lec -6 FIBROUS PROTEINS.pptx BIOCHEMISTRTY SLIDES

BIOCHEMISTRY SLIDES OF FIBROUS PROTEINS FROM LLIPPINCOTT

1. FIBROUS PROTEINS Lecture by Dr Sumera Saghir MBBS, M . Phil , PHD scholar Biochemistry

2. FIBROUS PROTEINS

3. Learning objectives At the end of the session You will be able to define and describe fibrous proteins Describe the synthesis and types of Collage proteins Collegenopathies Elastin protein and diseases due to deficiency of elastin protein Differentiate between globular & Fibrous proteins

4. FIBROUS PROTEINS/ Sclero proteins Fibrous proteins are highly elongated usually insoluble polypeptides composed of a single secondary structure element Primary component of skin, tendon, bone, connective tissues, etc Function as structural material mechanically strong & highly cross linked Have protective, connective or supportive roles

5. All fibrous proteins are insoluble in water, WHY???? Property conferred by a high concentration of hydrophobic amino acid residues both in the interior of the protein and on its surface. These hydrophobic surfaces are largely buried as many similar polypeptide chains are packed together to form elaborate supra molecular complexes.

6. : FIBROUS PROTEINS Theses are structural proteins ,these proteins form long fibres give strength to structures in cell and body.e.g Keratin, collagen, silk fibrion Sclero proteins

7. Any of various proteins (as collagen and keratin) that occur especially in connective and skeletal tissues, are usually insoluble in aqueous solvents, and are resistant to chemical reagents — called also albuminoid Main types of Albuminoids : Collagen: Most abundant protein in mammals and is found in connective tissues such as tendons, ligaments, skin, and bones

8. Elastin: Elastin is a protein found in elastic tissues such as skin, blood vessels, and lungs. It is responsible for the elasticity and resilience of these tissues, allowing them to stretch and recoil. Keratin: Keratin is a fibrous protein found in tissues such as hair, nails, and the outer layer of the skin (epidermis). It provides structural support and protection to these tissues and is highly resistant to mechanical and chemical damag

9. Keratin Keratin Mechanically durable and un-reactive protein of vertebrates up to 85% of protein Present in : horns, hair, nails & feathers α -keratins occur in mammals; β -keratins occur in birds and reptiles found in hair and nails It is a dimer having two subunits held together by covalent and non covalent interactions 30 keratin genes expressed in mammals

10. α -keratins are classified as relatively acidic (Type I) or basic (Type II) Keratins have complex quaternary structures (a) keratins are dimers composed of a Type I and Type II subunit (b) many dimers associate to form proto-filaments (c) proto-filaments dimerize to form proto-fibrils Micro-fibrils associate into macro-fibrils

11. α -keratin is rich in Cys residues and disulfide bridges The mechanical strength of horns, nails and skin depends upon the content of Cys Residues and the number of disulfide bridges. Extensive disulfide bridging accounts for the insolubility and resistance to stretching of α-keratins

12. Quaternary structure of Keratin

13. Examples The α -keratin helix is a right-handed helix, the same helix found in many other proteins. Collagen

14. Collagen Present in all multi-cellular organisms and the most abundant protein in vertebrates Extracellular protein that forms insoluble fibers of great tensile strength Major stress bearing component of connective tissue – bone, teeth, cartilage, tendon, skin, etc

15. Collagen is a glycoprotein containing galactose and glucose as the carbohydrate content. -

16. PRIMARY STRUCTURE of Collagen Rich in proline and glycine TRIPLE –stranded helix Glycine smallest a.acid – every third position - Gly -Pro-X ---- or (- Gly -pro- hyp --) Glycine fits into the restricted spaces where the three chains of the helix come together Proline / Hydroxyproline constitute about 1/6 Glycine accounts for 1/3

17. The glycine residues are part of a repeating sequence, – Gly –X–Y– Where X is frequently proline and Y is often hydroxyproline (but can be hydroxylysine . – Proline or a post-translational modification of Pro (4-hydroxyproline) Smaller amount of the modified amino acids, 3-hydroxyproline and 5- hydroxylysine also occur in collagen

18. 33% glycine 10% proline . Most remarkably, collagen contains 4-hydroxyproline (10%), 3-hydroxyproline ( 0.5%), and 5-hydroxylysine (1%), which do not occur in most other proteins.

