Topic 10 - Post-Translational Modifications, Prote 34c544a0cd594a07a0f64d9a8c507337.pdf

👩🏾‍🔬 Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis Define Proteome the entire complement of proteins that is or can be expressed by a cell, tissue...

👩🏾‍🔬 Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis Define Proteome the entire complement of proteins that is or can be expressed by a cell, tissue or organism Multiple post-translational modifications Collagen facts Most abundant protein in the body Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 1  Collagen in tendons and ligaments is loose, while in bone and cartilage, it's tight and rigid Also present in loose connective tissues Fibroblasts in connective tissue produce collagen Loose connective tissue has a branched, spaced-out structure; fibrous connective tissue is more compact Collagen Fibers Collagen's basic unit is tropocollagen, formed by cleaving procollagen with pro collagen peptidase Composed of three polypeptides, each about 1000 amino acids long, twisted into a rod-like structure Every third amino acid in the chain is glycine Hydrogen bonds between alpha chains stabilize the structure Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 2  collagen triple helix → non-extensible, non-compressible, and highly tensile Its structure remains rigid and unchanged Why is Glycine appearing at every 3rd amino acid advantageous non-polar nature creates tight, compact structures keeps collagen in a fibrous shape Glycine is the only amino acid small enough to fit in the middle of the helix, reinforcing its strength What is Osteogenesis Imperfecta? (autosomal dominant) family of genetic disorders that affect the bones making them weak and easily breakable. Causes bones to become weak —> prone to breaking Lack of type I collagen = primary cause Mutations in COL1A1 and COL1A2 genes prevent the production of type I collagen —> substitution of glycine with bulkier amino acids, altering collagen's triple helix structure Explain the process of Collagen Synthesis occurs in fibroblast cells signal sequences directs polypeptide chain into the E.R Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 3  the signal sequence is cleaved as the polypeptide enters the ER lumen hydroxylation of Proline + lysine residues (requires ascorbate + vitamin C) Glycosylation of selected hydroxylysine residues The peptides on the N terminal are not linked whereas on the C terminal side they are linked by disulphide bonds assembly of 3 Polypeptide hains into a procollagen triple helix Procollagen → Golgi Apparatus → assembled into secretory vesicles —> Secreted into extracellular matrix procollagen’s terminal ends are cleaved by Collagen Peptidases —> tropocollagen Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 4  Tropocollagen molecules covalently linked via aldol covalent cross-links, forming collagen fibrils by Lysyl Oxidase What is Scurvy: Connective tissue defect Scurvy is caused by insufficient hydroxylation of Proline + lysine residues due to lack of vitamin C and ascorbic acid results in fatigue, weakness, poor wound healing, anaemia and gum disease Connective tissue becomes defective —> instability of collagen Treatment → Vitamin C intake Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 5  Ehlers-Danlos syndrome (EDS) Mutations in genes affects extracellular peptide cleavage and alter collagen fibril cross-linking Results in altered stability and functionality of fibers Fibers become more extensible and lose their rigidity Skin can become loose and stretchy Define “Proteolytic cleavage” breaking peptide bonds to remove part of the protein processing after translation Proteins synthesises on free ribosomes are destined for… cytosol, or posttranslational import into organelles Proteins synthesised by ribosomes on the rough ER are destined for… Protein destined for plasma membrane secretory pathway via co-translational insertion What is co-translational insertion Co-translational insertion is the synthesis of polypeptides on ribosomes at the border of the ER such that the resulting polypeptide is contained within the ER What are the 4 requirements for Protein Sorting A signal, intrinsic to the protein A receptor that recognizes the signal A translocation machinery Energy Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 6  Protein targeting to peroxisomes (membrane bound organelle that undergoes chemical detoxification and lipid metabolism) 1) A signal, intrinsic to the protein Peroxisome targeting sequence (PTS) —> serine - lysine - leucine (SKL) Usually present on the C-terminus of the protein 2) A receptor that recognizes the signal and which directs it to the correct membrane Cytosolic receptor protein: PTS1R PTS1R binds to the SKL signal. PTS1R escorts the catalase tetramer. The destination is the Pex14p receptor on the peroxisome membrane. Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 7  3) A translocation machinery 14 Pex proteins create a transport channel through the peroxisomal membrane. A translocation machinery facilitates the transport of the PTS1R complex across the membrane. The PTS1R complex is transported across the membrane and then separates in the lumen of the peroxisome. 4) Energy to transfer the protein to its new place → ATP hydrolysis needed to allow recycling of the PTS1R receptor Protein targeting to mitochondria proteins contains a signal sequence - ensure it goes to the right organelle chaperons become associated with protein in cytoplasm (requires ATP) chaperons unfold proteins so it can fit through membrane Protein attaches to “Protein Translocator Complex” and moves into mitochondria Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 8  as it enters, chaperons are still bound to prevent premature folding Original chaperon released, another class binds signal peptidase removes signal sequence protein folded into final shape for its function. Translocation and Synthesis of Secretory Proteins/Membrane Proteins Signal Sequences: N-terminal amino acid sequence central region rich in hydrophobic residues, crucial for binding to the SRP protein Signal Recognition Particle (SRP): Necessary receptor to bind signal peptides on proteins headed for the endoplasmic reticulum (ER) Recognizes the signal peptide and the ribosome during protein targeting to the ER. Membrane Proteins Insertion into ER membrane is also required for delivery of membrane proteins destined for membranes Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 9  Approximately halfway through the protein's translation, there is an anchor sequence that specifically allows hydrophobic amino acids to associate with the lipid membrane. After this anchor sequence, the remainder of the membrane protein is synthesized, completing its insertion into the lipid membrane. Other Functions of the Endoplasmic Reticulum, other than the normal functions Hydroxylation of selected Lys and Pro residues Glycosylation Formation of S-S bonds What is N-linked Glycosylation and where does it take place N-linked glycosylation = Sugars are added on an Asparagine side chain (a reaction involving an amino group) Occurs in ER Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 10  Why is glycosylation of proteins important? Correct protein folding + stability Facilitates interactions with other molecules Deficiencies in N-linked glycosylation lead to severe inherited human disease: Congenital disorders of glycosylation (CDG) Where does formation of S-S Bonds take place Endoplasmic Reticulum Cysteine residues = most common amino acid affiliated with disulphide bonds Facilitated by disulphide isomerase Disulphide bonds are formed in the ER lumen Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 11  Disulphide bonds attributes to stability of proteins What happens if there are folding problems? What happens if mis-folding cannot be corrected? Protein may be returned to cytosol for degradation Protein may accumulate to toxic levels in the ER resulting in disease Processing of Preproinsulin Insulin consists of several components before entering the endoplasmic reticulum (ER): signal sequence, A chain, B chain, and C chain. Prior to entering the ER, the signal sequence is cleaved off, transforming proinsulin into insulin. Inside the ER, disulphide bonds form In the Golgi apparatus, the C chain is cleaved off —> final form of insulin. Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 12 Topic 10 - Post-Translational Modifications, Protein Targeting and Collagen Biosynthesis 13