Flashcards in MGD S6 - Protein processing and targeting in cells Deck (43):
What is the difference between constitutive and regulated secretion
Give examples of each.
Constitutive secretion - Continuous packaging and release of proteins from GA via exocytosis
Eg. Serum Albumin, Collagen
Regulated secretion - Proteins packaged into vesicles but only released in response to a signal
Explain the Protein secretion pathway in the RER.
Hint: 9 steps
1. Free ribosome begins protein synthesis
2. Hydrophobic N-terminal signal sequence produced
3. Signal recognition particle (SRP) recognises signal sequence and binds
4. Protein synthesis stops
5. GTP-bound SRP directs ribosome to SRP receptors on RER cytostolic surface
6. SRP dissociates
7. Protein synthesis continues, feeding protein into RER via pore in membrane (peptide translocation complex)
8. Signal sequence is removed by signal peptidase once the entire protein is finished
9. The ribosome dissociates and is recycled
List the protein modifications made in the RER to newly synthesised proteins and the enzymes involved
Signal cleavage (signal peptidase)
Disulphide bond formation (protein disulphide isomerase)
N-Linked Glycosylation (Oligosaccharide-protein transferase)
List the protein modifications made in the Golgi bodies to newly synthesised proteins and the enzymes involved
O-Linked glycosylation (glycosyl transferase)
Trimming and modification of N-Linked oligosaccharides
Further proteolytic processing (some proteins only)
Describe the process of N-Linked glycosylation
The oligosaccharide is built on a Dolichol phosphate carrier molecule (long chain hydrocarbon which inserts into membrane with phosphate protruding into ER lumen)
Oligosaccharide then transferred to the amide group of asparagine.
Describe the process of O-Linked glycosylation
Modification of -OH groups on serine and threonine
Glycosyl transferase builds a sugar chain from nucleotide sugar substrates
What enzymes are involved in proteolytic processing of proteins
(eg. Amino peptidase, carboxypeptidase)
How and where are pre- and pro- segments of a protein removed?
N terminal Pre segment (signal sequence) is removed via proteolytic modification in the ER
Further proteolytic modification to remove the Pro- segment takes place in the Golgi Apparatus
Give two examples of a proteolytic modification sequence that removes the pre- and pro- segments from a protein
Hint: looking for names of proteins in the sequence, not enzymes
Preproalbumin ---> Proalbumin ---> albumin
Preproinsulin ---> proinsulin ---> insulin
What are the major polypeptide 'segments' in preproinsulin? (List in order from N terminus)
Signal sequence and A, C and B peptides
What is the first step in post translational modification of preproinsulin?
Removal of signal sequence via signal peptidase to form proinsulin
Describe the tertiary structure of proinsulin
How is proinsulin processed into insulin?
3 Disulphide bonds are formed, 2 between the A and B peptide and 1 between two points on the A peptide
Endopeptidase in the trans golgi cleaves out the 'C' peptide
This is the active form of insulin
How is the C peptide of preinsulin useful to treatment of diabetics?
Provides a good marker for measuring levels of endogenous insulin
What is the basic unit of a collagen polypeptide and what does this unit contain?
3 amino acids
Glycine - X - Y
X and Y variable but commonly Proline or Hydroxy proline
What is collagen made up of?
3 helix chains forming a left handed superhelix
What are the physical features of a collagen triple helix?
Nen-extensible, non-compressible, high tensile strength
Describe the roles of proline and hydroxyproline in the collagen molecule
Provides correct geometry for extended alpha helix chain conformation
Prevents peptide from assuming another shape (Beta sheet)
Increase the amount of interchain bonds
What enzyme is responsible for hydroxyproline formation and what cofactors does it require?
Requires Vitamin C and Fe(2+) ions
How is scurvy related to weakening of collagen?
Low vitamin C reduces activity of prolyl hydroxylase and so less hydroxyproline residues are formed
Leads to weakened tropocollagen triple helices
In what form are tropocollagen subunits originally synthesised? What are the functions of the accessory peptide sequences?
Pre - signal sequence
Pro - N and C terminal peptides that prevent collagen formation inside the cell
How does collagen form fibres after being modified in the Golgi Apparatus?
Procollagen secreted via exocytosis
Extracellular Procollagen peptidase cleaves N and C terminal peptide sequences
Collagen subunits form covalent crosslinks
Lysine residues are oxidised by lysyl oxidase to aldehyde derivatives that spontaneously form aldol crosslinks
What cofactors does the enzyme lysyl oxidase need to function?
Vitamin B6 and Cu(2+) ions
How do proteins enter the nucleus?
Through nuclear pores
With the help of Importin
Protein requires a nuclear localisation sequence in their primary sequence to bind to importin
How does Importin shuttle proteins across the nuclear membrane and how is it recycled?
