Innate Molecules (Innate Sensors, Complement, MHC Molecules, Antigen Processing) Flashcards
Innate Sensors, Complement, MHC molecules
Give some basic information about TLRs.
3 domains
- extracellular N-terminal
- middle helix transmembrane
- intracellular C-terminal
homeostasis: exists as monomer or (weak) dimer
activation: homodimers (eg TLR4) or heterodimers (eg TLR1/2)
dimerisation = stability = signalling
leucine rich repeats (LRR): The variability in the number, sequence, and orientation of LRRs among TLRs determines their ligand specificity
TIR domain interacts w signalling molecules
TLRs can be intracellular or extracellular
Note some differences between human and mouse TLRs.
TLR10 is human exclusive - recognises triacylated lipopeptides
TLR11, 12, and 13 are mouse exclusive:
- 11 and 12 recognise profilin: found in certain protozoan parasites
- 13 recognises ribosomal RNA
mice express TLR8 but it seems to be non functional
Give some more detail about the TIR domains of TLRs?
toll/interleuokin-1 receptor
~ 200 aa
intracellular
these often have weak, transient interactions until TLRs self-associate
- this creates a scaffold that facilitates signal transduction = immune response or cell-death
What are the TLR adaptor proteins?
- MyD88
- TRIF
- TIRAP/MAL
- TRAM
- TRAF3
TLR signalling largely divided into MyD88-dependent and TRIF-dependent pathways
MyD88: utilised by all TLRs
= activates NK-kB and MAPKs for induction of inflammatory cytokine genes
TRIF is recruited to TLR3 and 4
= promotion of alternative pathway that leads to activation of IRF3, NK-kB, and MAPKs for induction of type I IFK and inflammatory cytokine genes
What is the function of NF-kB?
inducible TF
regulates inflammation directly by increasing production inflammatory cytokines, chemokines, and adhesion molecules
also regulates cell proliferation, apoptosis, morphogenesis, and differentiation
Give some detail about TLR 1/2 and 2/6.
1/2 detect triacyl lipopeptides
2/6 detects diacyl lipopeptides
= highly expressed cell wall components in gram+ bacteria
can also detect alarmins -> endogenous ligands eg heat shock proteins
signal through MyD88 (TIRAP/Mal facilitates connection to receptor)
downstream signalling triggers SEAP promoter = secretion of alkaline phosphatase (ALP) or induce production of pro-inf cytokines eg TNF a, IL, IFN
– ALP dephosphorylates peptidoglycan, previnting binding to receptors
Give some detail about TLR3.
detects viral dsRNA, and polyI:C (dsRNA analogue)
homodimerisation allows for signalling via TRIF/TRAF3 to activate IRF3 (TF) further downstream
strong inducer of type I interferon (IFN)
expression of TLR3 increases with age (may be due to immune response too strong for children to handle?)
Give some detail about TLR4.
detects LPS
- uses MD-2, an accessory protein, to assist in detection and dimerisation
extracellular LBP binds LPS monomer -> delivery to soluble or membrane CD14 -> transfer LPS to TLR4/MD-2 complex = homodimerisation -> dimerisation of TIR -> binding of MyD88
this activates TF NF-kB and MAPK -> transcription of pro-inf cytokines
endocytosis of LPS-TLR4/MD-2 complex leads to TRIF/TRAM-dependent pathway -> induction of IRF3 and IFNs
Give some detail about TLR5.
detects flagellin
main extracellular receptor interacting w human intestinal microbiota
- expressed in monocytes and immature DCs
- specifically expressed on basal side of intraepithelial cells (IECs)
Why is TLR5 specifically expressed on basal side of IECs and what does activation do?
intraepithelial cells
separation from luminal contents, prevents uncontrolled inflammation by symbiotic microbes
activation of TL5 decreases epithelial barrier resistance and reduced expression of tight junction proteins and produce chemokines eg IL-8
–> makes space for, and attracts immunes cells
Give some detail about TLR7 and 8?
