Protein and protein Dystrophin

Protein structures, Detail about protein dystrophin DMD and BMD primary structures, secondary structures, tertiary structures, Quaternary structures, functions of proteins ,
different sub types of protein structures, dystropins proteins structures , locations of it in chromosomes, chromosomal abnormalities, facts of Duchenne Muscular Dystrophy

1. Protein And Protein Dystrophin Hari Sharan Makaju M.Sc. Clinical Biochemistry 1 St 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

17. 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

18. 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.

19. β- turn

20. 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

21. 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

22. 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

23. 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

24. TERTIARY STRUCTURE Collagen

25. 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

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

27. 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

28. 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

29. Unity and Diversity of Protein

30. Protein dystrophin

31. 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)

32. 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

33. 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

34. 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

35. 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.

36. 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 .

37. 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 Ca 2+ ZZ domain predicted to form the coordination sites for divalent metal cations such as Zn 2+ 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

38. 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.

39. 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

40. 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

41. Dystrophin-Glycoprotein Complex (DGC) Dystrophin-associated proteins can be divided into three groups based on their cellular localization: Extracellular - α- dystroglycan Transmembrane - β- dystroglycan , sarcoglycans , sarcospan 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.

42. 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)

43. 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.

44. 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

45. 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 lymphoblastoid 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

46. 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

47. 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

48. 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 ,

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

50. 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

51. 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

52. 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.

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

54. 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

55. 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.

56. 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

57. 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.

58. 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.

59. 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

60. 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

61. 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

62. 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.

63. Allelic Variants

64. 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.

65. 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 Brains Brain 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

66. Thank you

67. `