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197 Cards in this Set

  • Front
  • Back
Describe the IFN system
Ancient system
Can be found in primitive vertebrates
Uses Type 1 IFN (a/B)
What does IFN do?
**Combat viral infection through cellular resistance
Can also play a role in cell growth, differentiation and immunomodulation
What are the 3 types of IFN?
Type I: IFN-a/B
Type II: IFN-y
TYPE III: IFN-delta
What is Type I IFN used for?
Anti-viral IFN
Fight pathogen invasion
Also plays a role in tumour dev'p
What is Type II IFN for?
Immunomodulation
What does Type III IFN do?
Plays almost a redundant role with Type I
Which cells produce Type I IFN?
All cells: fibroblasts, epithelial cells, leukocytes, macs, pDCs
What induces the production of type I IFN?
PAMPs (virus, dsRNA, LPS)
Where is Type I IFN encoded in humans?
Chr 9 (no introns)
How many genes are in IFN-a? What is the size of the genes?
More than 15 hu genes
16-27 kDa in size
How many genes does IFN-B have? What is the size of the genes?
1 gene
28-35 kDa
What produces Type II IFN?
Specialized cells: T cells, macs
What induces the production of type II IFN?
Mitogens
Ag
IL-2
Where in the human genome is IFN-y encoded?
Chr 12
How many genes does IFN-y encode?
What is its size?
How many aa is it?
1 gene, 3 Introns
20-25 kDa
146 aa long
What are the effects of type I IFN?
Antiviral
Cell growth
Ptn Synthesis
Apoptosis
Recruitment of adaptive immunity
Ag Presentation
What do MxA, 2'-5' OAS and APOBEC all do in type I IFN?
Directly involved in antiviral activity
What does MxA do?
Captures viral structural components, traps them around the ER and stops viral assembly
What does 2'-5' OAS do?
Triggers degradation of viral RNA
What does APOBEC do?
Anti-retroviral activity
How does type I IFN exercise its antiviral activity?
Does this through its recpetors
Gets 100s of genes transcribed
-->Central mediators of antiviral resistance are transcribed
Whatdoes Type I IFN make to control cell growth?
PKR: main inhibitor of cell growth
If you stop cell growth, viral growth dec
What else does PKR affect?
Ptn synthesis
-> PKR decreases Ptn synthesis
What does type I IFN induce to control apoptosis?
Noxa
TRAIL
p53
Caspase 8
These all regulate apoptosis, which is caused by IFN
Why is Type I IFN involved in the recruitment of adaptive immunity?
Increases adaptive immunity when T cell show up, so they can immediately respond to APC
-->MHC I caused by Type I IFN
What genes are involved in the recruitment of adaptive immunity?
RANTES
IP-10 (CXCL10)
How is IFN activated?
IFN regulatory factor (IRF1) binds regulatory element on promoter and causes IFN-secretion
IRF-3 and IRF-7: main inducers of Type I IFN
-IRF-3 and 7 are normally inactive in the cell cytoplasms, but when they're P, they become activated, dimerize and go to the nucleus
-There, they bind to IFN virus-responsive element (IFVRE)
-Kinases that P them belong to the IKK family: IKK-e and TBK-1
Does IKK-e or TBK1 signal through NF-kB?
No they don't and they don't P IKKs
What do IKK-e and TBK1 recognize?
C-terminal of IRF-3 and IRF-7
When do these kinases trigger their P?
After viral activation
What is the structure of the IFN-B enhancesome?
ATF-2 -- c-Jun-- IRF3A -- IRF7B--IRF3C -- IRF7D --p53 -- RelA
What is important about the binding sites of IFN-B?
Binding sites overlap .: all factors must bind co-operatively to DNA (the DNA is mostly linear)
What happens if one of the factors is deleted?
Cannot make any IFN-B at all
.: Needs to be highly specific and activated all 3 paths (not just NF-kB etc)
What is required to get T of IFN-B?
