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MGD > Post translation modification of proteins. > Flashcards

Post translation modification of proteins. Flashcards

0
Q

What proteins are synthesised on ribosomes on the RER?

A

Those destined for secretory pathway

Membrane proteins

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1
Q

What proteins are synthesised on cytosolic ribosomes?

A

Proteins that remain in the cytosol

Proteins that are transported into organelles post translation

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2
Q

What is the targeting sequence of a protein destined for the lumen of the RER?

A

One or more basic amino acids followed by 6-12 hydrophobic amino acids, located at the N terminus.

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3
Q

Is the signal for a protein destined for the lumen of the ER cleaved?

A

Yes

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4
Q

What is the signal for a protein destined for the mitochondrial matrix?

A

Amphipathic helix of 20-50 residues - hydrophobic side and side containing arginine and lysine (charged) at the N terminus
May also have internal ‘stop transfer’ - hydrophobic.

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5
Q

Is the signal that directs a protein into the mitochondria cleaved?

A

Yes.

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6
Q

What signal directs a protein into the peroxisome?

A

S K L at C terminus (serine, lysine, leucine)

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7
Q

Is the signal that targets a protein to the peroxisomes cleaved?

A

No

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8
Q

What signal targets a protein to the nucleus?

A

An internal signal - either a cluster of five basic amino acids or two smaller clusters separated by roughly ten amino acids. Must be on the surface of the folded protein.

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9
Q

How do proteins arrive in mitochondrial matrix?

A

Maintained unfolded by chaperones (MSF, mitochondrial import stimulation factor)
Signal binds receptor
Fed through pore in outer membrane (translocase of the outer membrane, TOM), and through adjacent channel in inner membrane (translocase of the inner membrane, TIM)
Target signal cleaved by matrix processing protease

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10
Q

Is the signal that targets a protein to the nucleus cleaved?

A

No

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11
Q

Is energy required for targeting proteins to the mitochondrial matrix, and if so what for?

A

MSF (mitochondrial import stimulating factor) uses ATP to maintain the protein in an unfolded form.
mHsp70 (mitochondrial heat shock protein 70) uses ATP hydrolysis to drive translocation.

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12
Q

What causes pyruvate dehydrogenase deficiency?

A

Mutation at codon 10 of nuclear matrix targeting signal (N-MTS) of the E1alpha subunit
Results in arg to pro substitution
Removal of one hydrophobic residue from hydrophobic face of amphipathic helix
Proline is a helix breaker.
Reduced uptake of protein into mitochondrial matrix.

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13
Q

How are proteins transported to the nucleus?

A

Folded cargo protein with nuclear localising signal (NLS) binds importin (carrier protein)
Enters nucleus via nuclear pore
Ran-GTP binds importin, cargo protein displaced by conformational change, importin bound to Ran-GTP recycled to cytoplasm.
Hydrolysis of GTP releases Ran-GDP, Pi and importin.

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14
Q

What is the point of retaining the signal sequence in a protein targeted to the matrix?

A

Allows re-importing when nucleus is reformed following cell division.

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15
Q

What is Swyer syndrome?

A

Loss of mutation of nuclear localising signal (NLS) means sex determining region Y (SRY) protein not targeted to nucleus.
Required for testis differentiation.
XY but outwardly female.

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16
Q

What causes:
Leri-Weill dischondrosteosis
And
Langer mesomelic dysplasia?

A

Mutation in nuclear localisation signal of SHOX transcription factor.
SHOX required for skeletal development - leads to short stature.

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17
Q

How are proteins targeted to peroxisomes?

A

Peroxisomal import receptor binds folded proteins with peroxisome targeting sequence.
Peroxisomal import receptor integrates with translocon and opens it.
Peroxisome targeting sequence dissociates.
Receptor returns to cytoplasm - requires hydrolysis of ATP.

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18
Q

What is rhizomelic chrondrodysplasia punctata?

A

Mutation in Pex7 (receptor for a set of peroxisomal enzymes)

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19
Q

What cells undergo regulated secretion?

A

Neurocrine cells - neurotransmitters
Endocrine cells - hormones
Exocrine cells - digestive juices

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20
Q

How are organelles distributed in a polarised cell such as a pancreatic acinar?

A

Nucleus and RER at basal end of the cell.

Secretory granules closer to lumen of the duct.

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21
Q

What is SRP?

A

Multidomain riboprotein
Mediates a three way interaction between a receptor in the ER, the ribosome and the signal peptide.
Allows proteins to enter the lumen of the ER as they are being synthesised.

