13. Post Translation Modification Of Proteins

Question 1

What are the two type of post translation modification?

Answer 1

1. Proteolytic cleavage

2. Chemical modification

Question 2

What is Proteolytic cleavage?

Answer 2

breaking peptide bonds to remove part of the protein

Question 3

What is Chemical modification?

Answer 3

addition of functional groups to amino acid residues

Question 4

Which proteins are synthesised on free ribosomes?

Answer 4

Protein destined for cytosol, or posttranslational import into organelles

Question 5

Which proteins are synthesises by ribosomes on the RER?

Answer 5

Protein destined for membrane or secretory pathway via co- translational insertion

Question 6

What is required for protein sorting?

Answer 6

• a signal (address), intrinsic to the protein
• a receptor that recognizes the signal and which directs it to the correct membrane
• a translocation machinery
• energy to transfer the protein to its new place

Question 7

Which proteins re targeted for secretion?

Answer 7

• Extracellular proteins
• Membrane proteins
• Vesicular proteins (lysosomes,
endosomes etc)

Question 8

What are the two types of secretion from cells?

Answer 8

• Constitutive secretion

* Regulated secretion

Question 9

Which secretion type is not regulated?

Answer 9

Constitutive secretion - happens all the time

Question 10

Give examples of cell which use regulated secretion?

Answer 10

1. Endocrine cells – secreting hormones
2. Exocrine cells – secreting digestive juices
3. Neurocrine cells – secreting neurotransmitters

Question 11

What is a signal sequence?

Answer 11

A signal is a short peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway.

Question 12

Describe the structure of a signal sequence

Answer 12

• N-terminal aa sequence • 5- 30 amino acids in length
• Central region rich in hydrophobic residues
• Able to form a-helix

Question 13

What does the pre part of preproalbumin mean?

Answer 13

“Pre” part of preproalbumin defines the signal sequence which is removed during processing

Question 14

What is the signal recognition particle?

Answer 14

The signal recognition particle (SRP) is an abundant, cytosolic, ribonucleoprotein (protein-RNA complex) that recognizes and targets specific proteins to the endoplasmic reticulum

Question 15

What is the SRP composed of?

Answer 15

Composed of 6 proteins and a short piece of RNA

Question 16

What does the SRP recognise and bind to?

Answer 16

Recognises the signal peptide, the ribosome and the receptor that is resent in the ER membrane

Question 17

Describe the synthesis of secretory proteins and their translocations across the ER membrane

Answer 17

1. Signal sequence on nascent polypeptide is recognised by the SRP
2. SRP binds to the polypeptide and ribosome
3. Translation halted by binding of SRP
4. SRP recognised by SRP receptor/docking protein on ER membrane
5. In making the interaction with the docking protein, the SRP is released from the signal sequence
6. This removes the inhibitory constraint on translation so translation continues
7. The signal sequence then interacts with a signal sequence receptor (SSR) within a protein translocator complex
8. The ribosome becomes anchored to this pore complex, through which the growing polypeptide chain is extruded.
9. The signal sequence is cut by the enzyme signal peptidase
10. Once translation is finished, the ribosome is detached and goes back into the cytoplasm to find more mRNA

Question 18

What are the functions of the endoplasmic reticulum?

Answer 18

• Insertion of proteins into membranes
• Specific proteolytic cleavage
• Glycosylation
• Formation of S-S bonds
• Proper folding of proteins
• Assembly of multisubunit proteins
• Hydroxylation of selected Lys and Pro residues

Question 19

What is N-linked glycosylation?

Answer 19

Addition of sugars to the amino group on an asparagine residue

Question 20

Why is glycosylation of proteins important?

Answer 20

• needed for Correct protein folding
• needed or Protein stability
• Facilitates interactions with other molecules

Question 21

What do deficiencies in N-linked glycosylation lead to?

Answer 21

Deficiencies in N-linked glycosylation lead to severe

inherited human disease: Congenital disorders of glycosylation (CDG)

Question 22

What type of bond formation occurs n the ER lumen?

Answer 22

Disulphide bonds

Question 23

Between which groups do disulphide bonds form?

