Protein Processing

Question 0

Give two examples of proteins secreted by constitutive secretion.

Answer 0

Collagen and serum albumin

Question 1

What is constitutive secretion?

Answer 1

A continuous process

Proteins are packaged in vesicles and released continuously by exocytosis

Question 2

Give features of regulated secretion

Answer 2

Proteins released in response to a signal eg hormone

Proteins packaged into vesicles but not released until signal is received

Question 3

Give an example of a protein secreted by regulated secretion

Answer 3

Insulin

Question 4

Give the nine steps of the protein synthesis pathway

Answer 4

1. Free ribosome initiates protein synthesis from mRNA molecule
2. Hydrophobic N-terminal signal sequence is produced
3. Signal sequence of newly formed protein is recognised and bound to by the signal recognition particle (SRP)
4. Protein synthesis stops.
5. GTP-bound SRP directs the ribosome synthesising the protein to SRP receptors on cytosolic face of the ER
6. SRP dissociates
7. Protein synthesis continues and the newly formed polypeptide is fed into the ER via a pore in the membrane (peptide translocating complex)
8. The signal sequence is removed by a signal peptidase once the entire protein has been synthesised
9. The ribosome dissociates and is recycled

Question 5

List the three protein modifications that can occur in the ER and the enzymes needed to do them

Answer 5

Signal cleavage - signal peptidase

Disulphide bond formation - protein disulphide isomerase

N-linked glycosylation - oligosaccharide-protein transferase

Question 6

Give protein modifications that can occur in the Golgi.

Answer 6

O-linked glycosylation - glycosyl transferase

Trimming and modification N-linked oligosaccharides

Further proteolytic processing

Question 7

Describe N-linked glycosylation

Answer 7

The oligosaccharide is built up on a Dolichol phosphate carrier molecule sitting in the membrane
The oligosaccharide is then transferred onto the amide group of asparagine

Question 8

What is a Dilochol Phosphate carrier molecule?

Answer 8

A long chain hydrocarbon molecule that inserts into the membrane with its phosphate group protruding
Involved in N linked glycosylation

Question 9

What is O-linked glycosylation?

Answer 9

Modification of hydroxyl groups on serine and threonine.

Glycosyl transferase builds up a sugar chain from nucleotide sugar substrates.

Question 10

Which enzymes carry out proteolytic processing?

Answer 10

Endoproteases which are sequence specific

Exoproteases - amino peptidase and carboxypeptidase

Question 11

What is proteolytic removal of the N-terminal known as and where does it occur?

Answer 11

Pre-segment removal

Occurs in the ER

Question 12

Where does the removal of the pro-segment occur?

Answer 12

Golgi apparatus

Question 13

What happens first, removal of the pre- or pro-segment?

Answer 13

Pre

Question 14

Why is glycosylation of proteins important?

Answer 14

Correct protein folding
Protein stability
Facilitates interaction with other molecules

Question 15

What is glycosylation?

Answer 15

The attachment of carbohydrate groups to proteins via glycosidic linkages

Question 16

What is a proprotein?

Answer 16

An inactive precursor of a protein

Question 17

What is a preproprotein?

Answer 17

A protein containing both a signal peptide and propeptide

Question 18

Why are disulphide bonds added to some proteins?

Answer 18

Improves stability - especially for extracellular proteins because conditions outside of the cell may be harsher

Question 19

How is mature insulin formed?

Answer 19

Preproinsulin is synthesised - an inactive, single polypeptide

Signal sequence is removed by signal peptidase and 3 disulphide bonds are formed - pro insulin

Proteases recognise pairs of basic amino acid residues and cut it into three peptides - A, B and C.

Mature insulin is composed of A and B held together by disulphide bonds.

C peptide is secreted with insulin.

Question 20

What is a basic unit of collagen called?

Answer 20

Tropocollagen

Question 21

What are the three main amino acids in collagen?

Answer 21

Glycine, proline and hydroxyproline

Question 22

How are the polypeptides in collagen arranged?

Answer 22

Three polypeptides

Left-handed triple helix

Question 23

What properties does the arrangement of polypeptides in collagen lead to?

Answer 23

Non-extensible/compressible

High tensile strength

Question 24

How is collagen produced while within the cell?

