Post-translational Processing of Proteins

Question 1

Explain proteolytic cleavage.

Answer 1

Breaking peptide bonds to remove part of the protein

Question 2

Explain chemical modification.

Answer 2

Addition of functional groups to amino acid residues.

Question 3

Why is extracellular post-translation processing of a protein sometimes needed?

Answer 3

Because you don’t want the protein to be active within the cell. An example is collagen fibres.

Question 4

Where are proteins destined that are synthesised on free ribosomes?

Answer 4

For the cytosol or post-translational import into organelles.

Question 5

Where are proteins destined that are synthesised by ribosomes on the rER?

Answer 5

For the membranes or secretory pathway via co-translational insertion.

Question 6

Give 4 required steps for protein sorting.

Answer 6

A signal that is intrinsic to the protein.
A receptor that recognises the signal and directs it to the correct membrane.
A translocation machinery like a channel or a vesicle.
Energy to transfer the protein to its new place.

Question 7

Give three types of proteins that are targeted for secretion and examples of each.

Answer 7

Extracellular proteins (insulin, pepsin)
Membrane proteins (integral, channel)
Vesicular proteins (lysosomes, endosomes)

Question 8

Give the two types of secretion and briefly outline their function.

Answer 8

Constitutive secretion - happens all the time and is a slow continuous secretion.
Regulated secretion - secreted on signal

Question 9

What do endocrine cells secrete?

Answer 9

Hormones

Question 10

What do exocrine cells secrete?

Answer 10

Digestive juices, serous, mucous, sebum.

Question 11

What do neurocrine cells secrete?

Answer 11

Neurotransmitters

Question 12

What is a lot of rER in a cell an indication of?

Answer 12

A lot of secretion of extracellular proteins, membrane proteins or vesicular proteins.

Question 13

What is a signal sequence?

Answer 13

An N-terminal amino acid sequence that is around 5-30 amino acids long. The central region of the signal sequence is hydrophobic and it is able to form an alpha-helix.

Question 14

What does the ‘Pre’ part of Preproalbumin mean?

Answer 14

It refers and defines the signal sequence of the Preproalbumin that is then removed during processing.

Question 15

Explain what a signal recognition particle (SRP) is.

Answer 15

It is a riboprotein complex that recognise and bings to the signal sequence of a protein.

Question 16

Outline the process of the synthesis of secretory proteins and their translocation across the ER membrane.

Answer 16

Ribosome binds to the mRNA and starts to translate it. The signal sequence at the N-terminal is synthesised and a SRP recognise it and binds to the signal sequence. The SRP binds to a SRP receptor on the ER membrane and a channel protein (translocon) on the ER membrane opens, the SRP now breaks off from the signal sequence. The peptide chain starts to move into the ER lumen and the signal sequence gets ‘stuck’ in the translocon due to its hydrophobic nature. A signal peptidase cleaves the signal sequence from the rest of the peptide chain and the rest of the peptide chain is transported into the ER lumen. It is no proalbumin for example instead of preproalbumin.

Question 17

Outline the process of the synthesis of a type 1 membrane protein at the ER.

Answer 17

The same process as with preproalbumin however a peptide chain destined to become a membrane protein has a hydrophobic chain somewhere in the middle of its entire length. The signal sequence is still being cut of but as the peptide chain is being transported into the ER lumen via the translocon it gets stuck due to its hydrophobic sequence called a stop-transfer anchor sequence. This part is then embedded into the ER membrane.

Question 18

Why is the formation of disulphide bonds important for a lot of proteins?

Answer 18

Proteins that are destined to be extracellular can be fragile. The disulphide bonds make the protein more rigid and less prone to damage. The disulphide bonds also folds the protein.

Question 19

What is N-linked glycosylation?

Answer 19

Sugars (carbohydrates) are added on an asparagine side chain. It is a reaction involving an amino group.

Question 20

Why is glycosylation of proteins important? (4 examples)

Answer 20

Correct protein folding to get correct function.
Protein stability
Facilitates interactions with other molecules. This is especially important for extracellular proteins and membrane proteins.
Deficiencies in N-linked glycosylation lead to severe inherited human diseases such as: Congenital disorders of Glycosylation (CDG) (Defective enzymes)

Question 21

What is the role of protein disulphide isomerase (PDI)?

Answer 21

A reduced substrate protein with SH-groups are being oxidised by a oxidised PDI. The oxidised PDI has a disulphide bond which becomes reduced by the hydrogens from the substrate protein. The reduced PDI leaves and the substrate protein becomes oxidised with two S- ions. These S- ions bind together to form a disulphide bond. This fold the proteins.

