MGD S6 - Protein processing and targeting in cells

Question 1

What is the difference between constitutive and regulated secretion
Give examples of each.

Answer 1

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
Eg. Insulin

Question 2

Explain the Protein secretion pathway in the RER.

Hint: 9 steps

Answer 2

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

Question 3

List the protein modifications made in the RER to newly synthesised proteins and the enzymes involved

Answer 3

Signal cleavage (signal peptidase)

Disulphide bond formation (protein disulphide isomerase)

N-Linked Glycosylation (Oligosaccharide-protein transferase)

Question 4

List the protein modifications made in the Golgi bodies to newly synthesised proteins and the enzymes involved

Answer 4

O-Linked glycosylation (glycosyl transferase)

Trimming and modification of N-Linked oligosaccharides

Further proteolytic processing (some proteins only)

Question 5

Describe the process of N-Linked glycosylation

Answer 5

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.

Question 6

Describe the process of O-Linked glycosylation

Answer 6

Modification of -OH groups on serine and threonine

Glycosyl transferase builds a sugar chain from nucleotide sugar substrates

Question 7

What enzymes are involved in proteolytic processing of proteins

Answer 7

Specific Endoproteases

Exoprotease
eg. Amino peptidase, carboxypeptidase

Question 8

How and where are pre- and pro- segments of a protein removed?

Answer 8

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

Question 9

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

Answer 9

Preproalbumin —> Proalbumin —> albumin

Preproinsulin —> proinsulin —> insulin

Question 10

What are the major polypeptide ‘segments’ in preproinsulin? (List in order from N terminus)

Answer 10

Signal sequence and A, C and B peptides

Question 11

What is the first step in post translational modification of preproinsulin?

Answer 11

Removal of signal sequence via signal peptidase to form proinsulin

Question 12

Describe the tertiary structure of proinsulin

How is proinsulin processed into insulin?

Answer 12

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

Question 13

How is the C peptide of preinsulin useful to treatment of diabetics?

Answer 13

Provides a good marker for measuring levels of endogenous insulin

Question 14

What is the basic unit of a collagen polypeptide and what does this unit contain?

Answer 14

Tropocollagen

3 amino acids

Glycine - X - Y

X and Y variable but commonly Proline or Hydroxy proline

Question 15

What is collagen made up of?

Answer 15

3 helix chains forming a left handed superhelix

Question 16

What are the physical features of a collagen triple helix?

Answer 16

Nen-extensible, non-compressible, high tensile strength

Question 17

Describe the roles of proline and hydroxyproline in the collagen molecule

Answer 17

Proline:
Provides correct geometry for extended alpha helix chain conformation
Prevents peptide from assuming another shape (Beta sheet)

Hydroxyproline:
Increase the amount of interchain bonds

Question 18

What enzyme is responsible for hydroxyproline formation and what cofactors does it require?

Answer 18

Prolyl hydroxylase

Requires Vitamin C and Fe(2+) ions

Question 19

How is scurvy related to weakening of collagen?

Answer 19

Low vitamin C reduces activity of prolyl hydroxylase and so less hydroxyproline residues are formed

Leads to weakened tropocollagen triple helices

Question 20

In what form are tropocollagen subunits originally synthesised? What are the functions of the accessory peptide sequences?

Answer 20

Preprocollagen

Pre - signal sequence

Pro - N and C terminal peptides that prevent collagen formation inside the cell

Question 21

How does collagen form fibres after being modified in the Golgi Apparatus?

Answer 21

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

Question 22

What cofactors does the enzyme lysyl oxidase need to function?

Answer 22

Vitamin B6 and Cu(2+) ions

Question 23

How do proteins enter the nucleus?

Answer 23

Through nuclear pores

With the help of Importin

Protein requires a nuclear localisation sequence in their primary sequence to bind to importin

Question 24

How does Importin shuttle proteins across the nuclear membrane and how is it recycled?

Answer 24

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

Question 25

What sequence do proteins destined for the mitochondrial matrix contain?

Answer 25

An amphipathic N terminal signal sequence of 10-80 amino acids

Question 26

Describe the process of of Mitochondrial targeting of proteins destined for the matrix

Answer 26

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

Question 27

How do proteins destined for the Inner membrane of the mitochondria prevent being passed into the matrix?

Answer 27

Additional signalling sequence

Question 28

How are lysosomal enzymes directed to lysosomes, where does this take place and what 2 enzymes are involved?

Answer 28

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:
N-acetylglucosamine phosphotransferase
N-acetylglucosamine phosphoglycosidase

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

Question 29

How are directed vesicles of lysosomal enzymes integrated with lysosomes?
How is the M6P receptor recycled?

Answer 29

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.

Question 30

What defect characterises I-cell disease and what does this defect result in?

Answer 30

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.

Question 31

Why is a retrieval pathway for ER proteins necessary?

What proteins might need to be retrieved?

Answer 31

Some resident proteins of the ER are sometimes lost when vesicles are pinched off and transported to golgi.

Two examples are:
Protein disulphide isomerase
Signal peptidase

Question 32

What is the mechanism of ER protein retrieval?

Answer 32

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.

Question 33

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.

Answer 33

Penicillin

Inhibits transpeptidase enzyme that forms cross links in cell wall. Osmotic pressure then causes cell lysis.

Question 34

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.

Answer 34

Rifampicin

Binds to bacterial RNA polymerase preventing transcription

Question 35

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.

Answer 35

Tetracycline

Competes with tRNA at A site of bacterial ribosome

Question 36

Give an example of an Antifolate drug and explain how it has antibiotic/anti-cancer action.

Answer 36

Methotrexate

Impairs synthesis of tetrahydrofolate which is essential for DNA synthesis. Competitively inhibits dihydrofolate reductase (DHFR)
Applies to both bacterial and fast replicating mammalian cells

Question 37

What are the 4 common targets for antibiotic action?

Answer 37

Bacterial cell wall synthesis
Bacterial transcription
Bacterial Protein synthesis
Inhibition of Tetrahydrofolate synthesis

Question 38

What are the 5 major methods of acquisition of resistance to an antibiotic or drug?

Answer 38

High rate of division
Decreased influx of drug
Increased efflux of drug
Increased transcription of target
Altered target

Question 39

How does high rate of division confer resistance to an antibiotic or drug?

Answer 39

Higher rate of mutation which will increase incidence of positive mutations which are selected for and resistance is developed quickly.

Question 40

How does decreasing rate of influx into a cell confer resistance to an antibiotic or drug?

Answer 40

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.

Question 41

How does increase efflux out of a cell confer resistance to anti-cancer drugs?

Answer 41

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

Question 42

How does increased transcription of a target confer antibiotic or drug resistance to a cell?

Answer 42

Increase production of drug target to overwhelm drug

Question 43

Hw does an altered target confer antibiotic or drug resistance to a cell?

Answer 43

Lowers the affinity of the drug for it, hence reducing drug effectiveness.