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CORE Block 4 > Co- and post-translational protein processing > Flashcards

Co- and post-translational protein processing Flashcards

1
Q

Define protein targeting.

A

Process by which proteins are transported from their site of synthesis to their proper site of function

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2
Q

What are the general mechanisms of protein targeting?

A
  1. recognition of protein via a signal
  2. interaction with receptor at target site
  3. NTP-dependent translocation across target membrane
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3
Q

Where are proteins targeted to post-translationally?

A

nucleus, mito, or peroxisome

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4
Q

Where are proteins targeted to co-translationally?

A

plasma or other membranes, , ER, secretory vesicles or lysosomes

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5
Q

If a protein does not have a targeting signal, where will it end up?

A

It will remain in the cytosol.

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6
Q

Walk through the co-translation of secreted proteins.

A
  1. Signal sequence at N-term of translated protein is recognized by SRP (signal recognition particle) and is bound
  2. SRP binds SRP receptor GTPase when GTP is bound to both SRP and its receptor.
  3. Both GTPs are hydrolyzed, and the signal sequence enters the now open translocon
  4. translation continues through the translocon into the lumen of the ER
  5. a signal peptidase in the ER membrane may cleave the N-terminal signal sequence if it is not important for the protein’s function
  6. the protein will fold in the lumen, the translocon will close, and if there are not additional targeting sequences, the protein will be secreted from the cell
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7
Q

Describe the SRP

A

it is a complex of RNA and protein that has a hydrophobic stretch at the core that will enter the ER membrane

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8
Q

If a protein has no additional targeting sequence after its signal sequence is cleaved in the ER, what is the fate of the protein?

A

It will be secreted from the cell.

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9
Q

Describe co-translational targeting of ER-resident proteins.

A
  1. Same translation steps as proteins that are secreted,
  2. the protein will have additional N-term retention signal telling it to stay in ER, or it may have a C-term retrieval signal for proteins that move through the vesicular pathway as components on vesicles that are recycled.
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10
Q

What is the C-term ER retrieval sequence?

A

KDEL

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11
Q

Describe co-translational targeting of integral/transmembrane proteins.

A
  1. Translation is similar to that of proteins secreted from the cell, but proteins are only partially translocated through the ER membrane
  2. threading of the protein through the ER membrane stops due to stop transfer or membrane retention sequences in the protein
  3. non-ER destined membrane proteins bud off from the ER membrane in vesicles in response to a signal and go to their target membrane.
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12
Q

What is the default membrane for integral/plasma membrane proteins to end up in if there is no extra sequence?

A

plasma membrane

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13
Q

Describe the stop transfer sequence

A

It is a 22 amino acid sequence that spans the ER membrane as a single alpha helix which gets stuck in the membrane. Proteins destined for membranes will have this.

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14
Q

For transmembrane proteins synthesized in the ER, on which side of the membrane is the C- and N-terms of the protein?

A

They can be on either side, or both on the same side depending on the sequence of the protein

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15
Q

What are lysosomes?

A

membrane-enclosed compartments that contain hydrolytic enzymes required for intracellular digestion

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16
Q

Describe the lumen of the lysosome?

A

It is maintained at an acidic pH, because lysosomal enzymes are only active in acidic environments.

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17
Q

How is the lumen of the lysosome maintained at an acidic pH?

A

Proton pumps hydrolyze ATP to move H+ into the lysosome against its concentration gradients.

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18
Q

How are proteins targeted to the lysosome?

A
  1. proteins are translated in ER as they are for secreted proteins
  2. proteins are N-glycosylated in the ER
  3. terminal mannose is phosphorylated at C#6
  4. mannose-6-phosphate recognizes clathrin coated pits, which then lose their coat and bind to the late endosome
  5. low pH causes dissociation of hydrolase from the receptor
  6. phsophatase removes phosphate
  7. late endosome fuses with lysosome
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19
Q

Describe the enzymatic reactions involved in targeting proteins to the lysosome.

A

two steps to phosphorylate mannose:

  1. UDP-GlcNAc is transferred to mannose by phosphotransferase
  2. GlcNAc is removed by phosphoglycosidase, leaving behind just the phosphate on mannose
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20
Q

When considering protein translocation to the lysosome, what happens if there is a mutation in phosphotransferase or phosphoglycosidase?

A

The protein will go down the default pathway and be secreted from the cell.

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21
Q

What are the general mechanisms for post-translational targeting?

A

Overall the same as co-translational targeting:

  1. recognition of protein
  2. interaction with receptor
  3. NTP-dependent translocation across target membrane
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22
Q

Describe nuclear import.

