Dystrophin is a high molecular weight cytoskeletal protein that localizes to the cytoplasmic face of the sarcolemma. It has four domains - an actin binding domain, a central rod domain composed of spectrin-like repeats, a cysteine-rich domain, and a carboxy-terminal domain. Dystrophin forms the dystrophin-glycoprotein complex with other proteins like dystroglycans and sarcoglycans to connect the actin cytoskeleton to the extracellular matrix. Mutations in dystrophin cause Duchenne/Becker muscular dystrophy by disrupting this connection and leading to muscle degeneration.

1. Protein And Protein
Dystrophin
Hari Sharan Makaju
M.Sc. Clinical Biochemistry
1St Year
2076/3/30

2. Outline
 Protein
Introduction
Functions
Structures
 Dystrophin
Introduction
Components
Functions
Problems

3. Protein
Introduction :
 Greek word “Proteios” which means primitive or Primary
 The most abundant biological macromolecules
 Occurring in all cells and all parts of cells.
 Proteins also occur in great variety;
 Ranging in size from relatively small peptides to huge
polymers with molecular weights in the millions, may
be found in a single cell.
 Proteins are the molecular instruments through which
genetic information is expressed

4. Protein
Define:
 Proteins are the polymer of L-α- amino acid held
together by peptide bond.
In general, the term protein is used for molecules
composed of over 50 amino acids.
 Protein contains Carbon, Hydrogen, Oxygen, and
nitrogen as the major components while Sulphar
and Phosphorous are minor constituents
Structure and functional unit of cells.

5. Peptide bond in protein
• Partial double bond
character
• Rigid and planar
• Uncharged but polar

6. Protein –Functions
 Proteins exhibit enormous diversity of biological function
 Proteins function as:
Enzymes: biological catalysts
Regulators of catalysis: hormones
Transport and store i.e. O2, metal ions sugars, lipids,
etc.
Contractile assemblies:
Muscle fibers
Sensory:
Rhodopsin nerve proteins

7. Protein –Functions
 Cellular defense
Immunoglobulins
Antibodies
 Structural
Collagen
Dystrophin (intracellular)
Silk, etc.
 Function is dictated by protein structure!!

8. Protein Structure

9. Primary Structure
 Primary structure of a protein refers to the covalent
structure of a protein .
 It includes amino acid sequence and location of
disulfide (cystine) bonds
 The most important element of primary structure is
the sequence of amino acid residues.
 Primary structure of proteins is important because:
 Many genetic diseases result in proteins with
abnormal amino acid sequences, which cause
improper folding and loss or impairment of normal
function.
 Example of Primary Structure: Insulin

10. Secondary Structure
 The conformation of polypeptide chain by twisting or folding.
 Generally stabilized by repeating pattern of hydrogen bonds.
 Rigidity of peptide bond determine the types of secondary
structure.
 Types of Secondary structures:
α-helix
β-sheet
β-bend (β-turn)
 Free rotation is possible about only two of the three covalent
bonds of the polypeptide backbone:
 the α-carbon (Cα) to the carbonyl carbon (Co) bond
 the Cα to nitrogen bond

11.  The Cα-N bond and Co-Cα bond can rotate, with bond angles
designated phi (Φ) angle and psi (Ψ), respectively.
 The peptide C-N bond is not free to rotate

12. Secondary Structure

13. α-helix
 First proposed by Linus Pauling and Robert Corey in 1951
 The polypeptide backbone of an α helix is twisted by an equal
amount about each α-carbon with a phi angle of approximately
−57 degrees and a psi angle of approximately − 47 degrees.
 A complete turn of the helix contains an average of 3.6
aminoacyl residues, and the distance it rises per turn (its pitch)
is 0.54 nm
 The stability of an α helix arises primarily from hydrogen bonds
formed between the oxygen of the peptide bond carbonyl and the
hydrogen atom of the peptide bond nitrogen of the fourth residue
down the polypeptide chain

14. α-helix

15. β-sheet
 Also first postulated by Pauling and Corey, 1951
 The polypeptide chain are nearly completely
extended and hydrogen bond are at the right angle to
the long axis of the polypeptide chain.
 Strands may be parallel or antiparallel
 phi = -119degrees, psi = +113degrees for parallel
Strands
 phi = -139degrees, psi = +135degrees for anti-parallel
strands

16. Parallel β-sheet
 The β-pleated sheet is described as parallel if the
polypeptide strands run in the same direction (as
defined by their amino and carboxy terminals.)
 Parallel sheets tend to have hydrophobic residues
on both sides of the sheets

