Protein Processing & Targeting in Cells

Question 1

What are the functions of the ER?

Answer 1

Insertion of proteins into membrane, specific proteolytic cleavage, glycosylation, formation of S-S bonds, folding proteins, assembly of multi-subunit proteins, hydroxylation of Lys & Pro residues

Question 2

Outline the protein secretory pathway

Answer 2

Protein synthesis on free ribosome, hydrophobic N-terminal signal produced, signal sequence recognised by SRP, GTP-bound SRP directs ribosome to SRP receptors on cystolic face of ER, protein fed into ER (peptide translocating complex), signal removed by signal peptidase, ribosome recycled

Question 3

What protein modifications can occur in the ER?

Answer 3

Signal cleavage (signal peptidase), disulphide bond formation (protein disulphide isomerase), N-linked glycosylation (oligosaccharide-protein transferase)

Question 4

What protein modifications occur in the Golgi?

Answer 4

O-linked glycosylation (glycosyl transferase), trimming and modification of N-linked oligosaccharides, further proteolytic processing (only for some proteins)

Question 5

Describe N-linked glycosylation

Answer 5

Oligosaccharide is built up on a dolichol phosphate carrier molecule sitting in the membrane. It’s then transferred to the amide group of asparginine

Question 6

What is dolichol phosphate?

Answer 6

Special long chain hydrocarbon molecule that inserts into the membrane with its phosphate group protruding

Question 7

Describe O-linked glycosylation

Answer 7

Modification of hydroxyl groups on serine and threonine. Glycosyl transferase builds up a sugar chain from nucleotides sugar substrates

Question 8

Describe the enzymes used for proteolytic cleavage

Answer 8

Endoproteases (sequence specific) and exoproteases (amino acid peptidase, carboxypeptidase)

Question 9

Outline the formation of the mature insulin molecule

Answer 9

Preproinsulin - cleaved by signal peptidase, proinsulin (contains A, B and C peptides). Endopeptidases cleave C peptide

Question 10

Describe the structure of triple-stranded collagen helix

Answer 10

Tropocollagen, (glycine-X-Y)n. 3 alpha chains, left-handed triple helix, proline residues, hydroxyproline residues

Question 11

What is prolyl hydroxylase used for and what does it require to function?

Answer 11

Forms the hydroxyproline residues on collagen. Requires vitamin C and Fe2+ ions. Allows more H bonds to stabilise triple helix

Question 12

What is scurvy the lack of?

Answer 12

Vitamin C - causes weak tropocollagen triple helices

Question 13

Outline the biosynthesis of collagen in RER

Answer 13

In the lumen of RER: Cleavage of signal peptide, Hydroxylation of selected proline and lysine residues, Addition of N-linked oligosaccharides, addition of galactose to hydroxylysine residues, chain alignment, Formation of S-S bonds, formation of triple helical procollagen from C- to N-terminus

Question 14

Outline the biosynthesis of collagen in Golgi

Answer 14

Completion of O-linked oligosaccharide chains by addition of glucose, transport vesicles, exocytosis, removal of N- & C-terminal propeptides, lateral association of collagen molecules, covalent cross-linking, aggregation of fibrils

Question 15

How are tropocollagen subunits synthesised?

Answer 15

Preprocollagen

Question 16

How is the formation of collagen fibrils inside cells prevented?

Answer 16

Pro-subunits with N and C terminal peptides are synthesised.

Question 17

Which reaction does lysyl oxidase catalyse and what does it require to function?

Answer 17

Lysine residues —> aldehyde derivatives

Requires vitamin B6 and Cu2+ ions

Question 18

What are the advantages of a triple helical structure?

Answer 18

Non-extensible, non-compressible, high tensile strength

Question 19

What is nuclear targeting?

Answer 19

Transport of proteins destined for the nucleus is done via nuclear pores. Proteins contain a nuclear localisation sequence (NLS). Involves the protein importin

Question 20

What are the steps of nuclear targeting?

