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Bioc 212 > Protein Folding in the Cell Lectures > Flashcards

Protein Folding in the Cell Lectures Flashcards

1
Q

What is PTM? (Post-Translational Modification)? Why is it useful?

A
  • Proteins can be chemically modified after translation
  • Important contributions to proteomic diversity and complexity and are essential for regulation of protein function and cellular signalling
  • Acts as a switch so that you can choose when there is a function etc.
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2
Q

What are the main types of PTM? And some different examples also.

A

1) Phosphorylation

2) Methylation

3) Acetylation

4) Glycosylation, Sumoylation, Ubiquitination

  • Cleaved into smaller peices by peptidases
  • covalent modification of N-terminus (co-translational)
  • covalent modification of side chainsL Introduce functional groups to proteins
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3
Q

What is the purpose of side chain modifications?

A
  • They are used for various cellular functions
  • Can change the surface or conformation of protein
  • Can create or block a binding site for other proteins
  • Many modifications are fast, so it is useful as switches
  • All modifications are mediated by enzymes
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4
Q

What is phosphorylation (PTM)?

A
  • Major regulatory mechanism
  • Phosphorylation is the addition of a phosphoryl (PO3) group to a molecule
  • There is phosphorylation on hydroxyl groups (S,T,Y)
  • adding the group changes the charge and the size
  • Kinases (enzymes) transfer phosphates from ATP (specific for side chain and surrounding peptide sequence)
  • Phosphatases remove phosphate
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5
Q

What are some characteristics of phosphorylation of S, T, and Y?

A

Kinase families:
- Ser/Thr kinases
- Tyr kinases
Dual specificity (Ser/Thr and Tyr)

Photophase families:
Ser/Thr photophatases
Protein Tyr phosphatases
Dual specificity (Ser/Thr and Tyr)

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6
Q

Describe phosphopeptide binding:

A
  • Phosphopeptides are modified self antigens which may induce an immune response
  • Specialized domains bind p-Ser, p-Thr or p-Tyr
    Thr allows interaction with other proteins through non-covalent bonds
  • Phosphorylation is required for binding
  • Surrounding polypeptide sequence also contributes
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7
Q

What is an example of phosphopeptide Binding?

A

Example: WD40 domain of Cdc4 with the Sic1 CPD peptide with pThr

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8
Q

What is the Acetylation of Lysine?

A
  • Lysine acetylation describes the transfer of an acetyl group from acetyl-coenzyme A (acetyl-CoA) to the primary amine in the ε-position of the lysine side chain within a protein, a process that leads to neutralization of the position’s positive electrostatic charge
  • Changes polarity (isopeptide bond)
  • Increase in size
    Signalling and metabolic effects
  • Lysine (K) acetyltransferases (KATS) and deacetylases (KDACs) Originally Histone acetyltransferases. They recognize specific sequences.
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9
Q

What is the different between methylation of Lysine vs Ariginine?

A
  • Methylation of Arginine involves the addition of 1 or 2 methyl groups to the guanidino group
  • methylarginines
  • Add size to K and R
  • Lysine can be mono, di, or trimethylated
  • Lysine methyltransferases (KMTs) and lysine demethylases (KDMs)
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10
Q

How are binding sites provided by PTM?

A

Different post translational modifications, such as phosphorylation, acetylation, and methylation provide new binding sites for proteins.

Specific domains bind Ac-Lys, Me-Lys and Me-Arg and surround sequences!

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11
Q

What is the native state of a protein? How is it stabilized?

A

The Native State is the completely folded conformation of a protein.

  • Most stable conformation of a protein
  • Structure is stabilized by hydrophobic contacts (exclusion of water)
  • Some domains also require a ligand partner to be stale
  • Cofactor (Haem, steriodm, etc) or another protein subunit
  • Native state can be in equilibrium with near-native folding intermediates
  • State of minimal energy: Folding is thermodynamically favoured

After the folding is complete, is when side chain modification usually take place.

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12
Q

What interactions are important for folding?

A

1) Hydrophobic interactions - many, strong
These interactions between secondary structures form the tertiary structures, they involve the side chains.

2) Hydrogen bonds - many, moderate
Stabilize secondary structure and are involved in peptide bonds.

3) Van der Waals interactions - many, weak

4) Ionic bonds - few, strong

5) Disulfide bonds - few, covalent (very strong)

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13
Q

What is a folding intermediate?

A

It is when a protein is not fully folded. They might have some secondary structure but the tertiary structure is incomplete.
Some hydrophobic side chains are exposed instead of buried.
More of the polypeptide is flexible and disordered.