detect GU-rich short ssRNA in endosomes/lysosomes
- signal through MyD88
- nuclear translocation of AP-1, NF-kB, and IRFs
- phosphorylation of IRFs promote induction of interferon stimulated response element (ISRE), w expression of IFN
== production of pro-inf cytokines
Give some detail about TLR9
detects CpG
- unmethylated cytidine-phosphate-guanine
- restricted to bacterial and viral DNA
- mitochondrial DNA following cell stress can activate TLR9
also recognises hemozoin
activated IFN and chemokines
upregulated co-stimulatory molecules on DCs (CD80)
What is the cGAS-STING pathway?
cyclic GMP-AMP synthase (cGAS) is a cytosolic receptor that detect cytosolic dsDNA, from bacteria, viruses, or damaged cell
- detection of dsRNA by cGAS to produce cGAMP from ATP and GTP
- cGAMP binds to STING dimer present on ER membrane and activates its signalling
- STING activates the kinase TBK1 to phosphorylate IRF3 which enters nucleus and induces expression of type 1 interferon genes
What is AIM2?
AIM2 recognizes dsDNA in the cytoplasm from viral or bacterial infection or mitochondrial DNA released from damaged cells
AIM2 binds to dsDNA via its HIN200 domain
The PYD of AIM2 interacts with the PYD of ASC (adaptor), leading to oligomerization of ASC
ASC recruits and activates pro-caspase-1 by forming a large multiprotein complex.
Active caspase-1 cleaves pro-IL-1β and pro-IL-18 into their mature, secreted forms
Caspase-1 also induces pyroptosis, a form of inflammatory cell death
What are NOD-like Receptors (NLRs)?
detect bacterial cell-wall peptidoglycans
NOD proteins reside in cytoplasm in inactive form
binding of bacterial ligands to NOD proteins -> dimerisation (like TLR) induces recruitment of RIP2 -> activates TAK1 > NF-kB activation
have a CARD domain (caspase recruitment)
What is the NLRP3 inflammasome?
detect heat shock protein 90 + co-chaperone
pyrin domain instead of a CARD domain
NLRP3 proteins remain as inactive monomers in cytoplasm
– potassium efflux induces dissociation of chaperones that keep NLRP3 in inactive conformation
NLRP3 form oligomers w ASC causing proteolytic cleavage of pro-caspase 1
– recruitment through interaction of ASC CARD and caspase-1 CARD
caspase 1 releases mature inflammatory cytokines such as IL-1 and IL-18 from their proproteins
What is the significance of ALUM in the context of the inflammasome?
Alum (aluminium hydroxide) is the most widely used adjuvant used in human vaccines as it is known to activate the NLRP3 inflammasome
How does C3b bind to the surface of a pathogen? (alternative pathway)
C3 protein is proteolytically processed to generate a beta chain and an alpha chain held together by disulfide bonds
thioester bond within TED (thioester containing domain) is protected from reacting
cleavage of C3 (by C3 convertase: C3bBb) = C3a + change of conformation of C3b -> allows thioester bond to react w a nucleophilic group on pathogen surface (NH2, OH)
How does MBL recognise pathogen carbohydrates? (lectin pathway)
mannose-binding lectin
MBL monomers form trimeric clusters of carbohydrate-recognition domains
complex w serine proteases
- MASP-1, -2, -3
MBL binds w high avidity to mannose and fucose residues
How do ficolins recognise pathogen carbohydrates? (lectin pathway)
ficolins have similar structure to MBL (trimeric) but have different carb-binding domain
complex w serine proteases
- MASP-1, -2, -3
ficolins bind oligosaccharides containing acetylated sugars
After recognition of carbohydrates, how does the lectin pathway progress?