Answer 20

Folded protein with NLS us bound by importin in the cytosol, complex binds to nuclear pore and translocates into the nucleus (energy dependent), protein is released, importin binds to GTPase protein (Ran), importins exported, Ran transported back to nucleus following hydrolysis of GTP

Question 21

What is mitochondrial targeting?

Answer 21

Proteins intended for the mitochondria. Unfolded when transported and contain N-terminal signal sequence.

Question 22

What are the steps of mitochondrial targeting?

Answer 22

Unfolded proteins are stabilised in cytosol (MSF), signal sequence recognised by TOM. Proteins needed in the matrix are transported by TIM (ATP needed and a membrane potential - DY), N-terminal sequence removed by MPP, protein then folds (ATP-dependent, chaperones such as mitochondrial Hsp70)

Question 23

What is lysosomal targeting?

Answer 23

Proteins destined for lysosomes such as lysosomal hydrolases

Question 24

How are lysosomal hydrolases targeted to lysosomes?

Answer 24

By the addition of mannose-6-phosphate (M6P) signal to N-linked oligosaccharides on the protein.

Question 25

What are the steps of lysosomal targeting?

Answer 25

Targeted for addition of M6P by presence of a signal patch, recognised by M6P receptors at the trans Golgi, vesicles pinched off. Once at lysosome acid pH causes dissociation of protein and receptor, receptor recycled. P group removed from protein

Question 26

What is I-cell disease?

Answer 26

Genetic defects in the N-acetylglucosamine phosphotransferase enzymes resulting in a lack of M6P addition to lysosomal targeted proteins

Question 27

What does I-cell disease cause?

Answer 27

Lysosomal hydrolases become mistargeted for secretion and results in large amounts of these proteins present in the blood and urine

Question 28

How can proteins that have escaped the ER be retrieved?

Answer 28

ER resident proteins have the sequence Lys-Asp-Glu-Leu (KDEL) near the C-terminus. If they are sent to the Golgi they will interact with KDEL receptors. These proteins are returned to the ER in transport vesicles

Question 29

What is the target of penicillin?

Answer 29

Bacterial cell wall.

Question 30

What is penicillin’s mode of action?

Answer 30

Inhibition of transpeptidase enzyme that forms cross-links in cell wall. Osmotic pressure causes cell lysis

Question 31

What is the target of rifampicin?

Answer 31

Bacterial transcription

Question 32

What is the mode of action of rifampicin?

Answer 32

Binds to bacterial RNA polymerase, thus preventing transcription

Question 33

What is the target of tetracycline?

Answer 33

Bacterial protein synthesis

Question 34

What is the mode of action of tetracycline?

Answer 34

Competes with tRNA at A site of bacterial ribosome

Question 35

What is the target of methotrexate?

Answer 35

Anti-folates in cancer therapy

Question 36

What is the mode of action of methotrexate?

Answer 36

Impairs the synthesis of tetrahydrofolate, which is essential for DNA synthesis, from folic acid. Competitive inhibitor for dihydrofolate reductase (DHFR)

Question 37

List a few general mechanisms by which cells can become resistant to an antibiotic or a drug

Answer 37

High rate of division, decreased influx, increase efflux, increased transcription of target, altered target

Question 38

What does a high rate of division do?

Answer 38

Means there’s a higher rate of mutation. Positive mutations such as drug resistance will be selected for and breed a population of drug resistant cells - uh oh!

Question 39

How do cells achieve decreased influx?

Answer 39

By expressing a reduced amount (or altered version) that reduces affinity of the carrier protein that allows the drug through the cell membrane

Question 40

Describe an example of increased efflux being used

Answer 40

P-glycoprotein (MDR1) is similar to CFTR and is responsible for efflux of toxic products from cells. In many cancers expression of P-glycoprotein is up-regulated, allowing efflux of chemotherapy drugs such as methotrexate

Question 41

What does an increased transcription of target do?

Answer 41

If drug targets a specific product of transcription (e.g. a ribosome) the cell can acquire resistance by increasing transcription of the target to overwhelm the drug