With this, there is a risk of aggregation. The hydrophobic regions prefer to be in contact with others.

The interactions between different unfolded proteins lead to insolubility.

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14
Q

When does protein misfolding take place? What are some consequences? What leads to it?

A

it will take place right after protein synthesis.

  • A required ligand may not be available
  • There could be a genetic mutation, which causes misfolded proteins and then a disease such as sickle cell anemia or cystic fibrosis.
  • Also may be caused by harmful environmental conditions (heat) lead to unfolding or misfolding of properly folded proteins
  • Could also be caused by Aging: Decrease efficiency of the protein quality control mechanisms –> lose of protein homeostasis, harmful aggregates of misfolded proteins (amyloid) -> neurodegenration (alzheimer, Parkinson, ALS, dementia)
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15
Q

What can genetic mutations lead to?

A
  • Genetic mutations can lead to changes in polypeptide sequences; amino acid substitution, insertion or deletion, or premature stop
  • If you substitute to a similar amino acid it may have little or no effect
  • If it effects greatly then it could disrupt the folding or function of the protein
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16
Q

What can allelic variations sometimes cause?

A

It can sometimes cause genetic disease

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17
Q

What is proteostasis?

A

Protein homeostasis (proteostasis) refers to an extensive network of components that acts to maintain proteins in the correct concentration, conformation, and subcellular location, to cooperaitvely achieve the stability and functional features of the proteome

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18
Q

What is at the centre of the protein quality control network?

A

Chaperones

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19
Q

What is the function of chaperones?

A
  • Molecular chaperones assist protein folding and prevent aggregation, without being part of the native state
  • Recognize exposed hydrophobic regions of folding intermediates
  • Constitutively expressed and essential under non-stress condition
  • Many chaperones are Heat Shock Proteins (HSP, eg. Hsp70) highly expressed after stress
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20
Q

What is an example of up-regulated transcription and what is an example of down-regulated transcription?

A

Transcription of heat shock proteins is up-regulated

Transcription of other proteins is down regulates

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21
Q

Does the expression of chaperones correspond to the level of unfolded and misfolded proteins? What controls this?

A
  • Cells control this
  • Yes they are tailored to go hand in hand
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22
Q

Which transcription factor mediates heat shock response?

A

HSF1 transcription factor

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23
Q

What is the difference between an Inactive/Active HSF1 transcription factor?

A
  • Inactive: monomeric
  • ActiveL trimer, recognized HSE (heat shock element) promoters
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24
Q

How is HSF regulated? (5 steps)

A
  1. Monomeric HSF1 is folded, but mimics unfolded protein and is bound by Hsp90
  2. After heat shock, unfolded proteins compete with HSF1 for Hsp90 binding
  3. Free HSF1 trimerizes and activates transcription
  4. Chaperones including Hsp90 are expressed and help fold or degrade unfolded proteins
  5. HSF1 is down-regulated by binding of excess Hsp90 to the monomer form
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25
Q

What is an example of an Inducible chaperone?

A

Heat shock proteins

  • Heat induces the transcription activation of specific genes and the expression of specific proteins
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26
Q

What is an example of constitutive chaperones?

A

Assisted protein folding
Proteins that facilitate the folding of others:
- Hold of stabilizing hydrophobic residues
- Assisting in the folding

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27
Q

What is the difference between ATP-dependent and ATP-independent chaperones?

A

ATP-dependent:
These chaperones are actively promoting folding. The substrate binding and release is regulated by ATPase cycles.

ATP-Independent:
These chaperones prevent aggregation and can catalyze some folding steps.

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28
Q

Where can there be cooperation between chaperones and between what kind?

A

There can be cooperation in the cytosol and endoplasmic reticulum. And between constitutive and inducible chaperones.

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29
Q

What are the three families of ATP-dependent chaperones? (different structures and ATPase cycles)

A
  1. Hsp70 Family
  2. Hsp90 Family
  3. Chaperonins (Hsp60)
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30
Q

Which chaperones are induced by Heat Shock Response? (in humans)

A

HSP70, HSP90, HSP60

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31
Q

Which chaperones are induced by ER Unfolded protein response? (in humans)

A

Bip and GRP94

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32
Q

Do HSP60 chaperones function like E. Coli GroEL?

A

Yes

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33
Q

Which group of chaperones is not constitutively (always) expressed?

A

HSP70

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34
Q

What is the different between ATP-bound and ADP-bound HSP70 Chaperones?