19. Quaternary Structure Collagen is composed of A repeating Gly – X – Y sequence the X position is typically Proline and the Y position is often hydroxyproline Three collagen polypeptides to wrap around one another forming a right-handed triple helix. Gly required at every 3rd position to allow close packing of subunits

20. A typical collagen molecule --- long, rigid structure in which three polypeptides(referred to as “α chains ”) are wound around one another in a rope-like triple helix Three separate polypeptides, called chains (not to be confused with helices), are super-twisted about each other The super-helical twisting is right handed in collagen, opposite in sense to the left-handed helix of the chains.

21. Amino acid sequence and composition Proline residues Introduce sharp bends in the – chains (because R group of proline is part of a cyclic ring), which helps tight wrapping of these chains around one another. Such tight wrapping enhances strength of the triple helix

22. Glycine residues Play an indirect role in permitting extremely tight inter winding of the -chains. The winding is so compact that there is little space available towards the interior, only the smallest amino acid, i.e. glycine , can be accommodated at that place .

23. Hydroxyproline and hydroxylysine : - Hydroxylation of proline and lysins Collagen contains hydroxyproline ( hyp ) and hydroxylysine ( hyl ), which are not present in most other proteins. – The hydroxylation is, thus, an example of post translational modification. Hydroxyl group in turn can participate in the formation of an additional hydrogen bond. Formation of a large number of hydrogen bonds is thus possible, which gives enormous collective strength. -

24. Hydroxyproline is important in stabilizing the triple-helical structure of collagen It maximizes interchain hydrogen bond formation

25. 4-hydroxyproline Prolyl hydroxylase is the enzyme that converts proline to 4-hydroxyproline requires ascorbic acid (vitamin C) for activity; Deficiency dietary vitamin C leads to scurvy

26. Scurvy is characterized by skin lesions, blood vessel fragility, poor wound healing and is ultimately fatal 4-hydroxyproline is responsible for stabilizing collagen structure stabilizing is via hydrogen bonds to 'bridging' water molecules located between individual subunits of the collagen fiber

27. Triple – helical structure Collagen, a fibrous protein, has an elongated, triple-helical structure that places many of its amino acid side chains on the surface of the triple helical molecule. The three polypeptide α chains are held together by hydrogen bonds between the chains This allows bond formation between the exposed R-groups of neighboring collagen monomers, resulting in their aggregation into long fibers.

28. Triple Helix while the triple helix is right-handed Gly at every third position contributes to tensile strength of helix

29. Collagen Fibrils Several common collagens form distinctively banded fibrils Type I collagens have a diameter of 100-2000 A Banding arises from packing of collagen molecules in the fibril hydrophobic interactions are the driving force for association of collagen triple helices into a fibril

30. Covalent cross-links Strength and insolubility of collagen fibrils is also due to intra- and intermolecular covalent cross-links not disulfide bonds as collagen is nearly devoid of Cys Cross-links are between Lys (and His) residues up to 4 residues can be involved in a single cross-link

31. Cross – link formation Lysyl oxidase – CU ++ containing enzyme – oxidatively deaminates some of lysyl and hydroxylysyl residues The reactive aldehydes that result – form covalent cross linkage – mature collagen fibers These covalent bonds cross-link the individual polypeptides Give strength and rigidity. .

32. Menkes syndrome Cu++ is required by lysyl oxidase , for covalent cross-links to strengthen collagen fibers kinky hair and growth retardation, due to dietry deficiency of the copper

33. Formation of cross -links in collagen lysyl oxidase is irreversibly inhibited by a toxin from plants in the genus Lathyrus , leading to a condition known as lathyrism .

34. Lathyrism Lathyrus odoratus is a sweet pea that contains significant amounts of β- aminopropionitrile is an inhibitor of lysyl oxidase and prevents the cross linking of collagen fibrils Increased fragility of collagen fibrils leads to abnormalities of bones, joints and large blood vessels Aging increases the cross-linking of collagen Meat of older animals???? Old human beings???

35. Types of collagen The collagen super-family of proteins includes more than 25 collagen types TYPE 1 : Contains two chains called α1 and one chain called α2 (α1 2 & α2) makes up about 90 percent of the collagen in your body,tendons , skin, bones, cartilage, and connective tissues.

36. Type I: C ollagen degrades, it becomes most  apparent in your skin . You’ll likely start to notice  wrinkles , fine lines, and a loss of elasticity. TYPE 2: Type II collagen contains three α1 chains ( α1 - 3 ) Type II collagen is found primarily in cartilage. While its structure is also a triple-helix,

37. TYPE III This type of collagen is often found alongside Type I. It makes up muscles, organs, arteries, and some connective tissues in the liver, spleen and blood vessels, and internal organs, including the uterus.