Question 22

What happens if there are folding problems?

Answer 22

Proteins may be trapped in mis-folded conformation.
Proteins contain mutations.
Proteins may associate incorrectly with other sub-units-

Question 23

What is used to correct folding problems?

Answer 23

An endoplasmic reticulum chaperone protein. E.g. BiP, Calnexin and Calreticulin.

Question 24

What happens if mis-folding can’t be corrected?

Answer 24

Protein may be returned to cytosol for degradation.

Question 25

What happens if the cytosol can’t deal with all the mis-folded proteins that are returning?

Answer 25

Proteins might build up to a toxic level in the ER so the ER stops working. This can be caused by a single mutation.

Question 26

Outline the modifications made in the Golgi.

Answer 26

Sorting - phosphorylation (cis Golgi network)
Removal of Man (cis cisterna)
Removal of Man and addition of GlcNAc (medial cisterna)
Addition of Gal and NANA (trans cisterna)
Sulfation of tyrosine and carbohydrates (trans Golgi netw)

Question 27

Give an example of a protein that is not mature after modification made in the Golgi.

Answer 27

Collagen.

Question 28

What is the basic unit of collagen fibres?

Answer 28

Tropocollagen

Question 29

What are the characteristics of Tropocollagen?

Answer 29

Long rod-shaped protein
3 polypeptide alpha chains wrapped around each other. (NOT alpha helices!)
Glycine in every third position. (Gly-X-Y)
Triple helix
X and Y are usually proline and hydroxyproline.

Question 30

Where does glycine sit in the triple helix of tropocollagen and explain how it is possible for it to be there.

Answer 30

In the middle of the helix. This is due to it being the smallest amino acid. Any other amino acid would make the helix fall apart.

Question 31

Why is the hydroxyproline important in the tropocollagen?

Answer 31

Provides hydrogen bonds to stabilise the helix.

Question 32

Briefly outline the process of synthesis and modification of collagen in the ER.

Answer 32

The peptide chain enters the ER lumen and signal peptidase cleaves the signal peptide. Prepro alpha chain becomes a Pro alpha chains.
Hydroxylation of proline and lysine occurs to form hydroxyproline and hydroxylysine.
Glycosylation occurs where N-linked sugars are added.
Galactose is added to the hydroxylysine residues.

Question 33

What is needed for the hydroxylation of proline?

Answer 33

2-oxoglutarate, prolyl hydroxylase, oxygen, Fe2+ and vitamin C (ascorbate)

Question 34

How does a defiency in vitamin C result in a weak collagen fibre?

Answer 34

Proline is not hydroxylated into hydroxproline. Therefore no extra hydrogen bonds are formed in the triple helices of tropocollagen. This means that the tropocollagen becomes weaker.

Question 35

What is scurvy?

Answer 35

A deficiency in vitamin C. Weak connective tissue. Can be seen in the gum.

Question 36

Does tropocollagen exist intracellularly? If not, what precursor does?

Answer 36

No it doesn’t. The precursor is called Procollagen.

Question 37

What is the main structural difference between tropocollagen and procollagen? Why is this difference important?

Answer 37

Procollagen has a sequence of amino acids on each side of its chain (N-terminal and C-terminal) that do not form a triple helix. This makes it look like a candy wrapper. This is important because if the unwound N and C terminals weren’t there tropocollagen would bind to other tropocollagen inside the cell. Collagen fibres would form inside the cell and it would explode.

Question 38

How is pro collagen secretion done?

Answer 38

Glucose is added to O-links in the procollagen in the Golgi and it is then transported via a vesicle and transported out of the cell via exocytosis.

Question 39

Where is procollagen converted into tropocollagen?

Answer 39

Extracellularly by procollagen peptidases where the N-terminal and C-terminal are cleave off so there is only a triple helix.

Question 40

How do collagen fibres form?

Answer 40

By lateral association of tropocollagen by covalent cross-linkage. Lysyl oxidase plays a role here.

Question 41

What is Ehlers-Danlos syndrome?

Answer 41

A mutation in collagen type 5 or lysyl oxidase deficiency. This means that interconnective tissue becomes weaker and the junctions between dermis and epidermis get weaker as well. EDS can take many forms, skin, musculoskeletal or cardiovascular depending on what type it is.

Question 42

Why is collagen structurally stable?

Answer 42

Disulphide bridges.
Hydrogen bonds.
Covalent bonds.

Question 43

What are the different types of precursors of collagen?

Answer 43

Preprocollagen
Procollagen
and the base unit Tropocollagen