A

ATP-bound:
- no substrate peptide binding

ADP-bound:
- the substrate binding domain is closed tightly on the peptide. Binds short hydrophobic sequences.

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35
Q

Describe “HSP70 function with the help of co-chaperones”

A
  • Co-chaperones are proteins which contact chaperones to regulate their activity
  • Some can bind to polypeptide substrate themselves and are both chaperons and co-chaperones
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36
Q

What are the HSP70 co-chaperones?

A

1) DNAJ (HSP40) family promote HSP70 substrate binding

2) Nucleotide Exchange Factors (NEFs) promote substrate release

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37
Q

What are the 5 steps of the HSP70 Functional Cycle?

A

1) Hsp40-mediated delivery of substrate to ATP-bound Hsp70

2) Hydrolysis of ATP to ADP mediated by HSP40 results in closing of the alpha-helical lid and tight binding of the substrate by Hsp70

3) NEF catalyzes exchange of ADP to ATP

4) Opening of the alpha-helical lid, induced by ATP binding, results in substrates release

5) Released substrate with either folds to native state (N)

38
Q

What are some characteristics of DNAJ co-chaperones (for the HSP70 family)?

A
  • DNAJs regulate HSP70 function (recruit HSP70s to the complex or membrane)
  • Many of them have at least 53 genes in human cells
  • All have conserved J domain, bind transiently to HSP70, activate it to hydrolyze ATP and bind polypeptide. DO NOT bind substrate.
  • Have specific domains that attach DNAJ to a protein complex or intracellular membrane
  • Substrate-binding DNAJs are the most highly conserved
39
Q

What are characteristics about Nuclear Exchange Factors (NEFs) (HSP70 family)?

A
  • Nucleotide Exchange Factors (NEF) remove ADP from HSP70 and allow ATP to bind
  • NEF binding opens up HSP70 ATPase domain and weakens interactions with nucleotide
  • ATP binds when NEF dissociates
  • ATP-bound HSP70 to release polypeptide
  • Several NEF families in humans
40
Q

How does HSP70 chaperone help folding?

A
  • HSP70 binds hydrophobic regions of folding intermediates and prevents incorrect contacts from forming
  • Release of polypeptide from HSP70 provides chances for it to fold
  • Balance between DNAJs and NEFs supports an optional rate of HSP70 substrate binding and release
  • Substrate-binding DNAJs may provide additional assistance
  • Can form multi-chaperone complex with HSP90
41
Q

What are some characteristics about the HSP90 family?

A
  • HSP90 chaperones are homodimers, with 2 identical subunits joined at the C-termini - human
  • Hsp90: 2 x 90 kDa = 180 kDa
  • Dimer can open and close, like a nutcracker
  • ATP controls opening and closing of the dimer
  • Co-chaperone p23 stabilizes closed form
  • Though to bind polypeptides at late stages of folding
42
Q

Where do HSP90 chaperones bind?

A

They bind to hydrophobic and polar surfaces which stabilizes intermediate folded states.
Substrate is bound along the side of the subunits
Different substrates can bind to different sites on the sides - unlike HSP70s and Chaperonins.

43
Q

What are the 3 steps of the HSP90 Functional Cycle?

A

1) Substrate is bound weakly in the open nucleotide-free state

2) ATP binding allows dimer to close and bind substrate tightly

3) ATP hydrolysis to ADP compacts the dimer and releases substrate

44
Q

Describe the HSP70 and HSP90 cochaperone system:

A
  • Cytosolic HSP70 and HSP90 chaperones form a multi-chaperone system
  • They cooperate to them assist substrates
  • A substrate is released from HSP70 and bound by HSP90 in coordinator, and HOP assist with this complex formation!
  • There are many cochaperones that regulate, they provide flexibility, have folding and non-folding functions, and sometimes act on substrates.
45
Q

Do HSP70 and HSP90 have similar EEVD motif? What are they?

A

Cytosolic HSP70 and HSP90 are not homologous, but they have similar C-terminal sequence motifs:

  • HSP70: PTIEEVD-COO-
  • HSP90: MEEVD-COO-
46
Q

What is EEVD motif?

A

It is one “hotspot” for cochaperone binding, found at the extreme C terminus of cytoplasmic Hsp70s.

47
Q

What recognizes EEVD motifs? What is an example of it helping and making impact?

A

Tetra-trico-peptide repeat (TPR) domains
- They are adaptors to HSP70 and HSP90

Example:
Cochaperone HOP binds both families together of chaperones, and it can do this because it has TPR that recognized EEVD motifs.