A
  1. cargo protein is recognized and bound by importin protein
  2. cargo-importin complex enters the nucleus through the nuclear basket, along with Ran-GDP
  3. Ran-GDP encounters guanine exchange factor and gets a GTP
  4. Ran-GTP interacts with importin-cargo complex, binding importin and releasing cargo
  5. Ran-GTP-importin exits the nucleus and encounters a phosphatase, releasing importin from Ran-GDP and recycling both players
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23
Q

Describe the nuclear localization signal.

A

4-8 basic amino acids internal in the protein (not cleaved off during translocation

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24
Q

What phenomenon drives the translocation of proteins into the nucleus and the recycling of factors involved?

A

Concentration gradients and enzyme compartmentalization:

  • phosphatase is only in cytosol, and guanine exchange factor is only in the nucleus
  • Ran-GTP is higher in nucleus so it moves to the cytosol
  • Ran-GDP is higher in cytosol so it moves to the nucleus
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25
Q

Compare protein translocation to the nucleus and to the peroxisome.

A

They are similar, but there are no pore-like structures in the peroxisome membrane like there are for the nucleus. The peroxisome targeting pathway is not well-known

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26
Q

Describe the PTS1 signal and what it stands for.

A

peroxisomal targeting sequence 1

  • uncleaved, C-terminal tripeptide motif
  • small residue, basic residue, hydrophobic residue
  • loosely conserved
  • these proteins are first fully translated in the cytosol ribosomes
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27
Q

Describe the PTS2 motif and what it stands for.

A

Peroxisomal translocation signal 2

  • cleaved, N-terminal nine amino acid motif
  • less frequent signal in most organisms than PTS1
28
Q

What are the two general mechanisms for targeting proteins to the peroxisome?

A
  1. signal sequence
  2. direct cytoplasm to peroxisome mechanism that requires chaperones (not well known)

(both energy dependent)

29
Q

What is the overview of protein targeting signals?

A
30
Q

List the common protein covalent modifications:

A
  1. ADP-ribosylation
  2. phosphorylation
  3. hydroxylation
  4. ubiqutiination
  5. glycosylation
  6. protein lipidation
31
Q

what type of protein covalent modification helps target proteins to cellular membranes?

A

protein lipidation

32
Q

What is the most common type of protein covalent modification?

A

glycosylation

33
Q

What are the functions of protein glycosylation?

A
  1. increase solubility (much more polar)
  2. protect against proteolysis by guarding termini and pushing proteases away
  3. influence spatial organization (can affect folding if added co-translationally)
  4. involved in recognition and antigenicity (proteins on cell surface)
34
Q

Describe N-glycosylation

A

linkage to amide group on asparagine in the sequence Asn-X-Ser/Thr

35
Q

Describe O-glycosylation

A

linkage to OH- groups in certain serine or threonine residues. Also on hydroxylysine of collagen

36
Q

How is the N-glycosidic linkage formed?

A

Uses a branched, preassembled oligosaccharide that is linked to dolichol phosphate and transferred en bloc (in its entirety) to the protein. Dolichol phosphate is highly hydorphobic and is embedded in the membrane.

37
Q

Describe the addition of sugars and reorganization of sugars in N-glycosylation.

A
  • preassembled block of sugars is added to dolichol phosphate on cytosolic side of ER
  • flippase moves dolichol phosphate and sugars to lumen side of ER, to which new sugars are added or taken away as the protein begins to be translated into the lumen
  • the sugars are then transferred onto the asparagine of the N-term that is inside the ER lumen
38
Q

N-glycosidation begins in the ER. Where does it finish?

A

In the golgi

39
Q

Describe the process of O-glycosylation.

A
  1. Uses UDP- or GDP-sugar
  2. occurs post-translationally in the golgi
  3. glucosyltransferases transfer sugars in a stepwise fashion
  4. can also occur in the cytosol, but this is a single sugar addition
40
Q

Where do proteins that are O-glycosylated with a single sugar in the nucleus end up?

A

They are normally targeted to the nculeus and have a similar affect on the function of the protein as does phosphorylation

41
Q

Describe the glycosylphosphatidyl inositol anchor

A

fatty acid chains of the anchor stick into the membrane. the polar components stick into the cytosol or lumen of the ER where the proten is anchored. these proteins will end up on the cell surface if they have no additional signal sequences

42
Q

Why are glycosylphosphatidyl inositol anchors used?

A

They are rapidly moved around different membranes, can be concentrated in the membrane.

43
Q

Describe myrositoylation and prenylation.

A

Both types of covalent lipidation anchors. Myrositoylation is co-translational and added to N-term of protein on inner surface of plasma membrane and is non-reversible. Prenylation is post-translational.

44
Q

How can protein folding be assisted?

A
  1. disulfide bond formation (covalent modiciation)
  2. chaperones
45
Q

Describe disulfide bond formation as a covalent modiciation.

A
  1. can be spontaneous in the proper environment
  2. linked with 3D folding of protein
  3. occurs in ER between cys residues which can be nearby or far apart.
  4. protein disulfide isomerase catalyzes the reaction
46
Q

How are disulfide bonds broken and reformed?