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22
Q

How does a protein targeted to the ER arrive in the lumen?

A

Ribosome starts to synthesise protein. Signal recognised by SRP (signal recognition particle)
Binds SRP receptor on ER membrane along with 2 GTP molecules.
Protein is synthesised through the translocon.
Signal sequence cleaved by signal peptidase.
Protein is folded in the lumen.

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23
Q

Is energy required for translocation of proteins into the lumen of the ER?

A

Yes - GTP is cleaved to release SRP

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24
Q

How are membrane proteins targeted?

A

Same as for proteins destined for lumen of ER.
Then a second hydrophobic sequence (stop transfer anchor sequence) prevents the protein moving any further into the lumen and hence anchors it in the plasma membrane.

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25
Q

List some (7) functions of the ER.

A 
Hydroxylation of lysine and proline
Glycosylation
Insertion of proteins into membranes
Formation of disulphide bridges
Folding of proteins
Proteolytic cleavage
Assembly of multisubunit proteins
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26
Q

Why is glycosylation of proteins important?

A

Increases stability (decreases access of proteases)
Interactions with other molecules
Correct folding of proteins

27
Q

Describe the process of N linked glycosylation.

A

Sugar (consisting of glucose, mannose and N-acetylglucosamine) is preassembled on dolichol phosphate.
Transferred to R group of asparagine (has amino group)
Occurs in ER

28
Q

Why are peptidyl-prolyl isomerases important?

A

Catalyse the interconversion of cis and trans residues of proline.
Needed to ensure folding of proteins occurs correctly, especially IgGs.

29
Q

How do disulphide bonds form in the ER?

A

Oxidised PDI (protein disulphide isomerase) oxidises substrate protein to form disulphide bond, and PDI is reduced to have to free thiol groups.

30
Q

PDI (protein disulphide isomerase) is normally resident in the ER. How is it retained in the ER and not secreted?

A

Has KDEL sequence (lysine, aspartate, glutamate, leucine) at the Ca terminus.
If the PDI arrives in the cis Golgi, KDEL signal binds KDEL receptor, transported back to ER in COPI coated vesicle.
Signal is retained - process can repeat.

31
Q

What causes PDI to bind KDEL receptors in cis Golgi, and dissociate in ER?

A

Lower pH in Golgi, higher ph in ER.

32
Q

What is the response of chaperone proteins to misfolded proteins?

A

BiP (binding immunoglobulin protein) binds to exposed amino acid sequences that would normally be buried in misfolded proteins, allows PDI to oxidise and reduce disulphide bridges to allow protein to be properly folded.
Calnexin and calreticulin bind oligosaccharides on incompletely folded proteins. They retain the proteins in the cell, and also up regulate the transcription of chaperones, and down regulate the translation of the misfolded protein.

33
Q

What is the result of misfolding of proteins that cannot be corrected?

A

Proteins accumulate to toxic levels.

Or return to cytosol for degradation - no protein.

34
Q

What are the components of the Golgi apparatus and their functions?

A

cis Golgi network. Phosphorylation of oligosaccharides on lysosomal proteins.
cis cisterna. Removal of man.
Medial cisterna. Removal of man, addition of N-acetylglucosamine.
Trans cisterna. Addition of galactose and N-acetylneuraminic acid
Trans Golgi network - sulphation of tyrosine residues and carbohydrates.

35
Q

What is the destination of secretions from Golgi?

A

Lysosomes, plasma membrane, secretory vesicles.

36
Q

How are lysosomal enzymes targeted?

A

Lysosomal enzyme (that’s been glycosylated-N linked) has phosphate group added to OH group on sixth carbon of mannose.
This is the signal - mannose 6-phosphate.
Requires N-Acetyl glucosamine phosphotransferase and phosphodiesterase.
UDP-n-Acetyl glucosamine (GlcNAc) loses phosphorous (energy input)
Occurs in the Golgi.

37
Q

What is I cell disease?

A

Fatal inherited condition - N acetyl glucosamine phosphotransferase deficiency means enzymes cannot be targeted to the lysosomes, lysosomes become bloated with undegraded material.

38
Q

How do enzymes that have been labeled with mannose 6-phosphate arrive in the lysosomes?

A

They bind mannose-6-phosphate receptors in the trans Golgi network and bud off into a clathrin coated vesicle.
Mannose 6-phosphate dissociates from its receptor in the low pH of the lysosomes.
Phosphate is cleaved in the lysosomes (by phosphatase)
Receptors are recycled back to the trans Golgi network.