38. TYPE IV Type IV collagen doesn’t form a fibrous triple-helix structure like Types I, II, and III. Instead, it creates a web-like pattern, found in the skin, liver, kidneys, and other internal organs.

39. Structural role of Collagen

40. ECM and vitreous humor of the eye In some tissues, collagen is dispersed as Gel that gives support to the structure, e.g extracellular matrix or the vitreous humor of the eye.

41. IN BONES Collagen of bone occurs as fibers arranged at an angle to each other so as to resist mechanical shear from any direction .

42. ORGANIZATION OF COLLAGEN on the basis of their functions and location in the body

43. Organization of Collagen into three groups, based on their location and functions in the body

44. 1.Fibril-forming collagens Types I, II, and III are the fibrillar collagens Rope like structure Characteristic banding patterns Regular staggered packing of individual collagen molecule

45. Fibril-forming collagens

46. Type I collagen Fibers are found in supporting elements of high tensile strength (for example, tendon and cornea), Type II collagen molecules are restricted to cartilaginous structures. Type III collagen are prevalent in more distensible tissues, such as blood vessels

47. 2. Network-forming collagens: Types IV and VII form a three-dimensional mesh, rather than distinct fibrils. For example, type IV molecules assemble into a sheet or meshwork that constitutes a major part of basement membranes

48. Network-forming collagens

49. Fibril-associated collagens : Types IX and XII bind to the surface of collagen fibrils, linking these fibrils to one another and to other components in the extracellular matrix

50. BIOSYNTHESIS OF COLLAGEN Precursors: Collagen is one of the proteins that functions outside the cell.

51. Biosynthesis of collagen Precursors formed in fibroblasts Secreted in extracellular matrix Enzymatic modification Collagen monomer aggregate

52. Transcription of Collagen Genes: Location : Nucleus of fibroblasts and other connective tissue cells. The process begins with the transcription of collagen genes (such as COL1A1, COL1A2) into messenger RNA (mRNA) within the nucleus of fibroblasts. Transcription is mediated by specific transcription factors that bind to regulatory regions of the collagen genes.

53. Translation of mRNA into Procollagen Chains: Location: Rough endoplasmic reticulum (RER) of fibroblasts. The mRNA molecules encoding collagen are transported from the nucleus to the rough endoplasmic reticulum (RER), where ribosomes translate them into procollagen polypeptide chains. These chains consist of three alpha chains (two alpha1 chains and one alpha2 chain) that form a triple helix structure.

54. Hydroxylation of Procollagen Chains: Location: Endoplasmic reticulum (ER) of fibroblasts. Within the endoplasmic reticulum, specific enzymes catalyze the hydroxylation of proline and lysine residues within the procollagen chains. This hydroxylation is essential for stabilizing the triple helix structure of procollagen .

55. Glycosylation of Procollagen Chains: Location: Endoplasmic reticulum (ER) and Golgi apparatus of fibroblasts. The hydroxylated procollagen chains undergo glycosylation , where sugar molecules are added to specific amino acid residues. This glycosylation process occurs in the endoplasmic reticulum and is further modified in the Golgi apparatus.

56. Formation of Triple Helix Procollagen : Location: Endoplasmic reticulum (ER) of fibroblasts. The three alpha chains of procollagen assemble into a triple helix structure within the endoplasmic reticulum, forming procollagen molecules. Procollagen Secretion: Location: Golgi apparatus and extracellular space. Procollagen molecules are packaged into transport vesicles in the Golgi apparatus and transported to the cell membrane. These vesicles fuse with the cell membrane, releasing procollagen into the extracellular space.

57. Summary Cleavage of Procollagen to Collagen: Location: Extracellular space. Once secreted into the extracellular space, procollagen molecules undergo enzymatic cleavage of their N- and C-terminal propeptides by procollagen peptidases. This cleavage step converts procollagen into mature collagen molecules. Collagen Assembly and Cross-Linking: Location: Extracellular space. Mature collagen molecules assemble into fibrils and fibers, guided by interactions between their triple helix domains. Cross-linking between collagen molecules occurs, forming stable covalent bonds that contribute to the strength and stability of collagen fibrils.

58. continued

59. Degradation of collagen Proteolytic activity of collagenase – part of metalloproteinase Normal collagens are highly stable molecules, having half-lives as long as several years. connective tissue is dynamic and is constantly being remodeled, in response to growth or injury of the tissue.

60. For type I collagen, the cleavage site is specific, generating three-quarter and one-quarter length fragments. These fragments are further degraded by other matrix proteinases into aminoacids .