Recruitment of cofactors
Describe the recruitment of the cofactors
In the IFN-B locus, there are 2 nucleosomes positioned up and downstream of the enhancesome
Enhancesome is free of nucleosomes and is exposed
There is a nucleosome on top of the TATA box that stops T initiation
When everything is in place, GCN-5 comes in and adenylates histones in the nucleosome
-Enhancement of A of H3 & H4 in nucleosomes and downstream on enhancesome
CBP recuited (after GCN-5 leaves)
CBP interacts withs IRF, NF-kB and AP-1
When all 3 are recruited, CBP gets a very stable interaction
CBP brings Poll II holoenz, SI/SNF complex
Histone is unwound and T start site is revealed
What are the master regulators of IFN transcription?
NF-kB
IRFs
What is the NF-kB pathway used for?
Central regulator of cell surviva, proliferation, apoptosis, innate immune response to viruses and bacteria, adaptive immunity
Describe the NF-kB pathway.
Activation
Signal transduction cascade (variable depending on the type of stimuli)
P of IKK so NF-kB can be transduced
Degradation of IKK
NF-kB dimers can go to the nucleus, recruit regulators
-->Get TF
--> Expression of many genes (including IK-Balpha)
How many gps of Rel/NF-kB TF and IkB family members are there?
What are they?
3
DNA binding, Non-DNA binding, Inhibitors
What are the DNA binding members?
p65, c-Rel
p52, p50
What are the more important members of the DNA binding gp?
Why?
p65/c-Rel
They are the activating members
Their C-terminal has an activation domain that can recruit CBP
What do all the DNA binding members have?
Rel-homology domain
What is the Rel homology domain important for?
Required for dimerization and DNA binding
What are p50/p52 important for?
Important for transactivation when dimerized with an activating member
->They cannot transactivate on their own
What do homodimers of p50/p52 act as?
Inhibitors
How do inhibitors work?
They bind the Rel homology domains by virtue of their Ankyrin repeats
They inhibit DNA binding ability of the other members and sequester them
What is the PEST domain on the inhibitors?
Sequence that targets ptns for degradation, but they aren't degraded everytime
Need P events for degradation to take place
Describe the non-DNA binding gp members
Hybrid of the DNA binding and inhibitor gps
They have long precursors
Have Rel-homology domain and Ankyrin repeats
What are the members of the non-DNA binding gp?
p105 and p100
What's the difference between the p100 and p105?
p105: precursor for p50
-->It is constitutively cleaved
p100: precursor for p52
-->highly regulated, only cleaved under special situations
-->Part of the non-canonical NF-kB path
What happens when the IKK complex P a ptn?
Gives the signal for Ub and ptn degradation
Describe the IkBalpha ptn
Signal domain reponse ---- Ankyrin repeats ---- PEST domain
What is the IkBalpha regulated by?
At the T level, regulated by NF-kB itself (negative fdbk loop)
What is the PEST domain of IkBa for?
Important for its degradation
Describe the cycle of IkBa
NF-kB acitvation makes NF-kB bind to the IkB promoter
When IkB is synthesized, NF-kB is inhibited
=> Shut off the signal
Negative regulator mechanism
Keep control of IR
Physiological fcts of NF-kB
What happens to mice with p65KO?
p65 = DNA binding (activating mb)
If p65 is KO: get massive apoptosis and liver degeneration, mouse dies
Physiological fcts of NF-kB
What happens to mice with RelB KO?
Rel B also part of DNA binding
RelB expression is restricted to lymphoid tissues.
->RelB -/-dev'l normally until day 10
Then they start expressing thymic atrophy, splenomegaly, hyperplasia and *Dysfunctional immune response*
Physiological fcts of NF-kB
What happens to mice that are c-rel -/-?
C-rel is restricted to hematopoeitic cells
C-rel -/- have profound lymphocyte activation defects
What is C-rel important for?
Important mediator downstream of BCR and TCR
What happens to mice with a p50 -/-?