48
Q

What cochaperones aid TPR in its function?

A
  • TPR co-chaperones oftern have other domains which interact with substrate directly
49
Q

What is HOP?

A
  • Hsp70-Hsp90 Organizing Protein.
  • It functions as a co-chaperone which reversibly links together the protein chaperones Hsp70 and Hsp90
  • it has domains that specifically binds them
50
Q

What is FKBP52?

A
  • FKBP52 is an Hsp90-associated co-chaperone
  • Has HSP90-binding TPR domain
    and a PPIase domains
  • peptidyl-prolyl isomerase: chaperone specific to prolines
51
Q

What is CHIP?

A

CHIP is a co-chaperone that functions as an E3 ubiquitin ligase that links the polypeptide binding activity of Hsp70 to the ubiquitin proteasome system

CHIP binds Hsp70 through interactions between its N-terminal TPR domains and the C-terminal EEVD motif found on Hsp70

Can bind either HSP70 or HSP90 and has a uniquitin ligase domain that helps degrade proteins

52
Q

Many HSP90 substrates are signal transduction proteins. Such as kinases, receptors, transcription factors. Many also require HSC70. What is the exception that does not require it?

A
  • HSP90 binds kinases without needing HSC70
53
Q

Are HSP90 and HSC70 drug targets for cancer treatment?

A

Yes

54
Q

What is an example of a mutation in a chaperone and how we go back to normal growth?

A

The example is v-src.

c-src (cellular) is a normal kinase involved in signalling growth.

v-src is a mutant (viral) kinase that causes cancer.

1) v-src expressed in fibroblast (epithelial) cells which causes them to become cancerous.

2) treat cells with Hsp90 inhibitor.

3) Hsp90 cannot chaperone v-src anymore.

4) Therefore the cells will revert from cancer to normal growth.

55
Q

Describe the characteristics of Chaperonins (HSP60 family):

A
  • Chaperonins are large oligomeric complexes, with a typical double-ring structure
  • Homologs of human mitochondrial Hsp60 and Hsp10
56
Q

What is the difference in structure between the E.Coli GroEL and the E.Coli GroES cap co-chaperone?

A

GroEL: 2 rings x 7 identical subunits x 60 kDa = 840 kDa

GroES cap co-chaperone: 7 subunits x 10 kDa = 70 kDa

The top cap is the GroES. And then the GroEL makes up the bottom. It has 4 different sections, from top to bottom; 1) substrate-binding domain
2) ATP-ase domain
3) ATP-ase domain
4) substrate-binding domain

57
Q

Describe GroEL Cavity: down position vs up position?

A
  • Rings are identical and work in alternating cycles

Down position (no nucleotide): (substrate-binding)
Subunits around the ring bind to hydrophobic polypeptide

Up position (ATP-bound): (GroES)
Subunits bind to GroES cap instead of the substrate.
A large cavity with a polar surface is formed.
Substrate is released inside cavity: enclosed but no longer bound.

58
Q

What do the GroEL subunits posses?

A
  • Each subunit has an ATPase domain and a substrate-binding domain
  • The ATPase domain is the interface with the opposite ring (upside down)
59
Q

In the GroEL, what is the movement of the substrate binding domain controlled by?

A

The movement of the substrate binding domain is controlled by the ATPase in both rings.

60
Q

Describe the GroEL functional cycle in one ring:

A
  • Chaperone that can bind misfolded protein when not bound to ATP or ADP
  • Bound to ATP and then there is a conformational change and then a bigger space
  • in the next step there is a hydrophillic interior
  • misfolded protein has time/space to fold in itself
  • Now ADP and GroEL is released and the folded protein is also released
  • It has cylinders but only need to know what happens to one of them
61
Q

How does GroEL help folding?

A
  • Substrate is enclosed inside polar cavity, this provides a chance to fold. The confinement promotes folding by favouring more compact conformations.
  • ATP hydrolysis acts as a timer for substrate release
62
Q

Does Human mitochondrial HSP60 function like GroEL?

A

Yes

63
Q

What are three characteristics about human chaperonin in cytosol?

A
  • TRiC (TCP1 Ring Complex)
  • does not have cap co-chaperone
  • long substrate-binding domains form the cavity themselves
64
Q

What is the importance of protein degradation? What is the major route?

A

Degradation is a key part of protein folding quality control, and also an essential regulatory and homeostatic mechanism.

Ubiquitin-mediated degradation by proteasome in cytosol is the major route.