61. Collagen diseases -- collagenopathies Several rare heritable disorders of collagen are known brittle bone disease can arise from a single point mutation in collagen of bone hyperextensibility of joints arises from lessening of cross linking in collagen of ligaments Osteoarthritis and atherosclerotic plaques arise from disruption of collagen in cartilage Defects in any step in synthesis Ehlers – danlos syndrome (EDS) Osteogenesis imperfecta (OI)

62. Epidermolysis Bullosa It is a heritable disease in which glycosylation of collagen is impaired and the skin characteristically blisters. the skin breaks and blisters as a result of minor trauma. The dystrophic form is due to mutations in COL7A1, affecting the structure of type VII collagen. .

63. Alports syndrome The Alport syndrome (hereditary nephritis) genetic disorders (both X-linked and autosomal ) Mutations in several genes encoding type IV collagen fibers affecting the basement membranes of the renal glomeruli, inner ear and eye The main presenting sign is hematuria, accompanied by ocular lesions and hearing loss, and patients may eventually develop end stage renal disease

64. Scurvy Affects the structure of collagen. a deficiency of ascorbic acid (vitamin C) not a genetic disease. Its major signs are bleeding gums, subcutaneous hemorrhages, and poor wound healing. Defective synthesis of collagen due to reduced activity of the enzymes prolyl and lysyl hydroxylases , which require ascorbic acid as a cofactor

65. Menkes disease Deficiency of copper results in defective cross-linking of collagen and elastin by the copper-dependent enzyme lysyl oxidase

66. E ffect of Aging and Disease on Collagen Metabolism Age related change is that the collagen of old animals and humans have more covalent cross links than those of the young. Also the amount of collagen, relative to the proteins of parenchymal cells, increases with age.

67. Ehlers— danlos syndrome (EDS) Heterogeneous group of connective tissues disorder Lysyl hydroxylase or procollagen peptidase Mutation in a.acid sequence of collagen types 1,3,5 Most common type 3 Mutant collagen not secreted Type 3 component of arteries The vascular form, due to defects in type III collagen, is the most serious form of EDS because it is associated with potentially lethal arterial rupture.

68. ELHERS DANLOS Syndrome (EDS) The classic form of EDS, caused by defects in type V collagen, is characterized by skin extensibility and fragility and joint hypermobility

69. Osteogenesis imperfecta (OI) Brittle bone syndrome ( easily fractured even without trauma) ,hearing loss , and blue sclerae . Heterogeneous group disorder Retarded wound healing Rotated and twisted spine – humped back ( khyphotic ) Type 1 -- Osteogenesis imperfecta tarda defects In type α 1 and α 2 chain

70. Types of osteogenesis imperfecta T ype I : the most common form, mild bone fragility,hearing loss , and blue sclerae . Type II: the most severe form, is typically lethal in the peri -natal period as a result of pulmonary complications . In utero fracture , Pulmonary hyperplasia in utero /neonatal period Mutation in pro α 1 pro α 2 of type 1 replacement of glycine residue by AA with bulky side chai n .

71. Type III is also a severe form. It is characterized by multiple fractures at birth, short stature , spinal curvature leading to a “humped-back” ( kyphotic ) appearance , and blue sclerae .

72. ELASTIN

73. Elastin Elastin is an extracellular matrix protein that lends elasticity and resilience to tissues such as the arteries, lungs, tendons, skin, and ligaments, rubber like properties Elastic fibers ----Elastic fibers consist mainly of elastin protein and other associated molecules, including fibrillin microfibrils .

74. Elastin Lungs , large arteries and elastic ligaments Can be stretched to several times their normal length

75. Structure of elastin Amino Acid Composition: Elastin is composed primarily of non-polar amino acids, particularly glycine , alanine, and valine , which account for over 90% of its amino acid residues. Rich in proline and lysine (play crucial roles in the formation of cross-links within the protein.) little quantity of hydroxyproline and hydroxylysine

76. Structure of Elastin Insoluble protein Precursor tropoelastin Linear polypeptide --- 700 AA

77. Hydrophobic Domains: Elastin contains hydrophobic domains rich in glycine and alanine residues. These hydrophobic regions facilitate the close packing of elastin molecules, contributing to the insolubility and stability of the protein.

78. Cross-Linking Domains: These cross-links, which include lysine-derived desmosine , are crucial for stabilizing the elastin network and conferring elasticity to tissues.

79. Tropoelastin : The precursor to elastin is tropoelastin , a soluble monomeric protein synthesized by fibroblasts and other connective tissue cells. Tropoelastin molecules undergo extensive post-translational modifications, including removal of signal peptides and glycosylation , before being incorporated into elastic fibers.