1. Fully folded protein binds to alpha and beta importin
2. resulting complex binds to the nuclear pore and translocated into the nucleus in an energy dependent mechanism
3. Nuclear protein released and improtin binds to Ran (a small GTPase) which causes the protein being transported to dissociate
4. Importins are exported from nucleus and can be re-used
5. Ran is transported back into nucleus following hydrolysis of GTP
What sequence do proteins destined for the mitochondrial matrix contain?
An amphipathic N terminal signal sequence of 10-80 amino acids
Describe the process of of Mitochondrial targeting of proteins destined for the matrix
1. Mitochondrial proteins transported to mitochondrial unfolded, they are stabilised in cytosol by molecular chaperones such as Mitochondrial Import Stimulation Factor (MSF).
2. Signal sequence is recognised by proteins on mitochondria's outer membrane.
3. A protein import channel is formed, known as TOM (Translocase of the Outer Membrane) proteins are transported through.
4. TIM proteins (Translocase of the Inner Membrane) transport proteins into matrix via use of ATP and membrane potential.
5. Protein signal sequence is cleaved by Mitochondrial processing peptidase (MPP) and the protein folds via an ATP dependent process assisted by chaperones such as Hsp70
How do proteins destined for the Inner membrane of the mitochondria prevent being passed into the matrix?
Additional signalling sequence
How are lysosomal enzymes directed to lysosomes, where does this take place and what 2 enzymes are involved?
Lysosomal hydrolases have a signal patch that signals for the addition of a Mannose-6-phosphate group
This takes place in the Golgi and involves:
These M6P groups are recognised by M6P receptors that bind at the trans golgi face and vesicles of M6P-M6P receptor complex are pinched off for transport to lysosymes
How are directed vesicles of lysosomal enzymes integrated with lysosomes?
How is the M6P receptor recycled?
Vesicle enters lysosome and acidic pH causes dissociation of protein and M6P receptor.
M6P receptor is carried from lysosome via transport vesicle back to golgi for re-use.
Protein is dephosphorylated so it doesn't return to golgi with the receptor.
What defect characterises I-cell disease and what does this defect result in?
Genetic defects in N-acetylglucosamine phosphotransferase results in:
Lack of M6P addition to lysosomal hydrolases
Mistargetting of lysosomal hydrolases for secretion
Large amounts of lysosomal hydrolases found in blood and urine.
Why is a retrieval pathway for ER proteins necessary?
What proteins might need to be retrieved?
Some resident proteins of the ER are sometimes lost when vesicles are pinched off and transported to golgi.
Two examples are:
Protein disulphide isomerase
What is the mechanism of ER protein retrieval?
ER resident proteins have a 'KDEL' sequence near the C terminus that interact with KDEL receptors (binding enhanced by low pH) in the golgi and are then transported back to ER in transport vesicles.
ER resident proteins dissociate with receptor once delivered to ER (aided by neutral conditions) and KDEL receptors are transported back to golgi body.
One target for antibiotics is the bacterial cell wall, give an example of an antibiotic that targets the bacterial cell wall and explain how it has an antibiotic effect.
Inhibits transpeptidase enzyme that forms cross links in cell wall. Osmotic pressure then causes cell lysis.
One target for antibiotics is bacterial transcription, give an example of an antibiotic that targets bacterial transcription and explain how it has an antibiotic effect.
Binds to bacterial RNA polymerase preventing transcription
One target for antibiotics is bacterial protein synthesis, give an example of an antibiotic that targets bacterial protein synthesis and explain how it has an antibiotic effect.
Competes with tRNA at A site of bacterial ribosome
Give an example of an Antifolate drug and explain how it has antibiotic/anti-cancer action.
Impairs synthesis of tetrahydrofolate which is essential for DNA synthesis. Competitively inhibits dihydrofolate reductase (DHFR)
Applies to both bacterial and fast replicating mammalian cells
What are the 4 common targets for antibiotic action?
Bacterial cell wall synthesis
Bacterial Protein synthesis
Inhibition of Tetrahydrofolate synthesis
What are the 5 major methods of acquisition of resistance to an antibiotic or drug?
High rate of division
Decreased influx of drug
Increased efflux of drug
Increased transcription of target
How does high rate of division confer resistance to an antibiotic or drug?
Higher rate of mutation which will increase incidence of positive mutations which are selected for and resistance is developed quickly.
How does decreasing rate of influx into a cell confer resistance to an antibiotic or drug?
For drugs which must be taken up by target cells the carrier proteins allowing the drug access may be altered or reduced in amount so less of the drug is allowed through the membrane and have less effect.
How does increase efflux out of a cell confer resistance to anti-cancer drugs?
P-glycoprotein (multidrug resistance proteins 1 (MDR1) is a protein similar to CFTR (Cystic fibrosis transmembrane conductance regulator) that is responsible for efflux of toxins out of a cell.
In many cancers MDR1 expression increases to increase efflux of cancer drugs
How does increased transcription of a target confer antibiotic or drug resistance to a cell?
Increase production of drug target to overwhelm drug