No developmental abnormalities
BUT --> they have increased susceptibility to bacterial infection
AND--> Increased RESISTANCE to viral infection
Which members are more important in mediating acute immune responses?
p50 and C-rel
What are p65 and RelB important for?
p65: liver development
RelB: development of the hematopoeitic system
What are the 3 subunits of IKK complex?
a, B and y
Are these 3 subunits redundant?
No, KO each one and you get a different result
What happens if you KO IKKa?
Mice die at birth with multiple developmental defects
IKKa not required for activation of NF-kB and degradation of IkB by proinflam stimuli (only true for canonical path)
What happens to mice that are IKKB -/-?
Mice progressivle died cuz of liver degeneration and apoptosis (like with p65 -/-)
-Also have reduced basal NF-kB activity and impaired cytokine induced kinase activity
What happens to IKKy -/- mice?
Male IKKy-//- mice died in utero
IKKy -/- female mice developed patchy skin lesions with massive granulocyte infiltration and hyperproliferation and increased apoptosis of keratinocytes
Describe the canonical NF-kB path (classical path)
TACI/BCMA/TNFRa
Signal through TRAF2 which sgnals to the IKK complex (Entire complex)
Signals IkBa which complexes with p50 and p65 which go to the nucleus after IkBa is P and goes to the proteasome
Describe the non-canonical path (alternative path)
BAFFR/CD40
(Triggered by different stimuli)
Signal through NIK (different kinase, not TRAF)
NIK usually unstable, but stabilizes once it binds its ligand and P p100
Processing, than activates p52 and RelB, whicha activate different set of factors
Are both these paths completely separate?
No, there is cross talk betwen the 2 paths
NIK can also activate the classical path
What is the role of TRAF3?
Usually keeps NIK inactive (also can keep TRAF2 inactive)
Why do viruses go after the NF-kB path?
This path is important for cell surival and growth, increased ptn production
Virus wants to use this
How do the following viruses subvert this path?
HIV
HTLV
EBV
HIV: Tat ptn can bind IKKy. Also acts through PLC to activate NF-kB
HTLV-1: Tax ptn -> activates NF-kB
EBV: activates NF-kbB through non-canonical path
What are the IFN regulatory factors (IRFs)?
Family of TFs
Regulate IFN and IFN-stimulated genes
Bind IFN stimulated response element (ISRE)
What does IRF-1 do?
Activator of IFN-B
What does IRF-2 do?
Inhibitor of IFN-B
What do IRF 3 and 7 do?
Main regulators of Type I IFN
What do IRF 1,5,9 do?
Participate in regulation of Type I IFN, but play different roles
What do IRF 4 and 8 do?
Involved in lymphoid development
What does IRF 6 do?
Repressor
What do viral IRFs do?
Repressor of host IR
What are the key mediators of IFN production?
IRF 3 and IRF 7
Describe the IRF-3 operon
DNA binding domain --- PRO --- IAD --- RD
What is the RD?
Rel homology domain
What does the RD do?
Allows dimerization
Allows DNA binding and interation btw binding parts
What needs to happen to the C-term of the IRF operon (RD)?
Why?
P
P allows the structure to open up and DBD unmasked by inhibitory domain
Highly polar PO4 gps change C-term
-Expose hetero/homo dom
What does the DBD recognize?
ISRE
-Has a NLS that is required for its transloation when its active
Describe the IRF-7 operon
DBD--- CAD --VAD--ID ---RD
What is CAD?
Constitutive Activation Domain
What is VAD?
Viral activation domain
What does virus inducible P of IRF 3 at the C terminus regulate?
Cytoplasmic-nuclear translocation: nuc localization seqwuence will become exposed
Ass't with CBP/p300 co-actiator
Trasactivation of virus responsive promoters
Proteasome mediated degradation: when signal is done and gene is T, IRF3 will be degraded
What form of IRF3 appears after viral infection?
Why?
Heavier form of IRF3 appears becuase it gets P
Where is IRF3 when its not activated?
Mainly cytoplasmic, but shuttles in and out of nucleus
Where is IRF 3 after viral activation?