- activated MASP-2 associated w MBL or ficolin cleaves C4 -> C4a + C4b
–> C4b binds microbial surface through binding of its thioester bond to a nucleophilic group - C2 is cleaved by MASP-2 to C2a + C2b
–> C2a complexes w C4b = a C3 convertase - C4bC2a cleaves C3 -> C3a and C3b
–> C3b binds microbial surface or C3 convertase
–> one molecule of C4bC2a can cleave up to 1000 molecules of C3
What is C1 composed of? (classical pathway)
C1q: recognition of antigen bound antibodies
C1r and C1s: cleave C4 and C2
C1q:Cr1_2:C1s_2
How is C1 activated? (classical pathway)
- multiple Ig (typically IgG1 and IgM) are needed for C1 activation, these bind to C1q
- movement of Fc domain opens up C1q-binding sites
- the number, and position, of Ig will determine the opening, thus, the strength of the pathway
- gC1q will rotate inward (heads), leading to opening of the CLR (stalk)
- C1rC1s will pass from inactive 8 form to active S-shaped conformation
After activation of C1, how does the classical pathway progress?
autocatalytic activation of C1r and C1s in turn cleaves C4 and C2 into larger (C4b, C2a) and smaller (C4a, C2b) fragments
–> C4bC2a = a C3 convertase
C4bC2a cleaves C3 -> C3a and C3b
–> C3b binds microbial surface or C3 convertase
What is tick over in the alternative pathway?
how the alternative pathway begins
spontaneous hydrolysis of the thioester bond in TED due to instability in plasma (aqueous)
–> resulting C3(H2O, like C3b) binds factor B
factor D cleaves factor B into Ba + Bb <- still bound to C3(H2O)
C3(H2O)Bb complex = a C3 convertase
this cleaves C3 into C3a and C3b -> C3b rapidly inactivated unless it binds cell surface
–> factor B binds noncovalently to C3b on a cell surface and is cleaved to Bb by factor D
What is properdin? (alternative pathway)
glycoprotein found in plasma that is mainly produced by leukocytes and can positively regulate AP activity by stabilising C3bBb convertase
eg activated neutrophils at site of inflammation
C5a generation induces increased properdin secretion ie positive feedback loop
How does bacterial PGN enter the cell to activate NOD1 and NOD2 receptors?
- some bacteria can deliver PGN into the host cell cytoplasm through its secretion system
- uptake of outer membrane vesicles (OMVs) released by Gram-negative bacteria facilitates internalization of PGN
- extracellular PGN fragments can enter the host cell through endocytosis and transported to the cytosol through lysosomal membrane transporters
What is iC3b?
inactivated C3b
produced when C3 is cleaved by complement factor I
cannot form convertases, but can still act as an opsonin and anaphylatoxin
stimulates phagocytosis via macrophages and DCs through binding to CR3 or CR4
stimulates phagocytosis of circulating pathogens via tissue resident macrophages through binding to CRIg
How do C3a and C5a increase vascular permeability?
activate mast cells which release TNFa
C5a more active than C3a
How can C5a enhance the phagocytosis of pathogens?
When only C3b coated on bacterium binds to CR1, bacteria are not phagocytosed
C5a can activate macrophages to phagocytose by binding to C5aR1
How is C5 convertase formed in the classical and lectin pathways?
some of the C3b fragments generated by C4bC2a C3 convertase bind to it, forming C4bC2aC3b = C5 convertase
How is C5 convertase formed in the alternative pathway?
some of the C3b fragments generated by C3bBb C3 convertase bind to it, forming C3bBbC3b = C5 convertase
How is the membrane attack complex formed (MAC)?
formed during the terminal pathway of complement activation
- C5b, generated by C5 convertase, binds C6 and C7
- C5b67 complexes bind to pathogen membrane via C7
- C8 binds to complex and inserts into cell membrane
- C9 molecules bind to the complex and polymerise
- Up to 16 C9 molecules bind to form a pore in the membrane
How is the complement system regulated?
- C1 inhibitor (C1INH) dissociates C1r and C1s from the active C1 complex
- DAF, C4BP, and CR1, can displace C2a from C4bC2a complex
– then C4b bound to any of these is cleaved by a soluble protease I to inactive forms, C4d and C4c - CD59 prevents final assembly of MAC on host cells at the C8 to C9 stage
What is MHC?
major histocompatibility complex
discovered in mice (H-2), then 20 years later in humans (HLA) as genetic locus responsible for rapid graft rejection
large genomic regions that determine “transplantation antigens”
thousands of alleles that are matched by tissue typing to avoid rejection
MHC molecules detect infections inside cells, cancers, and extracellular pathogens
high polymorphisms mostly driven through a molecular arms race w pathogens
What are classical MHC molecules?