80. Fibrillin Microfibrils : Fibrillin microfibrils provide a scaffold for the deposition and organization of elastin molecules within elastic fibers. Tropoelastin – secreted in extracellular space Interact – specific glycoprotein microfibrils – fibrilin --- scaffold onto which tropoelastin deposit

81. Elastin-Cross Linking: The final step in elastin assembly involves the cross-linking of tropoelastin molecules to form mature elastin fibers. Lysyl side chain – oxidatively deaminated – lysyl oxidase – allysyl residue Three allysyl and one lysyl – desmosine cross link

82. Desmosine cross -link in elastin.

83. Role of alhpa-1 antitrypsin in elastin degradation Alpha 1 antitrypsin – protein in blood and body fluids ( antiproteinase ) Inhibit proteolytic enzymes (proteases or proteinases ) Alpha 1 AT inhibit neutrophil elastase – alveolar walls and other tissues Most Alpha 1 AT -- synthesized in liver

84. Emphysema from alpha 1 AT deficiency Single purine base mutation GAG ----- AAG Substitution of lysine for glutamic acid at position 342 of protein Decrease secretion of alpha 1 AT by liver causes the normally monomeric AAT to polymerize within the ER of hepa tocyte s , resulting in decreased secretion of AAT by the liver.

85. Methionine 358 in AAT is required for the binding of the inhibitor to its target proteases . Smoking causes the oxidation and subsequent inactivation of the methionine , there by rendering the inhibitor power less to neutralize elastase . Smokerswith AAT deficiency,, have a considerably elevated rate of lung destruction and a poorer survival rate than nonsmokers.

86. T/M Deficiency can be reversed Weekly I/v administration of alpha 1 anti- tripsyin

87. Difference between Collagen & elastin

88. Clinical Correlates of Elastin deficency Deletions in the elastin gene (have been found in approximately 90% of subjects with the Williams- Beuren syndrome , a developmental disorder affecting connective tissue and the central nervous system, supravalvular aortic stenosis often found in this condition. Fragmentation or, alternatively, a decrease of elastin is found in conditions such as pulmonary emphysema, cutislaxa , and aging of the skin.

89. Fibrillin It is a glycoprotein, which is essential for the formation of elastic fibers found in connective tissue. It is secreted into the ECM by fibroblasts and becomes incorporated into the insoluble microfibrils . Microfibrils are fine fiber-like strands 10 to 12 nm in diameter which provide a scaffold for the deposition of elastin in the ECM. Fibrillins are large glycoproteins (about 350 kDa ) t hat are major structural component of these fibers.

90. Clinical correlates of Fibrilin Marfan syndrome is a relatively prevalent inherited disease affecting connective tissue It affects eyes ( eg , causing dislocation of the lens,known as ectopia lentis ), skeletal system (most patients are tall and exhibit long digits [ arachnodactyly ] and hyperextensibility of the joints)

91. causing weakness of the aortic media, leading to dilation of the ascending aorta). Mutations in the gene(on chromosome 15) for fibrillin- 1

92. Marfan Syndrome Clinical appearance: long extremities short trunk chest is deformed fingers are long and thin Defect in collagen crosslinks Misalignment of collagen molecules in collagen fibrils

93. Fibrion protein Fibrion is a fibrous protein-based bio materials ( silk, keratin, elastin and resilin ) for tissue regeneration &repair. Fibroin is rich in Ala and Gly residues, permitting a close packing of sheets and an interlocking arrangement of R groups Silk proteins can be extracted from silk glands or silkworms cocoons

94. Silk fibrion Silk Fibroin Fibroin , the protein of silk, is produced byinsects and spiders. Its polypeptide chains are predominantlyin the conformation.

95. Globular/Fibrous proteins Globular Globular protein is soluble and tends to be involved in metabolic functions  e.g cellular messengers, enzymes etc  The shape of a globular protein is a sphere ,have a folded ball like structure. Fibrous   Fibrous proteins are a are insoluble and that helps in building up the structuralelements e.g  formation of tendons, connective tissues, and fibers. Fibrous proteins are long and narrow in shape and have a helical or sheet like structure.

96. Globular soluble in water example of globular protein is hemoglobin  They are made up of primary, secondary, tertiary, and sometimes also quaternary structures. Fibrous Insoluble in water examples of fibrous protein are elastin, collagen, actin, fibrin, myosin, keratin. the shape of elongated strands like rods and wires, of polypeptide chain which results in the formation of it’s sheet like structure  

97. Globular Have a weak intermolecular interaction between them intermolecular hydrogen bonding.  Fibrous Fibrous proteins have a strong intermolecular interaction between them.