Session 6 Flashcards
Provide an overview of the secretory pathway in mammalian cells
- Free ribosome initiates protein synthesis from mRNA molecule
- Hydrophobic N-terminal signal sequence is produced
- Signal sequence of newly formed protein is recognised and bound to by the signal recognition particle (SRP)
- Protein synthesis stops
- GTP-bound SRP directs the ribosome synthesising the secretory protein to SRP receptors on the cytosolic face of the ER
- SRP dissociates
- Protein synthesis continues and the newly formed polypeptide is fed into the ER via a pore in the membrane (peptide translocation complex)
- Signal sequence is removed by a signal peptidase once th entire protein has been synthesised
- The ribosomes dissociates and is recycled
Contrast the constitutive and regulated secretory pathways
Constitutive - secreted constantly (continuous process), proteins packaged into vesicles and continually released by exocytosis, e.g. Serum albumin, collagen
Regulated - secreted when necessary (in response to a signal), proteins packaged in vesicles and secreted when stimulus received e.g. hormones (insulin)
List protein modifications that can occur in ER and Golgi complex
ER:
Signal cleavage (signal peptidase)
Disulphide bond formation (protein disulphide isomerase)
N-linked glycosylation (oligosaccharide-protein transferase)
Golgi:
O-linked glycosylation (glycosyl transferase)
Trimming and modification of N-linked oligosaccharides
Further proteolytic processing (some proteins only)
Distinguish between N-linked and O-linked glycosylation of proteins
N-linked:
Oligosaccharide is built up in a dolichol phosphate carrier molecule sitting in membrane
Dolichol phosphate is a special long chain hydrocarbon molecules that inserts into the membrane with its phosphate group protruding
The oligosaccharide is then transferred to the amide group of Asparagine
O-linked:
The modification of the hydroxyl groups on Serine and Threonine
Glycosyl transferase builds up a sugar chain from nucleotide sugar substrates
Describe the role that proteolytic processing plays in the formation of important secreted proteins
Proteolytic removal of the N-terminal signal sequence is a very early event occurring in the ER (removal of the ‘pre-segment’)
Further processing can occur in some proteins in the Golgi (removal of the ‘pro-segment’)
E.g. Preproalbumin –> proalbumin –> albumin
Outline the formations of the mature insulin molecule
Preproinsulin - contains signal sequence, A,B,C peptides
Signal sequence is cleaved by signal peptidase
Proinsulin - contains A,B,C peptides
Endopeptidases cleave C peptide
Insulin - contain A,B peptides, joined by 3 disulphide bridges, active form
(C peptide is a good marker for measuring levels of endogenous insulin in diabetes)
Describe the structure of the triple-stranded collagen helix and provide an overview of collagen biosynthesis
Basic unit of collagen is tropocollagen
Primary sequence (Glycine-X-Y) every third position, mostly proline or hydroxyproline in X and Y position
Made of 3 polypeptides (3x a-chains)
Left-handed triple helix - non compressible, high tensile strength
Proline residues - correct geometry for extended a-chain conformation (prevents peptide from assuming another shape)
Hydroxyproline residues (formed from proline residues by prolyl hydroxylase –> increases amount of inter chain H bonds)
Prolyl hydroxylase requires Vit. C and Fe2+ ions for activity
Scurvy is due to low Vit. C –> weak tropocollagen triple helices
Tropocollagen subunits are synthesised as preprocollagen (pre –> hydrophobic signal sequence marks protein for secretion, pro –> subunits synthesised with N and C terminal peptides - prevents formation of collagen fibres inside cells)
Procollagen is secreted by exocytosis
Procollagen peptidases cleave the N and C terminal peptides (extracellularly)
Collagen subunits (tropocollagen) form covalent cross-links
Lysine residues –> aldehyde derivatives by lysyl oxidase –> form aldol cross-links)
Lysyl oxidase requires Vit. B6 and Cu2+ ions for activity
Outline the mechanisms involved in targeting proteins to several different cellular compartments
Nuclear targeting:
- A fully folded protein with a NLS (nuclear localisation sequence) is bound by Importin a and B in the cytosol
- The resulting complex binds to the nuclear pore and translocates into the nucleus in an energy dependent mechanism
- Once inside the nucleus the nuclear protein is released and the Importins bind to a small GTPase protein known as Ran
- Importins are exported from the nucleus and can be recycled to transport more nuclear proteins
- Ran is transported back to the nucleus plowing hydrolysis of GTP
Mitochondrial targeting
Lysosomal targeting
I-cell disease
Retention of proteins within ER
Describe the structure and mode of action of selected antibiotics and growth inhibitors
Penicillin - targets bacterial cell walls, inhibits transpeptidase enzyme that forms cross links in cell wall, osmotic pressure causes cell lysis
Rifampicin - targets bacterial transcription, binds to bacterial RNA polymerase, preventing transcription
Tetracycline - targets bacterial protein synthesis, competes with tRNA at A site of bacterial ribosome
Methotrexate - targets anti-folates (cancer therapy), impairs synthesis of tetrahydrofolate from folic acid (essential in DNA synthesis), inhibits dihydrofolate reductase (DHFR)
Provide and overview of the general mechanisms by which cells can become resistant to an antibiotic or drug
High rate of division - high rate of mutation, positive mutations (drug resistance) are selected for and breed a drug resistant population
Decreased influx - cells express an alternate version that reduces affinity of carrier protein that allows drug through membrane
Increased efflux - expression of P-glycoprotein is up-regulated so cells increase efflux of chemotherapy drugs
Increased transcription of target - increasing transcription of target can overwhelm drug
Altered target - specific target of drug acquires a mutation, lowering the affinity of the drug for it