39
Q

Does the transfer of enzymes to lysosomes require energy?

A

Not directly, but phosphotransferase requires ATP indirectly.

40
Q

What is O linked glycosylation?

A

Attachment of sugar to the OH group of serine or threonine.

Occurs in Golgi

41
Q

What undergoes o linked glycosylation?

A

Proteoglycans - extracellular matrix and mucus secretions.

42
Q

By what means is collagen secreted?

A

Constitutive

43
Q

By what means is insulin secreted?

A

Regulated secretion.

44
Q

What is secreted by constitutive secretion?

A 
Collagen
Immunoglobulins
Prohormones
Albumin
Fibronectin
Laminin
Receptors
45
Q

What is secreted by regulated secretion?

A 
Insulin
Trypsin
Glucagon
ACTH
Alpha MSH
Beta Endorphin
Beta lipotropin
46
Q

How abundant is collagen?

A

Most abundant bodily protein (25-35%)

47
Q

What cells synthesise collagen?

A

Fibroblasts.

48
Q

What is the structure of tropocollagen? How does this affect function?

A

Triple helix - right handed
Three alpha chains, each 1000 amino acids long
300nm rod shaped protein, 1.5nm diameter
Every third amino acid is glycine. Many prolines and hydroxyprolines.
Not extensible or compressible, high tensile strength.

49
Q

How is tropocollagen stabilised?

A

Hydrogen bonds between alpha chains

50
Q

What chains are present in type 1 collagen?

A

Two alpha 1 chains and an alpha 2 chain.

51
Q

What alpha chains are found in type two collagen?

A

Three alpha one chains.

52
Q

What alpha chains are present in type three collagen?

A

Three alpha one chains.

53
Q

What alpha chains are present in type four collagen?

A

Two alpha one chains and an alpha two chain

54
Q

What alpha chains are found in type five collagen?

A

Two alpha one chains and an alpha two chain.

55
Q

How is procollagen synthesised?

A

Synthesised on ribosomes associated with ER. Enters lumen of ER as prepro alpha chain.
Signal cleaved by signal peptidase - pro alpha chain.
Proline and lysine residues are hydroxylated by prolyl hydroxylase and lysyl hydroxylase
Undergoes N linked glycosylation at the C terminus
Addition of galactose to hydroxylysine.
alpha chains align and disulphide bonds form at C terminus - 250 aas
Triple helical procollagen forms from C to N terminus.
Glucose is added to galactose on hydroxylysine residues.
Then secreted - exocytosis.

56
Q

prolyl hydroxylase requires oxygen, iron and ascorbate - vitamin C
Associated with PDI in the ER
OH groups increase H bonding, increases stability of helix
Deficiency of vitamin C decreases strength of tropocollagen - scurvy.

A

Describe the action of prolyl hydroxylase

57
Q

How does procollagen form collagen?

A

C and N terminal peptides cleaved - forms tropocollagen.
By procollagen peptidases - extracellularly.
Lateral association into 50nm wide fibrils - (banding!), then cross linking of tropocollagen - covalent.
Fibrils associate into a collagen fibre.

58
Q

How are tropocollagen molecules cross linked?

A

Lysine residues are oxidised (lysyl oxidase) to form aldehyde derivatives.
They spontaneously form covalent aldol cross links

59
Q

What is required for the activity of lysyl oxidase?

A

Vitamin B6 and copper ions.

Present extracellularly

60
Q

What is Ehlers Danlos syndrome?

A

Either mutation in type 5 collagen

Or lysyl oxidase deficiency.

61
Q

What explains the banding observed in collagen?

A

Dark bands - gaps between tropocollagen, heavy metal stain

Pale bands - no gaps.

62
Q

How is insulin synthesised?

A

Disulphide bonds form between cysteine residues - occurs in ER
Connecting peptide cleaved - occurs post Golgi.

63
Q

What enzymes cleave proinsulin?

A

PC3 endopeptidase cleaves internally to the peptide that is removed, after two Arg residues
PC2 endopeptidase cleaves C terminus end of peptide that is removed
Carboxypeptidase cleaves Arg residues from amino end of insulin.

64
Q

How can proteolytic processing yield different products?

A

Different amounts of enzymes in different locations.

65
Q

Why is proteolytic processing common in the secretory pathway?

A

Can give rise to very short products - enkephalins 5aas - too short to enter ER by cotranslation
Secreted enzymes would be dangerous if active outside of the cell.
Multiple products from the same polypeptide - saves energy.
Doesn’t activate insulin receptor.

MGD (6 decks)

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