18. Anti-Parallel β-sheet
 The β-pleated sheet is described as anti-parallel if the
polypeptide strands run in opposite directions.
 Antiparallel strands are often the same polypeptide chain
folded back on itself, with simple hairpin turns or long runs
of polypeptide chain connecting the strands.
 Antiparallel sheets usually have a hydrophobic side and a
hydrophilic side

20. β-bend (β-turn)
 β-Bends reverse the direction of a polypeptide chain, helping it
form a compact, globular shape.
 Found on the surface of protein molecules, and often include
charged residues.
 β-Bends are generally composed of four amino acids
Proline and Glycine are prersent in β-bends.
 Stabilized by the formation of hydrogen and ionic bonds.

21. β-turn

22. TERTIARY STRUCTURE
 The three dimensional arrangement of protein structure
is referred as tertiary structure.
 Hydrophobic side chains are buried in the interior,
whereas hydrophilic groups are generally found on the
surface of the molecule.
 The polypeptide chain with its regions of secondary
structure, α-Helix and β-Sheet further folds to achieve
the tertiary structures
 The tertiary structure of a globular protein is made up
of structural domains

23. TERTIARY STRUCTURE
 Domains are the fundamental functional and three-
dimensional structural units of a polypeptide.
 The core of a domain is built from combinations of super-
secondary structural elements (motifs).
 Folding of the peptide chain within a domain usually
occurs independently of folding in other domains
An example of an all a-domain globin fold in the
enzyme lysozyme

24. Many proteins are composed of separate functional domains e.g.
bacterial catabolite protein (CAP). Protein domain: a segment (100 –
250 aa) of a polypeptide chain that fold independently into a stable
structure

25. TERTIARY STRUCTURE
 These higher levels of structure, classify proteins into two
major groups:
1.Fibrous proteins
 having polypeptide chains arranged in long strands or sheets.
 that provide support, shape, and external protection
 Examples:- α-Keratin, collagen, dystrophin and silk fibroin
2. Globular proteins,
 Having polypeptide chains folded into a spherical or globular
shape.
 most enzymes motor protein, immunoglobulin and regulatory
proteins are globular proteins
 Examples: Myoglobin, cytochrome c, lysozyme, and ribonuclease a

26. TERTIARY STRUCTURE
Collagen

27. TERTIARY STRUCTURE
 Interactions stabilizing tertiary structure
 Four types of interactions cooperate in stabilizing the tertiary
structures of globular proteins.
 Disulfide bonds, Hydrophobic interaction, Hydrogen bond &
Ionic interaction

28. Non-covalent bonds within and between chains are as important in
their overall conformation and function

29. Quaternary structure
 Quaternary structure refers to the arrangement of polypeptide
chains in a multi chain protein.
 The subunits in a quaternary structure must be in non covalent
association
 Provide the opportunity for cooperative binding of ligands (e.g.,
O2 binding to hemoglobin)
 Form binding sites for complex molecules (e.g., antigen
binding to immunoglobulin),
 Increase stability of the protein
Example :Hemoglobin, lactate dehydrogenase, Aspartate
transcarboxylase

30. Quaternary structure of Hemoglobin
 Composed of two identical dimers, (αβ)1 and (αβ)2
 The two polypeptide chains within each dimer are held tightly
together, primarily by hydrophobic interactions
 Ionic and hydrogen bonds also occur between the members of
the dimer

31. Unity and Diversity of Protein

32. Protein dystrophin

33. Dystrophin
Introduction:
 High molecular weight cytoskeletal protein and a member of the
β-spectrin/α-actinin protein family
 localizes to the cytoplasmic face of the sarcolemma
 Mediates interaction with extracellular matrix
 Dystrophin is predominantly hydrophilic throughout its entire
length and 31% of the amino-acids are charged (i.e. Arg, Asp,
Glu, His and Lys).
 Associates with many other proteins to form the dystrophin
glyco-protein complex (DGC)

34. Dystrophin
 Expressed in skeletal muscle but also in cardiac muscle as
well as in the brain
 Cytogenetic Location: Xp21.2-p21.1, which is the short (p)
arm of the X chromosome between positions 21.2 and 21.1

35. Dystrophin
 Structure:
 Rod-shaped protein, measuring about 150 nm
 molecular weight of 427 kDa , consisting of 3684
amino acids
 Gene contains 79 exons in which with a high rate
of alternate splicing on the C-terminus
 Dystrophin can be separated into four domains:
 actin binding domain
 central rod domain
 Cysteine-rich domain
 Carboxy-terminal domain