- highly polymorphic
- present peptides to T cells
- have wide tissue distribution:
– class I expressed in all nucleated cells
– class II expressed on professional and facultative APCs - are conserved among all jawed vertebrates
What are non-classical MHC molecules?
- differ in one or more of attributes of classical MHC molecules
- are derived from classical molecules at different times in evolutionary history
What is cross presentation, and in which cell does it occur?
exogenous antigens are processed and presented on MHC class I molecules
enables activation of CD8+ T cells where antigen source did not directly infect the presenting cell
cDC1 is cell that can most efficiently perform cross presentation
– cDC2 also can but to a much less degree
What is the structure of peptide binding regions of MHC class I?
peptides bound to class I molecules are 8-10 aa long
– longer peptides tied down at the ends and bulge in the middle
peptides have a few anchor residues which bind into deeper pockets which stabilise peptide binding
– because fewer variety of intracellular antigens
– peptides generated from proteosome
P2 binds pocket B - commonly a leucine
P9 binds pocket F - hydrophobic residue eg proline, leucine
What is the structure of peptide binding regions of MHC class II?
peptides lie flat and hang out the ends, w multiple anchor residues bound to more pockets
- focussed on a particular epitope on peptide it recognises, the rest doesn’t matter and hangs out the ends
- peptides generated from endocytic vesicles
pockets P1, P3, P6, and P9 are important for binding anchor residues
Give examples of non-classical class I-like molecules?
HFE
– sequesters iron from pathogens
– non-polymorphic, widely expressed
– in MHC or extended MHC
EPCR
– protein C receptor in clotting
– not polymorphic, on endothelial cells
– used by some malaria strains to bind to blood vessels
– genes outside of MHC
MR1
– present bacterial metabolites
– monomorphic, widely expressed
– genes outside of MHC
What are the classical class I molecules?
HLA-A, -B, -C
– present peptide to T cells
– NK ligand
– highly polymorphic, widely expressed
What are the non-classical class II-like molecules?
DM
– chaperone, peptide editor
– non-polymorphic
– expressed in APCs
DO
– binds to and inhibits DM
– non-polymorphic
– expressed in some APCs (B cells)
DQ2
– no known function
– monomorphic
– expressed in DCs in skin (Langerhans cells)
What is the structure of the MHC gene regions?
MHC organisation is similar among placental animals
– class I regions v different
– class II regions v similar
– class III are similar as they are not responsible for immune function
in humans there are also extended class I and II regions
class I and II genes separated by class III, thought to be evolutionary mistake
many more non-classical class I molecules in mice than humans
What is the function of ubiquitin?
ubiquitin ligase creates chains of ubiquitin on target protein which result in different outcomes
– degradation
– signal transduction
– cell death protection
– DNA repair
different types of ubiqutination
– different bond types (phosphorylation, acetylation)
– branched
– homotypic, heterotypic
What is the constitutive proteasome?
found in most cells
AAA ATPases act as chaperones and unfold substrates
- four stacks of rings, each composed of 7 related proteins
- two identical rings of a subunits form gates
- two identical rings of b subunits include 3 threonine proteases
What is the immunoproteasome?
present in mTECs, DCs, T cells
IFNy upregulates class I and II presentation
for class I:
- upregulation of class I a chains, B2-microglobulin, TAP1, TAP2
- better C-termini peptides for class I molecules
What is the thymoproteasome?
present in cTECs, important for positive selection of T cells
– have beta5t subunit
– produces self peptides from digested self proteins
What is the role of cytosolic proteases after peptides have undergone proteasome degradation?
trims peptides to optimal length for optimal antigen presentation, N terminal or C terminal extensions
eg tripeptidyl peptidase II (TPPII)
What is the structure of TAP?
ATP-binding cassette (ABC) transporter
TAP1 and 2 (heterodimer) each composed of a:
– tapasin-binding domain
– transmembrane peptide-binding domain
– nucleotide-binding domain (NDB)
each ATP binding site is split between the NDBs of TAP 1 and 2
the two NDBs must come together to hydrolyse one ATP
What is the function of TAP?
important for class I presentation, transports peptides from cytosol into ER where they can be loaded onto MHC class I molecules
the C-terminal specificity of the TAP corresponds w the specificity of the class I molecule at pocket F
– the immunoproteasome produces peptides that fit the specificity of the class I molecule at pocket F
so TAP can only transport peptides produced by the immunoproteasome
What are the steps of the antigen presentation pathway for class I molecules (steps 1-5)?