Answer 24

Tropocollagen produced as preprocollagen. - Pre marks protein for secretion - Pro prevent collagen from forming fibres within the cell Pro collagen is secreted by exocytosis

Question 25

How is collagen formed once pro collagen is outside of the cell?

Answer 25

Procollagen peptidases cleave N and C terminal peptides (the pro-segment) Tropocollagen form covalent cross links Aldehyde residues are produced from lysine via the enzyme lysyl oxidase. They can form cross links

Question 26

What does lysyl oxidase require to work?

Answer 26

Vitamin B6 and Cu 2+ ions

Question 27

Describe nuclear targeting

Answer 27

Proteins that need to enter the nucleus contain a nuclear localisation sequence which is contained in the primary sequence. Importin alpha and beta bind to the NLS in the cytosol. Complex translocates to the nucleus - energy dependant process. Nuclear protein is released within the nucleus and Importins bind to a small GTPase protein (Ran) Importins are exported from the nucleus and can be recycled Ran is transported back to nucleus following hydrolysis of GTP.

Question 28

Are mitochondrial proteins folded or unfolded?

Answer 28

Unfolded

Question 29

What type of sequence do proteins destined for the mitochondrial matrix contain?

Answer 29

Amphipathic N-terminal signal sequence (10-80 amino acids)

Question 30

Which two stages of mitochondrial targeting requires energy in the form of ATP?

Answer 30

Transport of the protein across the inner mitochondrial membrane by TIM proteins. Folding the protein into its native conformation once inside the matrix - helped by chaperones.

Question 31

In mitochondrial targeting, what does the protein MSF do?

Answer 31

It is a chaperone which stabilises the unfolded protein in the cytosol.

Question 32

In mitochondrial targeting, what does the protein TOM do?

Answer 32

Recognises the signal sequence on the protein and forms a protein channel to allow it through the outer membrane of the mitochondria.

Question 33

In mitochondrial targeting, what does the protein TIM do?

Answer 33

Transports the protein across the inner membrane of the mitochondrion.

Question 34

In mitochondrial processing, what does the protein MPP do?

Answer 34

Cleaves the signal sequence on the protein being transported in the matrix of the mitochondrion.

Question 35

What type of enzymes are targeted to lysosomes?

Answer 35

Lysosomal hydrolases

Question 36

Describe lysosomal targeting

Answer 36

A signal patch on the lysosomal hydrolase attracts M6P - binds to N-linked oligosaccharides on the protein. This occurs in the Golgi and involves two enzymes. M6P groups on the protein are recognised by receptors on the trans side of the Golgi and vesicles are pinched off for transport for transport to the lysosome. Once at the lysosome, the acid pH causes the protein and M6P to dissociated. Phosphate group is removed from the M6P to ensure that the protein doesn't return to the Golgi with it.

Question 37

What are the two enzymes needed to bind M6P to the lysosomal hydrolase? Which one can be deficient?

Answer 37

N-acetylglucosamine phosphotransferase - deficient | N-acetylglucosamine phosphoglycosidase

Question 38

What is I-Cell disease?

Answer 38

A genetic defect in N-acetylglucosamine phosphotransferase. Lack of M6P addition to lysosomal targeted proteins. Lysosomal hydrolases are mis targeted for secretion. They can have a high concentration in urine and blood.

Question 39

How are proteins returned to the ER from the Golgi?

Answer 39

ER resident proteins have the sequence Lys-Asp-Gly-Leu (KDEL) near the C-terminus. If these proteins are transported to the Golgi, they will interact with KDEL receptors, a process that is enhanced by the low pH there. ER proteins bound to the receptors are transported in vesicles. ER proteins dissociate from the receptors in the neutral pH within the ER. KDEL receptor is transported back to the Golgi.

Question 40

What does the triple helix structure of collagen allow?

Answer 40

High tensile strength Non-extensible Non-compressible

Question 41

Why does a lack of vitamin C lead to scurvy?

Answer 41

The enzyme prolyl hydroxylase allows hydrogen bonds to stabilise the triple helix Prolyl hydroxylase requires vitamin C and iron ions to do this Scurvy is due to weak tropocollagen triple helices.