple is associated with the cortical actin. The C-terminal domain associates with b-dystroglycan and with a- and b-syntrophin and dystrobrevin. nNOS is known to interact with the syntrophins as well as with caveolin.
Dysregulation of myospryn (large, muscle-specific protein kinase A anchoring protein) in DMD; binds dysbindin

4. 4/25/2017
Contraction Units
Costamer is an organizational unit (plaque) of the membrane, transduces contractile force from Z line to basement membrane.
Titin is 3 MDa protein
Abundance - actin > myosin > titin
Filaments - thin (actin), thick (myosin), titin, nebulin

5. Cytoskeletal Linkages at Sarcolemma
4/25/2017
Cytoskeletal Linkages at Sarcolemma
MLP: muscle LIM protein
Dystrophin serves as a bridge between the Z line and the basement membrane. Dystrophin serves as a key organizational element of the dystroglycan complex. Dystrophin serves as a mediator of signal activities within the cell. One might predict that some combination of these three features will become important in a unifying mechanism. Dystrophin has domains which bind actin, -dystroglycan, dystrobrevin, and syntrophins. The dystroglycan complex is part of the organization of the basement membrane, which includes other proteins including ion channels (Ca2+ for example) and pores (water for example). Dystrophin has domains that feature signal transduction elements, suggesting that key cellular activities may be influenced by dystrophin. These activities include but are not limited to second messenger systems, metabolic control, nuclear signaling, and gene expression.

6. Muscle Proteins and Diseases
4/25/2017
Muscle Proteins and Diseases
CMD: congenital muscular dystrophy CMT: Charcot-Marie Tooth
DCM: dilated cardiomyopathy AD-EDMD: autosomal dominant Emery-Dreifoss muscular dystrophy
FPLD: familial partial lipodysrtophy LGMD: limb girdle muscular dystrophy

7. Creatine Kinase and CKI
4/25/2017
Creatine Kinase and CKI
Cell damage
contents spill out
detected in blood
CKtotal U/L CKMB 0-5 ng/mL
Muscle damage CKtotal elevated
CK index (CKI) = CKMB/CKtotal (x 100 for %)
Skeletal origin CKI <0.03 (3%) or use CKMM
Cardiac origin CKI >0.06 (6%)
If 0.03 (3%) > CKI <0.05 (5%) follow with Tn biomarkers
CK: creatine (phospho)kinase Tn: troponin
Mosby’s normal range: M, U/L; F, U/L
Isozymes CK-BB (CK1, brain/lung); CK-MB (CK2, heart), CK-MM (CK3, skeletal muscle)
Isozymes also have isoforms (CK-MM; MM1, MM2, MM3)

8. 4/25/2017
In Situ Dystrophin
Normal dystrophin staining around the rim of muscle fibers
Absent dystrophin: Duchenne muscular dystrophy
Left: No staining around the rim of muscle fibers.
Right: No staining of most muscle fibers. One "revertant" fiber with dystrophin staining.

9. Western Blots Western blot of dystrophin from dystrophinopathies.
4/25/2017
Western Blots
Western blot of dystrophin from dystrophinopathies.
Lane 3: Normal; Dystrophin has normal size and amount.
Lane 1: Becker dystrophy; Dystrophin has reduced abundance but normal size.
Lane 2: Becker dystrophy; Dystrophin has reduced size and abundance.
Lane 4: Duchenne dystrophy; Almost no protein is present.
Lane 5: Duchenne outlier; Dystrophin has severely reduced abundance.

10. Review Questions What is dystrophin (domains, characteristics)?
4/25/2017
Review Questions
What is dystrophin (domains, characteristics)?
Where is dystrophin found (interactions, role)?
How do you assess muscle damage?
How do you assess dystrophin levels?