- translation of class I heavy chain into ER lumen through the Sec61 translocon complex, w co-translational N-linked glycosylation (one particular very important for quality control (QC))
- heavy chain binds to calnexin, a chaperone which prevents premature aggregation or misfolding, w glucosidases trimming N-linked glycan
- this trimming allows proper folding of heavy chain which loses calnexin and binds B2-microglobulin in the ER
– without this, the heavy chain is not stable and cannot present antigen - immature class I molecule (without peptide) associates with the peptide loading complex (PLC) on the ER lumen, includes:
- Meanwhile, TAP has pumped peptides into ER lumen
What are the steps of the antigen presentation pathway for class I molecules (steps 6-9)?
- ERAPs have trimmed the N-termini of peptides
- in the PLC, tapasin stabilises class I molecules without peptide and allows cycles in which peptides bind and fall off
– tapasin holds MHC molecule in a peptide-receptive conformation
– peptides w weak affinity bind and fall off - class I molecules bound to peptide w appropriate affinity are released from PLC
- released class I molecules are subject to another round of quality control by the UGT1-TAPBPR system, and sent either back to the PLC or to the GA and then to the cell surface
What is the function of ERAPs?
endoplasmic reticulum aminopeptidases
trims peptides bound to class I molecules to optimal length for optimal antigen presentation, specifically N-terminal extensions
eg ERAP 2, trims longer peptides before ERAP 1 refines them to optimal final length
What is the function of the UGT1-TAPBPR quality control system?
the class I molecule is released from the PLC through removal of the last glucose from the N-linked glycan, now it undergoes a second round of quality control
the lectin UGT1 binds the N-linked glycan and recruits tapasin via TAPBPR
– if the peptide falls of while tapasin is bound, then N-linked glycan is re-glucosylated and the class I molecule binds back to PLC
rinse and repeat until a peptide with high enough affinity is bound
What is the PLC
peptide loading complex
– Tapasin: chaperone that connects MHC class I molecule to TAP
covalently binds to ERp57
– Calreticulin: chaperone that stabilizes immature MHC class I molecule during peptide loading
– ERp57: thiol oxidoreductase that assists in folding and prevents improper disulfide bond formation in the heavy chain
– TAP1/TAP2: TAP transporter complex pumps peptides from the cytosol into the ER lumen, where they can bind to MHC class I molecules
Describe different methods used by viruses to evade the MHC class I antigen presentation pathway.
– HCMV US2 and US11 dislocate the MHC class I heavy chain into the cytosol where it is degraded by the proteasome
–HHV7 U21 diverts MHC class I molecules to the lysosome
– HIV Nef down-regulates MHC class I molecules from the PM, sequestering them in the trans-golgi network (TGN) through blockage of transport
What are the first steps for canonical antigen presentation on MHC class II molecules?
- class II a and b chains co-translationally glycosylated as they enter the lumen of the ER and then associate w invariant chain (Ii) trimer -> protects from peptide-binding in ER
2a. Ii-MHC class II delivered to PM and endocytosed in a clathrin coated vesicle which is targetted to an early endosome
2b. exogenous antigen binds receptor and is endocytosed in a clathrin coated vesicle where it targetted to an early endosome
- early endosome fuses to the MIIC which contains DM molecules
- proteases eg cathepsins digest Ii molecule to leave class II invariant peptide (CLIP) in the binding groove of class II molecules, and proteases digest antigen
What is the function of the ATP-dependent H+ ion transportation in the context of MHC class II presentation?
present in early endosomes, which makes the endosome more acidic as it matures
– deeper into the cytoplasm, the more acidic the endosome
this allows cathepsins to carry out their enzymatic activity
also promotes the exchange of CLIP for antigenic peptides w help from DM
Why is CLIP called a “universal binding peptide”?