36. Domain of Dystrophin
Actin binding domain (amino acids 14-240):
 actin-binding domain at the NH2 terminus
 alpha-actinin is a normal component of the actin filaments
in smooth and skeletal muscle
 Involved in cross-linking F-actin and thereby connecting the
filamentous elements of the cytoskeleton to the cell
membrane

37. Domain of Dystrophin
Central rod domain (amino acids 253-3040):
 The central rod domain is composed of 24 spectrin-like repeat.
 Each repeat unit is ~110 aa in size and forms a triple α-helical
bundles; a and b form the long helix while c forms the short helix.
 These α-helical coiled-coil repeats are interrupted by four proline-
rich hinge regions, so called hinge regions.
 In the normal dystrophin protein, repeat 19 and repeat 20 is
separated by hinge 3.

38. Domain of Dystrophin
Central rod domain (amino acids 253-3040):
 At the end of the 24th repeat is the fourth hinge region
that is immediately followed by the WW domain.
 separates the rod domain from the cysteine-rich and COOH-
terminal domains
 The WW domain is a recently described protein-binding
module found in several signaling and regulatory molecules.
 The WW domain binds to proline-rich substrates in an
analogous manner to the src homology-3(SH3) domain .

39. Domain of Dystrophin
The cysteine-rich domain
• Contains : two EF-hand motifs and ZZ domain
• EF-hand motifs
• Consist of two α-helices, linked by a short loop region that has
been implicated in calcium binding(intracellular Ca2+
• ZZ domain
• predicted to form the coordination sites for divalent metal
cations such as Zn2+
• The ZZ domain is similar to many types of zinc finger and is
found both in nuclear and cytoplasmic proteins.
• The WW domain along with two neighboring EF-hands binds
the carboxy-terminus of β-dystroglycan, anchoring the
dystrophin at sarcolemma

40. Domain of Dystrophin
Carboxy-terminal (CT)domain (amino acids 3361-3685)
 Contains two polypeptides that fold into α-helical coiled
coils similar to the spectrin repeats in the rod domain .
 Coiled coils are common protein motifs that are involved in
protein-protein interaction.
 The CT domain provides binding sites for dystrobrevin and
syntrophins, mediating their sarcolemma localization.

41. Dystrophin-Glycoprotein Complex (DGC)
 The Dystrophin-Glycoprotein Complex (DGC) is a multiprotein
complex
 Functions as a structural link between the sarcolemma-
cytoskeleton and the extracellular matrix .
 It aides in blood flow regulation, and in muscle fatigue recovery.
 A decrease in function of this protein complex causes muscle
fibers to become weakened and results in more susceptibility to
muscle degeneration and tissue death

42. Dystrophin-Glycoprotein Complex (DGC)
 The DGC regulates,
 the recruitment of Neuronal Nitric Oxide Synthases (nNOS)
 a signaling molecule important in muscle relaxation
 catalyzes the production of nitric oxide (NO)
 When muscle relaxation occurs, NO diffuses through muscles cells causing the
muscle to relax.
 nNOS has an effect on the DGC, which in turn, affects muscle fatigue,
vasodilation, and the structural integrity of the sarcolemma and the
cytoskeleton

43. Dystrophin-Glycoprotein Complex (DGC)
 Dystrophin-associated proteins can be divided into three groups
based on their cellular localization:
i. Extracellular -α-dystroglycan
ii. Transmembrane - β-dystroglycan, sarcoglycans, sarcospan
iii. Cytoplasmic - dystrophin, dystrobrevin, syntrophins, neuronal
nitric oxide synthase
 α-dystroglycan functions as a receptor for the extracellular ligands
such as laminin
 α-dystroglycan is tightly associated with β-dystroglycan, a
transmembrane protein that also interacts with dystrophin.

44. Sarcoglycan subcomplex
 Tightly associated with β-dystroglycan.
 Most prevalent form of sarcoglycan complex in skeletal muscle is
composed of four single-pass transmembrane proteins:
 α-sarcoglycan
 β-sarcoglycan
 γ-sarcoglycan
 δ- sarcoglycan.
 consensus phosphorylation sites for cyclic adenosine monophosphate
(cAMP)-dependent protein kinase, protein kinase C and casein kinase II
Dystrophin-Glycoprotein Complex (DGC)

45. Dystrophin-Glycoprotein Complex (DGC)
Sarcospan
 Small transmembrane protein that is tightly associated with
the sarcoglycans.
 The α-dystrobrevin/syntrophin triplet associates with
dystrophin and anchors neuronal nitric oxide synthase
(nNOS) to the sarcolemma.
 Syntrophins
 Function as modular adaptors that localize signaling molecules,
such as neuronal nitric oxide synthase (nNOS) , water channel
aquaporin-4 (AQP4) , ion channels , kinases , and transporters
at the muscle membrane in association with the DGC.