Answer 23

Disulphide bonds four between sulfhydryl groups in cysteine residues

Question 24

What is the role of protein disulphide isomerase (PDI) in the formation of disulphide bonds in the ER lumen?

Answer 24

• PDI is important in allowing the proteins to form the correct disulphide bonds
• If there are many cysteine residues, there are multiple possible combinations of disulphide bonds, the PDI ensures the correct pairs of disulphide bonds form so the protein fold correctly

Question 25

What happens if there are folding problems?

Answer 25

* Protein may be trapped in mis-folded conformation * Protein contains mutation resulting in mis-folding * Protein may be incorrectly associated with other sub-units

Question 26

Which protein in the ER attempt to correct the folding problems?

Answer 26

chaperone proteins

Question 27

What do chaperone proteins in the ER do?

Answer 27

They get misfolded proteins back to the correct folding state: • retain unfolded proteins in the ER * act as sensors to “monitor” extent of protein mis-folding * mediate increased transcription of chaperones * mediate reduction in translation

Question 28

What happens if mis-folding cannot be corrected?

Answer 28

• Protein may be returned to cytosol for degradation • Protein may accumulate to toxic levels in the ER resulting in disease > This may arise due to single mutation

Question 29

Which 2 organelles carry out post translation modification?

Answer 29

ER and golgi apparatus

Question 30

What is the basic unit of collagen?

Answer 30

Tropocollagen

Question 31

Describe the structure of collagen fibres

Answer 31

• 300nm rod-shaped protein • 3 polypeptides (a chains), each ~1000aa long • glycine in every 3rd position along each a chain ie (Gly-X-Y) n repeat • H-bonds between a chains stabilise structure • characteristic triple helix (right-handed) • mostly proline or hydroxyproline in X and some Y positions

Question 32

What are 3 physical properties of collagen?

Answer 32

* non-extensible * non-compressible * high tensile strength

Question 33

Why is glycine the only amino acid found in the middle of the helix?

Answer 33

Glycine is the only amino acid with a side chain small enough to fit in the middle of the helix

Question 34

How is collagen synthesised?

Answer 34

* Collagen alpha chains have a signal sequence that will target it to the ER for secretion * As each of the alpha chains of collagen are produced, they are entered into the lumen of the RER * The signal sequence is cleaved by signal peptidase - from preprocollagen a chains to procollagen a chains * Hydroxylation also occurs in the ER - addition of hydroxyl groups to proline and lysine residues * N linked glycosylation also occurs - addition of N linked oligosaccharides * Then there is addition of galactose to hydroxylysine residues * The 3 alpha chains come together due to disulphide bonds between cysteine groups of the 3 procollagen chains - forming the triple helix * Then winding of the helix occurs * the procollagen is then transferred to Golgi apparatus where it is modified some more and packaged not a vesicle * the procollagen is then secreted out of the cell by exocytosis * in the extracellular space, N and C terminal pro peptides are removed by procollagen peptidases, forming tropocollagen * the tropocollagen molecules come together to form collagen fibrils which come together to form collagen fibres. •

Question 35

What is the function of prolyl hydroxylase?

Answer 35

To add hydroxyl groups to proline residues. Hydroxylation allows increased H-bonding to stabilise triple helix

Question 36

What is Prolyl hydroxylase associated with?

Answer 36

associated with PDI in the ER

Question 37

What does Prolyl hydroxylase require for activity?

Answer 37

requires vitamin C and Fe++ ions for activity

Question 38

What causes scurvy?

Answer 38

Deficiency of vitamin C. | Scurvy is due to weak tropocollagen triple helices

Question 39

Why is tropocollagen secreted before final processing occurs?

Answer 39

Assembly of the mature collagen fibres would cause the cell to explode!

Question 40

Describe how cross-linking among tropocollagen forms?

Answer 40

Strong covalent cross links between lysine residues in individual fibrils form due to Lysol oxidase.

Question 41

What causes Ehlers-Danlos syndrome (EDS) ?

Answer 41

Mutation in collagen type V or lysyl oxidase deficiency