Lecture 4 - Antigen Presentation I

Question 1

Describe the structure of MHC molecules

Answer 1

MHC class I:
• α1, α2, α3 (heavy chain)
• β2 microglobin
• α1 & α2 form binding cleft

MHC class II:
• α1, α2
• β1, β2
• α1 & β1 form binding cleft

Binding cleft:
• Two α-helices
• β sheet forms ‘floor’

Question 2

Compare the binding cleft of MHC class I and II

Answer 2

MHC I:
• Shorter, more enclosed
• Binds shorter peptides of relatively specific lengths
• “Hamburger”

MHC II:
• More open cleft
• Binds longer peptides
• “Hot dog”

Question 3

Compare CD4 and CD8 interactions with MHC

Answer 3

CD4:
• Interacts with conserved β2 on MHC II

CD8:
• Interacts with conserved α3 on MHC I

Question 4

Give an overview of the various pathways of antigen processing in APCs

Answer 4

– MHC I pathway –

1. Endogenous, cytosolic protein degraded in proteasome
2. Peptides move into ER through TAP dimer
3. Peptides loaded onto MHC class I in ER
4. MHC+peptide transported in vesicle to cell surface through secretory pathway
5. MHC:peptide complex remains at the cell surface until peptide dissociates

– MHC II pathway –

1. MHC II binds Ii chain in ER
2. Trafficked to MIIC
3. Endolyososome with degraded exogenous peptide fuses with MIIC
4. Invariant chain broken down in MIIC by Cathepsin S and HLA-DM
5. Exogenous peptides loaded onto MHC class II
6. MHC II:peptide trafficked to cell surface

Question 5

Compare constitutive antigen presentation and AP in the presence of an infection

Answer 5

Constititive:
• Presentation of self-peptides

Infection:
• Viral peptides presented
• “Altered self” proteins presented
etc.

Question 6

What is TAP?

Describe special functional features

Answer 6

Transported Associated with Peptide loading
Pore in the ER membrane through which cytosolic proteins move into the ER
• ATP dependent
• Preferentially transports 9-12 aa long peptides w/ little regard for peptide sequence

Minimum size: 8 aa
Maximum size: 40 aa
Average size: 9-12 aa

• NB in mice, TAP preferentially transports peptides with hydrophobic residues at the C-terminus (not in humans)

Question 7

Describe the structure of the proteasome

Answer 7

26S
Regulatory caps
Four rings
Each ring made up of 7 subunits
Central channel
Three subunits (B1, B2, B3) are catalytic
The catalytic subunits are Threonine proteases

Question 8

Describe the function of the proteasome

Answer 8

1. Proteins polyubiquinated by ubiquitin ligases
2. Polyubiquinated proteins targeted to the proteasome
3. Proteins degraded into small peptides by Threonine proteases

Question 9

What is the source of MHC class I presented peptides constitutively?

Answer 9

DRiP hypothesis:
Defective Ribosomal Products
• The process of protein synthesis is defective (up to 30% of translated proteins are defective)
• Misfolded or incomplete polypeptides are poly-ubiquinated
• DRiPs are a major source of proteasomal substrates

Viral Ags are presented very soon after infection, despite the reasonably long half life of viral proteins. This was a quandary.

The hypothesised solution is that the mainsource of antigenic peptides for presentation is not functionally, properly folded peptides, but rather polypeptides that are defective in some way.

Question 10

What are Immunoproteasomes?

Answer 10

1. In viral infection, IFN-γ expression is up-regulated
2. IFN-γ stimulates the transcription of certain subunits of the proteasome
3. This ‘immunoproteasome’ preferentially produces peptides that are transported into the ER by TAP (i.e. more efficient at generating antigenic peptides)

Question 11

What are the effects of IFN-γ?

Answer 11

• Increased expression of ‘immunoproteasome’ subunits

• LPG2
• LPG7
• MECL-1
• PA28
• Increased MHC I expression
• Increased TAP expression
• Increased ERAAP expression

Question 12

What is the fate of peptides generated by the proteasome / immunoproteasome?

Answer 12

The vast majority of peptides formed in the proteasome are degraded further into amino acids by Aminopeptidases in the cytosol

Only a few (1/100) make it into the ER for loading onto MHC I This process is occurring at a rate such that this is more than enough for Ag presentation to T lymphocytes

Question 13

Describe the structural features of peptide generated by the proteasome

Answer 13

C-terminal residues:
• ‘Fixed’ by the proteasome
• Hydrophobic residues
• Recognised by TAP

N-terminal residues:
• ‘Flexible’, i.e. more variable
• Not so much determined by the proteasome
• Residues trimmed in the cytosol by aminopeptidases

Question 14

Describe the assembly of MHC I

Answer 14

• Entails the interaction of the Peptide loading Complex 
Protein loading complex:
 • Several ER chaperones:
- Calnexin
- Calreticulin
- ERp57
- Tapasin

• TAP
Some of the components are dedicated to antigen presentation, whilst some are multipurpose chaperones

Dedicated:
• TAP
• Tapasin
• ERAP

Multipurpose:
• Calnexin
• Calreticulin
• ERp57

Question 15

What is ERAP?