In the nucleus
Is P of IRF 3 sufiient ot cause IFN-B?
Yes (IRF3 (5D) )
What does CBP interact with?
The 3 helices of the IRF3 AD
(C-term region of IRF3 also interacts with this region)
What is the main difference btw IRF 3 and IRF 7 that makes them non-redundant?
IRF3 expression is constitutive
=>All cells at all ties have IRF-3 expressed in their cytoplasm
=>1st factor activated when the virus comes in

IRF-7 expression is induced
=>You need the 1st wave of IFN production so that the neighboring cells can detect the IFN
-->The IRF-7 will be part of the IFN-stimulated gene production
-->ITF-7 will then be preset in the cytoplasm
=>IRF-3 is part if the initial response and IRF-7 will be part of the secondary response/amplification response
What makes more IFN, IRF 3 or IRF 7?
IRF 7 makes sigificantly more
What does IRF 7 induce?
Production of IFN-B
What does IRF 3 induce?
Only certain types of IFN-a (can induce IFN-a2, but not IFN-a1)
Which cells have constitutive IRF 7 expression?
Macs and plasmaytoid DCs
Why don't all cells have constitutive IRF 7 expression?
IRFs do differet things.
IRF 7 can stumulate all types of type I IFN
IRF 3 can only stimulate certain types of type I IFN
What kind of an experiment could be designed to show the difference between the type of mac with constitutive expression of IRF 3 or IRF 7?
Take amac
Put a constitutively active form of IRF 3 or IRF7 into an adenovirus casette, then put gene in macs so macs can express active form of the IRFs
What kind of experiment can be done to see the big differences?
Use microarrays
Which genes are shared by both IRF 3 and 7 when you llok at a microarray of it?
PRR paths (TLR/NF-kB/JAK-STAT)
IFN-stimulated genes (ISF)
Chaperones
Stress release ptns
Ub path
What are 2 very different gees btw IRF3 and IRF 7?
Apoptotic response
Tumoricidal and immune fct
Which IRF can induce apoptosis?
IRF3
How does a cell detect infection?
Pathogen recognition receptors:
-TLRs
-NLRs
-DAI: DNA sensors, rec'z DNA in the cytoplasm
-RLRs: RIG-I, Mda5
What kind of a structure do the RLRs have?
Helical
What do RLRs rec'g?
Viral dsRNA (rec'z these using their helical structures)
What do NLRs recognize?
What do they signal through?
Re'z bact compartments
Signal through inflammasome for NF-kB
Where are TLRs mainly expressed?
Macs, DCs, monocytes and B cells
-->Regular cells don't express TLRs
Describe the TLR structure
Ectodomain: sigal peptide, LRR, cystein-rich domain
Transmb domain: single pass
Intracellular domain: TIR (toll-IL1 receptor) domain
What does the ectodomain do?
PAMP rec'g and binding
What does the TIR domain do?
Reruits adaptors
What does TLR3 reognize?
ds RNA
What does TLR4 rec'z?
LPS + other things
What is TLR4 the main mediator of?
Septic shock
What does TLR7 rec'z?
ssRNA
What does TLR8 rec'z?
ssRNA
small synthetic cmpds
What does TLR9 rec'g?
unmethylated CpG (on bacteria and viral DNA)
Which apoptotic gene does IRF-3 induce greater amounts of than IRF 7?
NOXA
What is the key place for apoptosis induction?
Mitochondria
Where does cyt C have to go for apoptosis?
From mito to cytoplasm
How does cyt C escape the mito?
Bax/Bak oligomerize
Get pore formation
Cyt C can get out
How are Bax/Bak usually kept inactive?
By binding anti-apoptotic members of the Bcl2 family (MCL-1)
What happens when the cell is stressed?
Get NOXA expression
Binds MCL-1
Displaces it from Bax/Bak
.: Bax/Bak makes an oligo to form porers to trigger cyt C release
-->NOXA aloe a help trigger olig of bax/Bak
End result: Cyt C release ad activation of apoptosis
.:IRF3 activates NOXA T and thats all it needs
What does active IRF-3 expression do to the NOXA promoter?
