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What are the 2 precursors of insulin?
1. preproinsulin (inside the cell → in a reduced environment)
- 2. proinsulin (oxidized in ER)
- - upon entering the ER lumen, it goes from a reduced to oxidized environment
- - disulfide bonds get oxidized
- • final: Insulin (secreted & oxidized)
- - releases the pro bit upon secretion
• AA involved in disulfide bonding = Cysteine
• cytoplasm: reducing
• extracellular (eg. plasma) + ER lumen: oxidizing
- • sulfides on cysteine tend to form disulfide bonds with each other (→ cystine) in an OXidizing environment
- - this imparts high stability to proteins
- • major protein of the human body
- • procollagen contains ~1500 AA residues; mature collagen contains ~1000
- • mature has 3 polypeptide chains called alpha chains
- • it’s a fibrous protein that doesn’t dissolve in water
- • they give structural integrity to tendons, cartilage, skin
Collagen Type I
- • found in Bone (bONE), Skin, Tendon
- • they have similar, not identical, AA sequences & are encoded by 1, 2 or 3 differernt genes
Collagen Type III
found in Reticulin (supporting mesh in soft tissues, also skin)
Collagen types I & III share:
1. collagen triple helix
2. modified amino acids (hydroxy-proline & -lysine)
What skin cell makes a large amount of both collagen I & III?
• collagen fibers (comprising fibrils) give structural rigidity to the skin
• problem though: don’t want fiber assembly INSIDE the fibroblast cell
What are the steps of collagen biosynthesis? (9)
- 1. Synthesis of polypeptide chain
- 2. Hydroxylation of proline & lysine
- 3. Glycosylation
- 4. Disulfide bond formation at C-terminus
- 5. Triple-helix formation
- 6. Secretion
- 7. Hydrolysis of propeptides (result = tropocollagen)
- 8. Assembly of fibril (result = collagen fibril)
- 9. Fiber assembly & formation of cross (X) links
• result = mature collagen fiber!
• Shit Head Gary Doesn't Talk Smack, He Asks for X •
Prolyl & Lysyl hydroxylase
• hydroxylate proline & lysine in step #2 of collagen formation to make hydroxyproline & hydroxylysine
• require the cofactor is ascorbic acid (a form of Vitamin C)
• example of postranslational modification
What causes scurvy?
lack of ascorbic acid (Vitamin C) which leads to a decrease in the hydroxylation of lysines & prolines in collagen which makes collagen unstable
damage & fragility of many tissues & organs such as the gums, as well as skin lesions & blood vessel fragility
Step #3 Glycosylation
- • hydroxylysines are attachment sites for simple sugars
- • at this point, the 3 pro alpha chains have been translated & modified
Step #4 Disulfide Bond Formation
• unlike simple proteins (which fold from N → C), procollagen begins to fold by disulfide bond formation in the C TERMINUS (zips up toward the N term)
• disulfide bond formation should be a clue that protein has moved from reducing location (cytoplasm) → oxidizing (ER lumen)
Step #5 Triple Helix Formation
• collagen adopts a 2ndary structure formed by H-bonding among the 3 chains, which involves the NH groups of glycine on one chain & the COOH group on an adjacent chain
• a glycine at every 3rd residue allows close packing of each helical segment (it's small size allows close approach of the chains in the triple helix)
• A MUTATION WOULD DISRUPT ASSEMBLY!
What causes collagen to form a triple helix rather than a standard alpha helix or beta sheet?
the steric repulsion between the prolines & hydroxyprolines
Step #7 Tropocollagen Formation
- • conversion of pro → tropo collagen is catalyzed by specialized propeptidases in the ECM
- • tropocollagen is still 3 collagen polypeptide chains complexed together
- • N & C-terminal propeptides are cleaved to make collagen alpha chains
Step #7 Spontaneous Fibril Assembly
• Tropocollagen spontaneously assembles into a fibril with gaps between each tropocollagen molecule
• these are important for bONE formation (collagen forms the matrix on which calcium phosphate is deposited during osteogenesis)
Which condition usually results from a mutation in type I collagen?
- • a severe forms of the mutation replaces collagen's glycine with a cysteine → interfering with the collagen triple helix
- • for a multi-subunit protein, all the chains have to be functional in order to get an overall functional protein
- • if you’re heterozygous for OI, you’ll still have bad collagen
What are the symptoms of Osteogenesis Imperfecta?
bone deformities, fragile blood vessels, brittle bones & teeth
Step #9 Maturation through X-links
collagen fibrils assemble into fibers, which are strengthened by cross-links between lysine or hydroxylysine
What enzyme helps catalyze the formation of cross links in mature collagen fibers?
lysyl oxidase oxidizes lysine to allysine
this forms a cross-link with unmodified lysines
What's the difference between lysyl hydroxylase & lysysl oxidase?
• lysyl hydroxylase (step #2): catalyzes hydroxylation of lysine to hydroxylysine [requires cofactor Vit C]
• lysyl oxidases (step #9): oxidizes NH3+ group at the end of the lysyl side-chain resulting in formation of modified amino acid residue → allysine
• allysine condenses with the side-chain of an unmodified lysine forming a covalent cross-links in mature collagen
What steps of collagen synthesis are completed inside the cell and when is the protein secreted?
• steps 1-5, up to triple helix formation occur inside the cell
• after the triple helix is formed, a procollagen molecule is secreted from the cell to undergo C & N-terminus hydrolysis (severing), spontaneous assembly into a fibril, then cross-linking & further final fiber formation
What are two causes of Ehlers-Danlos Syndrome?
- 1. Improper processing of collagen caused by lack of enzyme (eg. lysyl oxidase deficiency)
- - heterozygotes are usually okay because having 1 copy of a functional enzyme is usually sufficient; most defects occur with homozygous “recessive” mutations
- 2. genetic defects in the collagen genes
- - like what’s described in OI; heterozygosity is equally destructive as homozygosity
What are the symptoms of Ehlers-Danlos Syndrome?
hyperelastic joints, skin hyper-extendibility
Examples of transmissible spongiform encephalopathies:
- • Mad Cow disease
- • Scrapie (sheep)
- • Creutzfeldt-Jakob disease
- • Kuru
What causes Transmissible spongiform encephalopathies?
1. Inherit a missense mutation (eg. Creutzfeldt-Jacob disease)
2. Come into contact with "bad" form of the protein
• prion protein can exist in either a soluble or aggregated form; aggregated forms are disease causing
• classical C-J disease presents later in life, whereas new-variant disease presents at an average age of 29
True or False: different types of collagen arise from alternative splicing?
FALSE: different types of collagen are encoded by separate genes
What are the 2 modified amino acids present in collagen?
hydroxyPROLINE & hydroxyLYSINE
What determines the stability of the collagen helix?
Where is glycine found in collagen?
it's typically found at every third residue
What form of collagen is made after hydrolysis of procollagen?
Tropocollagen; these molecules spontaneously assemble to form a collagen fibril
What kind of bond holds the two peptide chains of collagen to one another?
disulfide bonds & non-covalent interactions