Answer 15

ERAP: Endoplasmic Reticulum Associated Protease

Endopeptidase

Trims peptides that have got into the ER that are longer than 10 aa residues

ERAP ensures that peptides are 9 aa in length (for optimal binding into the MHC I binding cleft)

This happens after peptide moves into the ER through TAP

Once the peptides are trimmed, they can be loaded into MHC I

Question 16

Describe what happens to MHC I:peptide complexes at the cell surface

Answer 16

Remain at the surface until peptide dissociates

The half life of the complex is directly proportional to the affinity of the interaction between the binding cleft and the peptide

The more stable the complex, the longer the half life and vice versa

Question 17

What is the PLC?

Answer 17

Protein loading complex

Question 18

What is the importance of the PLC?

Answer 18

• Helps form the MHC I
• Aids loading of peptide into MHC I
• Subjects the MHC I:peptide complexes to editing so that only complexes carrying peptides that confer a minimum stability leave the ER

Question 19

Describe the formation of “Good Quality” MHC I-peptide complexes in the ER

Answer 19

PLC subjects the MHC I-peptide complexes to editing so that only complexes of a minimum stability can leave the ER

Each MHC I molecule undergoes multiple rounds of peptide binding, dissociation and rebinding

Eventually a peptide of good enough ‘quality’ is found

Question 20

What are ‘high quality’ peptides?

Answer 20

• One that fits well into the binding groove:
• Contains the right anchor residues
• Lacks residues that hamper binding

Question 21

Describe the Ag that B cells recognise

Answer 21

B cells recognise Ag in its native conformation

i.e. no processing and presentation, as with T cells

Question 22

How do cytosolic proteins gain access to the ER for loading onto MHC class I?

Answer 22

Transmembrane TAP molecule in the membrane of the ER forms a channel, through which peptides can move

This process is ATP dependent

Question 23

At which times will peptide be presented in the context of MHC I on the surface of cells?

Answer 23

Constitutively
Peptide presentation is always displayed on the surface
When the cell is not infected, or undergoing cellular transformation (e.g. transformation to a cancer cell), self peptide will be presented

This enables the immune system to constantly maintain tolerance (i.e. determine what is ‘self’)

When infected or undergoing cellular transformation, the MHC I will present peptides that represent this ‘altered self’ state of the cell

Question 24

Why is the proteasome very important for the health of the cell?

Answer 24

Missfolded proteins aggregate to form highly toxic masses

This machinery recognises miss folded proteins and degrades them so that this doesn’t happen

Question 25

Describe how the proteasome determines the sequence of the peptide it produces

Answer 25

It plays a minimal role in the aa sequence of the peptides it produces

Question 26

What other functions in the cell does TAP perform?

Answer 26

None
The only function that has so far been ascribed to it is its role in Ag presentation (i.e. movement of peptides across the ER membrane, and role in the Protein Loading Complex)

Question 27

Peptides of which length are preferentially loaded into MHC class I?

Answer 27

9 aa long

Question 28

Differentiate between the 26S and the 20S proteasome

Answer 28

26S: catalytic core + regulatory caps

20S: catalytic core

Question 29

Which proteasome subunits are upregulated by IFN-γ signalling?

Answer 29

LMP2
LMP7
MECL1
PA28

These displace β1, β2 and β5 to form the immunoproteasome

Question 30

What is the function of the regulatory caps of the proteasome?

Answer 30

Recognise ubiquitin and unfold the protein

Question 31

What is PA28?

Answer 31

Subunits whose expression is upregulated by IFN-gamma

Replace the regulatory caps on the proteasome

Increase the rate at which peptides are released from the immunoproteasome

Question 32

Which subunits of the proteasome perform the proteolysis?

What type of proteases are these subunits?

Answer 32

β1, β2 and β5

These are threonine proteases

Question 33

Describe the function of the immunoproteasome

Answer 33

Probably more efficient at generating antigenic peptides, i.e. those that more efficiently:
• Bind MHC I binding pockets
• Transported by TAP

PA28:
• Increases the rate at which peptides are released from the immunoproteasome

However, even the peptides generated by the immunoproteasome will, for the most part, be degraded in the cytosol by aminopeptidases

Question 34

What determines the following on antigenic peptides:
• N terminus sequence
• C terminus sequence

Answer 34

N terminus: aminopeptidases in the cytosol or ERAAP in ER

C terminus: proteasome

Question 35

On which end do the cytosolic aminopeptidases cleave?

Answer 35

N terminus

Remember, C terminus is fixed by the proteasome