CLIP binds to the MHC class II groove in a non-specific manner, fitting into a wide range of MHC class II alleles
uses methionines as anchor residues, not high affinity binding so can be exchanged
How is antigen uptake and subsequent degradation performed in different cell types?
phagocytosis (macrophages)
macropinocytosis (DCs): Non-specific uptake of extracellular fluid containing soluble antigens
B cells (also DCs and macrophages) endocytosis
degradation by a variety of proteases, glycosidases etc that depend on cell type and organelle
digestion can destroy epitope, so v high in macrophages but lower in DCs and B cells
IFNy inducible lysosomal thiol reductase (GILT) reduces disulphide bonds to allow protein to be unfolded
What are the 5 steps of peptide loading in the MIIC?
Step 1: CLIP is bound in DR groove and P1 pocket, which prevents premature antigen binding
Step 2: DM (chaperone) interacts with DR -> triggers a conformational change
– during this transition, the N terminus of CLIP detaches from the groove, and DR rotates away from the P1 pocket, allowing it to become available for interactions with DM
Step 3: DM stabilizes empty DR, and DR protects the hydrophobic P1 pocket.
Step 4: Rapid binding of peptides to partially accessible groove; peptides that do not successfully compete with DR residues for P2 site and P1 pocket are not stably bound.
Step 5: Binding of the peptide N terminus reverses conformational changes and results in DM dissociation.
What is DO?
expressed in B cells, medullary thymic epithelial cells (mTECs), and certain DC subsets
bind strongly to DM as a competitive inhibitor of classical class II binding
Its exact function seems to still be a topic of ongoing research but one hypothesis says DO might have a role in negative selection in the thymus due to is selective expression pattern
How do bacteria evade the MHC class II antigen presentation pathway?
– M. tuberculosis secretes SapM (secretory acid phosphatase M) that can hydrolyze phosphoinositides, disrupting vesicle acidification and preventing effective peptide loading
– Helicobacter pylori: VacA has been found to interfere with proteolytic processing by B-cells and to specifically inhibit the antigen presentation by newly synthesized MHC class II
– S. typhimurium inhibits surface expression of class II molecules through ubiquitination, performed by T3SS effector proteins
What is the pathway for cytoplasmic cross presentation for class I molecules?
occurs in cDC1
the phagosome is created from the ER, so it contains many ER proteins, such as the sec61 complex, part of ER-associated protein degradation (ERAD)
– this complex is involved in translocation, movement of peptides out of ER and into cytosol which are targeted for degradation
– in this case, antigens translocated out of phagosome and into cytosol. this targets antigen to the immunoproteasome where the antigens will proceed through the MHC class I presentation pathway
What are CLRs?
C-type lectin receptors, PRRs that recognise different carbohydrates on bacteria, viruses, and fungi in a calcium-dependent manner eg
– Dectin-1: β-glucans (fungal cell walls)
– DC-SIGN: Mannose and fucose on pathogens (e.g., HIV, M. tuberculosis)
What are RLRs?
Rig-I-like Receptors which detect cytoplasmic viral ssRNA containg terminal 5’ triphopsphate
work w MDA5
– both have caspase activation and recruitment domains (CARDs) in tandem orientation
signal via MAVS (mitochondrial antiviral signalling proteins) on mitochondrial membrane
activation through IRF3/IRF7/NfkB
What is the function of type I interferons?
e.g., IFN-α and IFN-β
Induce expression of interferon-stimulated genes (ISGs) that inhibit viral replication and spread
Promoting RNA degradation and inhibiting protein synthesis in infected cells.
Enhance NK cells and CTLs to eliminate infected cells
Increase antigen presentation by upregulating MHC class I molecules on cells, improving recognition by CD8+ T cells
Potassium efflux causes dissociation of chaperones from NLRP3 proteins allowing formation of the NLRP3 inflammasome. What causes the potassium efflux?
high extracellular Ca2+ activates NLRP3 acting as particulate matter, i.e. triggering K+ efflux
introducing phagocytosis inhibitors strongly impairs both the efflux of K+ and NLRP3-dependent IL-1β secretion triggered by particulate matter