Induces it
What does active IRF7 do to macrophages?
Increases their killing ability
->IRF7 doesn't increase apoptosis but increased death of neighboring cells
What happens to cells infected with chimeric Adeno-F7?
Inhibit prolif of target cells
Is this process completely dependent on IFN?
What does this mean?
No, because when the chimeric insert is removed, the cell doesn't go back to basal lvl of type I IFN
.: IRF-7 induces very specific types of cells that don't need type I IFN
What kind of genes are involved in active IRF-7 expressig macs?
Genes and cytokine genes involved in AP, cross presentation and cell killing
What are the effects or IRF7 activation?
Release soluble factors
Inc AP
Recruit TH1 by secretion of IFNa/B, IP-10, RANTES
Recruit NK cells because of RANTES, IL-15, IL27
Release TRAIL and IFNa/B to kill infected cell
What is the distict role of IRF3?
In all non-immune cells
Triggers restricted, specific antiviral response
Strong pro-apoptotic response mediated by upregulation of NOXA
What is the distinct role of IRF7?
In specialized immune cells (pDCs and Macs)
Amplifies antiviral response by induction of multiple type I IFN species ad ISG
Stimulates IR fct (inc AP)
Inductio of anti-tumour fcts of macs
Weak apoptotic response
Describe TLR4 structure
Horse shoe structure
What happens whe a ligand binds its TLR? (usually binds at surface ad not at the groove)
Causes olig. in the cytoplasmic part
Brings 2 TIR domains in proximity and this starts the signalling
Which cells express all TLRs?
DCs
Monocytes/macs
What do B cells ad mast cells express?
Subset of TLRs
What do T cells express?
Some TLRs
Why do airways and intestinal epithelium express a subset of TLRs?
Sites of pathogen entry
Do neurons express any TLRs?
Yes (TLR 12 and 13, unknown fct)
Describe TLR signalling
Mainly through adaptors ad TIR domain
How many adaptors does TLR 4 have?
4adaptors:
MyD88
MAL
TRAM
TRIF
How many adaptors does TLR 3 have?
1: TRIF
How many adaptors does TLR 7/8/9 have?
1: MyD88
What happens once the TLRs signal through the adaptors?
Get recruitment of kinases,
Activate IKK and TBK1/IKKe that P the target T factors
Get antiviral and inflam response
What is TLR4 signalling always ass't with?
MD2 and CD14
What rec'z LPS that is shed by G- bacteria?
LBP (LPS Binding Ptn)
What on the host cell recognizes the LPS-LBP complex?
What does it do?
CD14
CD14 loads the LPS-LBP complex onto TLR4 and allows its dimerization
What hapens once TLR4 dimerizes?
TIR domains come into close contact and there is initiation of signalling cascade
Which adaptors are recruited following TIR domain activation?
MAL and MyD88
What is TLR4 signalling always ass't with?
MD2 and CD14
What rec'z LPS that is shed by G- bacteria?
LBP (LPS Binding Ptn)
What on the host cell recognizes the LPS-LBP complex?
What does it do?
CD14
CD14 loads the LPS-LBP complex onto TLR4 and allows its dimerization
What hapens once TLR4 dimerizes?
TIR domains come into close contact and there is initiation of signalling cascade
Which adaptors are recruited following TIR domain activation?
MAL and MyD88
What do the adaptors then recruit? What does it do?
IRAK-4, which P TRAF6
What is TRAF-6?
UB-lyase of TAK-1/TAB
What happens once the TAK/TAB complex is Ub?
Complex gets activated
MAPK cascade is activated
--->Get production of ATF2/c-JUN in the cytoplasm
What happens to TLR4 after this signaling path?
TLR4 gets translocated to the endosome
What does TLR4 recruit once its in the endosome?
Recruits TRIF through Adaptor TRAM
What do TRIF and TRAM cause?