A

broken by urea and mercaptoethanol, and removal of these agents with dialysis can rescue the disulfide bonds.

47
Q

Describe chaperones and in general how they work.

A
  • bind to nonnative state of other proteins and assist them to reach functional conformation
  • originally identified as proteins that increased in abundance in response to heat shock (need more help refolding denatured proteins under heat shock so more chaperones are expressed)
  • chaperones recognize hydrophobic surfaces of nascent proteins and form a reversible complex with them to prevent irresversible aggregation
48
Q

Describe the Hsp70 family of molecular chaperone proteins.

A
  • recognize hydrophobic stretch on protein’s surface
  • after binding, Hsp70 hydrolyze ATP and is released from the protein
  • repeated cycles of Hsp protein binding and release help the target protein to fold properly
49
Q

When do Hsp60 chaperones come into play?

A

When Hsp70 proteins can no longer cut it

50
Q

Describe the Hsp60 family of molecular chaperones.

A
  • misfolded proteins are captured in a barrel of Hsp60 machinery (isolation-chamber)
  • after ATP hydrolysis, Hsp60 machinery ejects proteins
  • repeated cycles to help protein folding
51
Q

Describe the unfolded protein response (UPR).

A

When many unfolded proteins are sensed by the cell, the response tells the cell to stop making more proteins and to then fix the problem.

  • unfolded proteins in the ER lumen bind protein BIP, which normally binds Ire1 monomers, allowing the monomers to dimerize
  • the dimer in the ER membrane of cytosolic side acts as an endonuclease to cleave Hac mRNA
  • Hac is then translated and functions as a transcription factor to increase the number of chaperones being expressed
52
Q

Why are peptide bonds cleaved?

A
  1. removal of initiating methionine
  2. removal of signal sequence: preproprotein -> proprotein
  3. conversion of proprotein to functional, native protein
53
Q

Describe the cleavage of insulin to form functional insulin.

A

preproinsulin is formed, initiating methionine and signal sequence is cleaved to form proinsulin in ER, disulfide bonds are formed, C-term cleavage takes place in the golgi, resulting in functional insulin protein

54
Q

Why are protein multimeric complexes sometimes necessary?

A

Many proteins must associate to form multimeric complexes in order to function, such as ribosomes or antibodies (heavy and light chains). On their own, the protein components of these complexes have no function.

55
Q

What are the repeated amino acid sequences for collagen?

A

gly-X-Y where X and Y are proline and hydroxyproline

56
Q

What type of secondary structure does collagen have?

A

lef-handed alpha-chain (not helix!!)

57
Q

Describe the tertiary structure of collagen

A

3 alpha chains coming together to form a right-handed triple helix.

58
Q

Describe the process of collagen synthesis.

A
  1. prepropeptide is synthesized in ER lumen
  2. methionine and signal sequence are cleaved
  3. all in the ER lumen, propeptide is covalently modified with hydroxylation of proline and lysine, N-glycosylation, and triple helix formation
  4. intracellular procollagen transported to golgi where O-glycosylation and packaging takes place. No additional signal sequence, so secreted to ECM
  5. extracellular procollagen has N and C terminal peptides cleaved
  6. tropocollagen undergoes helical aggregation
  7. collagen fibrils undergo cross-linking, resulting in mature collagen fibrils
59
Q

What allows for collagen to be very tightly coiled?

A

Every third amino acid is glycine

60
Q

Describe the generic scheme of the collagen alpha chain

A

consists of N-term, repetitive sequence, and C-term

61
Q

In collagen, why is proline hydroxylated, and what is the mechanism of hydroxylation?

A

Hydroxylation occurs because hydrogen bonding is required for tight coiling. It is catalyzed by ascorbate (vitamin C) and prolyl hydroxylase.

62
Q

Describe triple helix coiling in collagen synthesis.

A

cysteines starting at the C-terminus form disulfide bonds, bringing the alpha-chains together. Coiling moves from C-N terminus.

63
Q

What would happen to a misfolded alpha-chain in collagen synthesis?

A

It would be recognized as a misfolded protein and would be degraded. Thus, mutations in alpha-chain genes could cause issues in collagen.

64
Q

Why does tropocollagen aggregate, but procollagen does not?

A

Tropocollagen consists of procollagen minus the N- and C-termini. What is left over is very hydrophobic and therefore aggregates.

65
Q

Why do lysines on collagen alpha-chains undergo aldol condensation (another covalent modification)?

A

Helps to hold the triple helices close together

66
Q

What occurs when proline is not hydroxylated using vitamin A in collagen formation?

A

Scurvey

67
Q

How does co- and post-translational covalent modifications lead to functional diversity of a protein?

A

Allows for the products from a single gene to be modified in different ways that allow for different functions.

CORE Block 4 (8 decks)

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