48. Dystrophin Protein Isoform
 The isoforms are encoded by a range of different mRNA's which are
generated by three processes;
i. the use of different, unique and often tissue-specific promoters
ii. alternative splicing
iii. the use of different polyA-addition signals

49. Dystrophin Protein Isoform
1.The use of different, unique and often tissue-specific promoters
 Dp427l, Dp427c, Dp427m, Dp427p, Dp260, Dp140, Dp116 and Dp71
Name synoniem
protein
length
amino
acids
mRNA
promoter
located in
expression
Dp427l L-dystrophin 427 kDa 3,562
13,764
bp
5' Dp427c
lymphoblastoi
d
Dp427c
brain or C-
dystrophin
427 kDa 3,677
14,069
bp
5' Dp427m brain
Dp427m M-dystrophin 427 kDa 3,685
13,993
bp
5' of gene muscle
Dp427p P-dystrophin 427 kDa 3,681 14 kb 3' Dp427m Purkinje cells

50. Dystrophin Protein Isoform
• Dp71 is detected in most non muscle tissues including brain, kidney, liver,
and lung
• The remaining short isoforms are primarily expressed in the central and
peripheral nervous system
• Dp140 has also been implicated in the development of the kidney .
• Dp 260 is detected in retina

51. Dystrophin Protein Isoform
2. Alternative splicing:
 Dp140ab, Dp140b, Dp140bc, Dp140c, Dp71a, Dp71b and Dp71ab
 the alternatively spliced transcripts is:
a-types miss the exon 71 sequences,
b-types miss the exon 78 sequences and
c-types miss the exon 71-74 sequences.
The b-types have an alternative 31 amino acid C-terminus

52. Dystrophin Protein Isoform
3. Alternative polyA-addition sites:
 Dp40
 The normal 3'-terminal exon present in mRNA's derived from
the dystrophin gene is exon 79.
 The use of an alternative polyA-addition site, localized in
intron 70 of the dystrophin gene, was first reported
by Feener,

53. Dystrophin
Functions
 Provides the structural integrity link between the sarcolemma
and the cytoskeleton.

54. Dystrophin
Functions
 Serve as a molecular shock absorber that defines the
physiological level of force in the dystrophin-mediated force-
transmission pathway during muscle contraction /stretch, there
by stabilizing the sarcolemma.
Stochastic unfolding and refolding of
dystrophin central domain

55. Dystrophin
Functions
 Dystrophin aids in signaling pathways, such as nitric oxide
production, Ca2+ entry, and reactive oxygen species production
 The syntrophins and dystrobrevin are members of the cytoplasmic
complex of dystrophin, and serve as a scaffold for signaling
proteins

56. Dystrophin
Functions
 Research suggests that the protein is important for the normal
structure and function of synapses, which are specialized
connections between nerve cells where cell-to-cell
communication occurs.

57. The pathophysiology of dystrophin deficiency
 This diagram illustrates the scheme described by Steinhardt and co-
workers in mdx(X-linked muscular dystrophy) mice.

58. The pathophysiology of dystrophin deficiency
The two-hit hypothesis (two-hit theory) for myofiber damage and the effects of the functional
ischemia on muscular dystrophy and animal models

59. Fig. A flow diagram of the known
pathways by which the loss of
dystrophin or a severely truncated
dystrophin leads to the development
of cardiomyocyte death.

61.  Mutations in the dystrophin gene can cause truncated proteins that
get low productions levels, or the dystrophin protein isn’t produced
at all.
 Without this the complex cannot bind to F-actin and fulfill its role.
 There are hundreds of mutations associated with the dystrophin
gene in the majority of the exons and many of the mutations cause
a type of dystrophy.
 Duchenne muscular dystrophy (absent) and Becker muscular
dystrophy (truncated) are two of the most severe mutations.
Problems

62. Duchenne Muscular Dystrophy (DMD)
Facts
 DMD affects mostly males at a rate of 1 in 3,500 births.
 There are over 200 types of mutations that can cause any one of the
forms of muscular dystrophy.
 There are also mutations that occur within the same gene that cause other
disease types.
 DMD is the most severe and common type of muscular dystrophy.
 DMD is characterized by the wasting away of muscles.
 Diagnosis in boys usually occurs between 16 months - 8 years.
 Parents are usually the first to notice problem.
 Death from DMD usually occurs by age of 30.