65
Q

What marks a protein for degradation?

A

Poly-Ub chains mark the protein!

66
Q

What is Ubiquitin (Ub)?

A

Small 8 kDa protein (76AA) that can be covalently linked to lysine side chains of other proteins and to itself.

67
Q

Describe the Ubiquitin-Proteasome System (UPS):

A
  • Ubiquitination enzymes attach chains of uniquitin to substrate proteins
  • E3 Ub ligases select the substrates
  • Poly-Ub chains are recognized by receptors on proteasomes
  • The proteasome is a large protein complex that unfolds and degrades substrates
68
Q

What enzymes are involved in Ubiquitination?

A
  1. E1 activating enzyme attaches Ub to itself in a chemically reactive state, on a Cys side chain (thioester bond)
  2. E2 conjugating enzyme transfers Ub to its own Cys
  3. E3 ligase selects the substrate to be modified
  4. E3 triggers Ub transfer from E2 to Lys side chain on substrate
  5. E2-E3 adds more UB to Lys on previous Ub, to make Poly-Ub chain
69
Q

Describe Ubiquitination in K (Lysine/Lys/K)

A
  • Ub C-terminus carboxyl is covalently linked to side chain amine
    There is isopeptide bond
    Lysine, but not arginine or histidine
  • A substrate cap can have multiple ubiquitination sits, many but not all lysines, depending on accessibility
70
Q

Where can Ub C-term be linked?

A

Can be linked to Lys 63, 48, or 11 of another UB

71
Q

What targets protein for degradation by proteasomes?

A

Long Lys48 Poly-Ub chains target

72
Q

What two types of Ub linkage are not recognized?

A

Mono-Ub
Lys63 poly-Ub

But they still signal other things

73
Q

What is an E3 ligase?

A

Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation.

74
Q

Describe the diversity of E3 ligases:

A

In humans:
- Dozen genes for E1 enzymes
- 50 genes for E2 enzymes
- >600 genes for E3 enzymes
- Most proteasome subunits only have 1 gene
- Cells express different E3 enzymes fro each degradation situation, which all use the same proteasome
- More effective than expressing hundreds of different proteases with different specificites

75
Q

Describe what is meant by substrate selection by E3 ligases?

A
  • All proteins are continually degraded but at different rates
  • degradation of substrate is controlled by selectivity of E3 ligase not by the proteasome
76
Q

What are three examples of Substrate selection by E3 ligase? (different pathways)

A
  1. Quality control degradation of a misfolded protein
  2. Constitutive degradation of a native protein to control its level: N-End rule
  3. degradation of a native protein in response to a signal
77
Q

Describe this substrate selection (by E3 ligase ) pathway: Quality control degradation of a misfolded protein

A

A. Misfolded protein degradation regulated by the CHIP co-chaperone
a. TPR domain binds HSC70 or HSP90
b. E3 ligase domain binds E2

B. Chaperone, CHIP and E2 form complete E3 ligase complex

C. Chaperone-bound substrate is selectively ubiquitinated

78
Q

Describe this substrate selection (by E3 ligase ) pathway: Constitutive degradation of a native protein to control its level: N-End rule

A

A. All proteins are translated with N-terminal Met

B. Many proteins are processed by cleavage within their sequences, so a different residue becomes the N-terminus

C. Certain N-terminal residues are bound by N-end rule E3 ligases which uniquitinates the proteins

D. Arg, Lys, His, Phe, Trp, Tur, Leu, IIe: E3 ligases recognized the properties of the side chains (if there is one of these, automatically degraded all the time)

E. N-end rule degrades proteins rapidly, whether or not they are folded

F. Other proteins are longer lived, unless degraded by other means

If they are basic or large hydrophobic, they probably follow the N-End rule.

Another explination:
The N-end rule pathway is a proteolytic system in which N-terminal residues of short-lived proteins are recognized by recognition components (N-recognins) as essential components of degrons, called N-degrons.

79
Q

What is meant by N-End Rule Modifications? What are the two exceptions?

A
  • Some N-terminal residues are enzymatically modified to be recognized by N-end rule (but there are two exceptions)

1) Asp, Glu (acidic): Arg is added to N-terminus

2) Asn, Gln (amides): side chains converted to Asp, Glu by removal of amine. Then Arg is added to N-terminus.

  • Both need to be converted so then they can be added and then degraded.
80
Q

What are the 4 different N-end Rule Pathways?