Another signalling cascade is initiated through TRADD and RIP1, which also feeds into the TRAF-6/NF-kB path
(This is how you get early and late NF-kB activation)
How is TRAF3 recruited to TRAM?
Through endosomal signalling
What does TRAF3 do?
Signals to TBK-1 and IKKe (kinases) to activate IRF 3 and IRF 7
What does TLR 4 signaling induce?
NF-kB signalling
IRF signalling
AP-1 activation
What happens when all 3 of these systems are actvated?
Get inductionof IFN- genes
How does the TLR3 signal path go?
Similar to TLR 4 signalling
Works through TRIF, recruits TRAF 6 and get IKK/TBK to get IRF3/7 production
Which cells use RIG-I-like receptors?
The common cells
What are the 2 RLRs?
RIG-I
Mda5
What is LGP2?
Truncated form of the Mda5 regulator. Acts as an inhibitor
It is not mb bound. Resides in the cytoplasm
What kind of expression do RIGI and Mda5 have?
Constitutive expression in all cells
What induces the RLRs?
Type I and II IFN
Where are the RLRs localized?
Cytoplasm
Describe the Structure of RLRs.
2 CARD domains--- DExD/H box helicase ---- CTD/RD
What does the C-term domain of RLRs recognize? How?
dsRNA
Rec'z it through the helicase domain and 2 CARD domains that mediate ptn-ptn interactions and signal transduction
What do the CARD domains of the RLRs interact with?
MAVS (adaptor ptn)
What is MAVS?
Main transductor of signalling
-->Adaptor that all signalling components bind to
What is another adaptor molecule that is essential for IFN signalling by RIG-I?
IKK-y/NEMO
What does NEMO do?
Interacts with IKKe and TBK-1 through TANK
Whgat localizes MAVS to the outer mitochondrial mb?
TM domain
What happens once the RLR is activated?
CARD-CARD interactions recruit MAVS. which bring in TRAFs
What does TRAF 3 signal to?
IKKe and TBK1
-->Get production of IRF3/7
What does TRAF6 do?
Brings in TK kinasa
-->Get MAPK activation =>ATF-2/c-Jun
-->get removal of IKK complex => get NF-kB activation
What happens when TRADD and FAD are recruited?
Get activation of Caspases
What happens during the 2nd wave of IFN signalling?
IFN-a binds its receptors, recruits STATs which complex with IRF9
Forms ISGF3 complex
Get an antiviral response by activating production of IRF-7 when ISGF3 binds ISRE and also produce more IFN-a
What happens when type I IFN binds its receptors on neigboring cells?
Jaks cross-P each othr
Activated Jaks P receptors on tyrs
STATs dock and Jaks P them
STATs diss't from the receptor and dimerize through SH2 domains
Ass't with IRF9 to form the ISGF3 complex
ISGF3 complex binds ISRE and get T of ISGs in the nucleus
What kind of STATs are involved in Type I and III signalling?
STAT 1 & 2 (forms ISGF3 complex)
Antiviral activity
What STATs does type II IFN actiate?
Only STAT-1
-->antimicrobial immunity
What are ISGs?
IFN-inducible genes
What are ISGs for?
Antiviral immunity
What is ISG15 like?
Ubiquitin, but plays an antiviral role
What happens when ISG15 is conjugated to target ptns?
Seems to lead to a permanence of IFN response
Why is MxA an antiviral trap?
It is produced as a monomer, but it oligomerizes and traps viral components in the ER
What does OAS1 do, after being produced by ISRE?
The OAS1 monomer is inactive
Active when becomes a tetramer
Gets polyadenyulated
Activated RNAse L and lets it dimerize
What does RNAse L do after is dimerizes?
Chops RNA into small pieces and destroys viral RNA
Generates fragments tht can be rec'z by RLRs and trigger immunity
What does PKR do?
Its made as an inactive monomer
Becomes activev in the presence of viral RNA
Stops Tl by P of EIF-2a