63. Clinical Features Genotype of DMD
 Females carry the DMD gene on the X
chromosome.
 Females are carriers and have a 50%
chance of transmitting the disease in
each pregnancy.
 Sons who inherit the mutation will have
the disease.
 Daughters that inherit the mutation will
be carriers.
 The DMD gene is located on the Xp 21 band
of the X chromosome.
 Mutations which affect the DMD gene.
 96% are frameshift mutations
 30% are new mutations
 10-20% of new mutations occur in the
gametocyte (sex cell, will be pass on to
the next generation).
 The most common mutation are repeats of
the CAG nucleotides.

64. Genotype of DMD
 During the translocation process, a mutation occurs.
 Mutations leading to the absence of dystrophin
 Very Large Deletions (lead to absence of dystrophin)
 Mutations causing reading errors (causes a degraded, low
functioning DMD protein molecule)
 Stop mutation
 Splicing mutation
 Duplication
 Deletion
 Point Mutations

65. Clinical Features Phenotype of DMD
 Delays in early childhood stages involving muscle use, in
42% of patients.
 Delays in standing alone
 Delays in sitting without aid
 Delays in walking (12 to 24 months)
 Learning difficulties in 5% of patients.
 Speech problems in 3% of patients.
 Leg and calf pain.
 Mental development is impaired.
 Memory problems
 Carrying out daily functions

66. Clinical Features Phenotype of DMD
 Increase in bone fractures due to the decrease in bone
density.
 Increase in serum CK (creatine phosphokinase) levels up to 10
times normal amounts.
 Wheelchair bound by 12 years of age.
 Cardiomyopathy at 14 to 18 years.
 Few patients live beyond 30 years of age.
Reparatory problems and cardiomyopathy leading to
congestive heart failure are the usual cause of death

68.  Loss of the middle section of domain 2 causes a very mild phenotype. If domain 2
only provides ‘size’ then deletions may be predicted to have minimal impact.
 Deletions around exons 43 - 53 cause Becker muscular dystrophy. Phenotypic
variability suggests that environmental factors may play important roles in clinical
progr ession.
 Domain 3 and the proximal region of domain 4 are apparently essential - loss leads
to Duchenne muscular dystrophy.
 Loss of the terminal portion of domain 4 is associated with mild Becker muscular
dystrophy.

69. Allelic Variants

71. References
 Robert k. Murray, D.K.Granner ,P.A.Mayes & Victor W.Rodwell Harpers
illustrated biochemistry 26th edition
 Lippincot - Marks' Basic Medical Biochemistry A Clinical Approach
 Thomas M.Devlin , textbook of Biochemistry with clinical correlation
5th edition
 Lehninger Principle of Biochemistry 4th edition
 Pamela C. Champe Richard A. Harvey, Denise R. Ferrier Lippincot
illustrated Biochemistry 4th edition
 https://ghr.nlm.nih.gov/gene/DMD
 https://www.dmd.nl/DMD_home.html
 The Dystrophin Complex: structure, function and implications for
therapy,Q. Gao and E. M. McNally, Compr Physiol. 2015 July 1; 5(3):
1223–1239. doi:10.1002/cphy.c140048.
 Function and Genetics of Dystrophin and Dystrophin-Related Proteins
in Muscle, Blake et al (2002); Physiological Reviews, 82: 291-329.

72. References
 Bailey Nichols 1, Shin’ichi Takeda 2, and Toshifumi Yokota 1,3,
Nonmechanical Roles of Dystrophin and Associated Proteins in Exercise,
Neuromuscular Junctions, and BrainsBrain Sci. 2015, 5, 275-298;
doi:10.3390/brainsci5030275
 Shimin LeShimin LeMiao YuLadislav Hovana,Dystrophin As A Molecular Shock
Absorber November 2018ACS Nano 12(12) DOI: 10.1021/acsnano.8b05721
 Venus Ameen and Lesley G. Robson ,Experimental Models of Duchenne
Muscular Dystrophy: Relationship with Cardiovascular DiseaseThe Open
Cardiovascular Medicine Journal, 2010, 4, 265-277

73. Thank you

74. `