A

1) N-terminal N, convert to D; if Q, convert to E

2) If N-terminal D or E, add N-terminal R

3) If N-terminal R,K,H,F,W,Y,L,I, ubiquitinate the protein

4) if something else, leave the protein alone

81
Q

Describe this substrate selection (by E3 ligase ) pathway: degradation of a native protein in response to a signal

A
  • Each case has different E3 ligase recognition mechanism
  • This example is CHIP mechanism:
    CHIP interactions with chaperones are transient (not bound together forever. Have relatively fast binding and release.
    If they are balance between chaperones for a long time they are more likely to form a complex with CHIP and be ubiquitinates, but the substrates bound for a short time are likely to escape ubiquitination.
82
Q

How is degradation regulated by SCF E3? (4 steps) What is it?

A
  • SCF complexes are a class of E3 ubiquitination ligase that facilitate proteasomal degradation through the catalysis of ubiquitinylation.

1) E3 ubiquitin ligase complex (Skp1/Cullin/F-box)

2) Scaffold binds E2 and substrate-binding (F-box) protein

3) F-box protein binds phosphorylated substrate

4) Substrate is presented to the E2 for ubiquitination

83
Q

How is degradation regulated by phosphorylation?

A
  • Many F-box proteins recognize phosphorylated peptide sequences
  • Phosphorylation by kinase is used as a signal for degradation
  • De-phosphorylation then prevents degradation
  • SCF ligases degrade native, functional proteins to stop their function.
84
Q

What are F-box proteins?

A

F-box proteins are proteins containing at least one F-box domain. The first identified F-box protein is one of three components of the SCF complex, which mediates ubiquitination of proteins targeted for degradation by the 26S proteasome

85
Q

What is the proteasome?

A
  • Responsible for general protein degradation in the cytosol and nucleus, and from the endoplasmic reticulum
  • Large oligomeric complex with 20s core particular and two 19s regulatory particles (caps)
  • Core and 2 caps form complete 26s proteasome
86
Q

What are the two proteasome subunits?

A

1)
20s core:
2 outer rings of 7 similar alpha subunits, and 2 inner rings of 7 similar beta subunits
3 of the beta subunits have protease activity on the inside surface
19s cap attaches to outer ring

2)
19s Regulator (cap):
base with 6 AAA-family ATPase subunits *AAA: family of ATP-dependent proteins with many different functions
A lid with non-ATPase subunits
- Poly-Ub receptors
- deubiquitinases (DUBs)

87
Q

What is the function of Ub receptors (proteins)?

A
  • Increase efficiency of targeting
  • Select only K48 chains
  • Protect against premature DUB activity
88
Q

What are two types of Ub receptors:

A

1) Intrinsic receptors: the cap subunits Rpn10, 13 bind poly-Ub

2) Extrinsic (shuttling) Ub receptors
- Separate from proteasome
- bind poly-Ub through Ub-Associated domain (UBA)
- have Ub-like domain (UBL) that is recognized by cap

89
Q

What is the function of proteasome cap?

A
  • Lid Ub receptors bind poly-Ub or UBL domains of shuttling receptors
  • DUBS remove Ub chains
  • Base unfoldase passes substrate into core
90
Q

What is the function of proteasome core?

A
  • Cavity inside core is small and narrow, proteins have to stay unfolded
  • 3 active subunits in each Beta-ring, 6 sites total
  • Once cuts at basic AAs, one cuts at acidic AAs, and one cuts at hydrophobic AAs
  • Peptides diffuse out and are digested into amino acids by peptidases
91
Q

What is the function of proteasome? (4 steps)

A

1) 19s cap recognizes Poly0Ub, or UBL domain of shuttling receptor

2) DUBS remove poly-Ub from substrate and pass it to base

3) The base subunits use ATPase activity to unfold substrate and feed it inside 20s core

4) The proteolytic beta subunits cleave substrate into short peptides of amino acids

92
Q

What is an 8 steps summary of the Ubiquitin-Proteasome system?

A

1) Ub is activated by E1 and transferred to E2 on Cys side chains

2) E3 selects substrate polypeptide and transfers UB from E2 to Lys side chains in the substrate

3) E2/E3 attaches more Ub onto Lys48 of the previous Ub, to make poly-Ub chain

4) Poly-Ub is bound by shutting receptor with UBL domain

5) 19s cap lid binds poly-Ub, or UBL domain of shuttling receptor

6) DUB removes poly-Ub

7) 19s base ATPase unfolds substrate

8) Proteasome core cleaves at basic, acidic, and hydrophobic sites

Bioc 212 (11 decks)

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