LMP299Y1 Lecture Notes - Nod2, Nod1, Frameshift Mutation

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16 Dec 2013
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LMP October 15, 2013
NOD proteins in mucosal defense against bacteria: implications for Crohn’s disease
Epithelial layer line mucosal surfaces; on mucosal side, have macrophages (defense cells in general);
major component of lumen of intestine is bacteria (commensals)
TLR recognize bacterial and viral products, trigger signalling
TLR on luminal side of epithelium? Bad because always active due to presence of commensal bacteria
same products produced by both pathogenic and commensal bacteria, so signalling will always be active
Defense mechanism in cytosol if bacteria found in cytosol, IT IS DEFINITELY PATHOGENIC
Location of defense mechanisms is important!!!
NOD-like receptors expressed in all sites of the body
Recent research has identified key link between NOD receptor and inflammatory disorders of
the intestine
Mechnikov father of innate immunity
Receptors important for detecting microbes first were identified in 1995, were TLRs
Sensing systems of microorganisms
PRMs = pattern recognition molecules
Molecules of complement that detect specific microorganisms all secreted molecules that play specific
role in detection and handling of microorganisms OUTSIDE CELLS key defense organism at level of
whole organism
At level of cell signalling, have TLRs, NLRs, RLRs, etc. that lead to cell signalling
The NOD-like receptor family
TLRs are homogenous group; domain organization is conserved VERSUS NLR, all lack transmembrane
domain and have ATP/GTP binding domain whose function is unclear and flanked on C-terminus by
leucine-rich repeat domain
At N-terminus, depending on sub-category of NLR, have different domains, which are responsible for
inducing different types of cell signalling
Common motifs in domain organization research in the last year have shown that NLRs have this very
interesting ability to detect either microbial motifs and an example is peptidoglycan from bacteria or
danger signals (detection of microbes or danger associated with infection or tissue damage; ex. Host
DNA or histones; some molecules released by dying cells, mitochondrial DNA, evolved to be detected by
danger signals important in case of tissue inury where have tissue damage and this dmage can be
cured by different means)
NOD1 and NOD2
Looking for molecules able to detect intracellular bacteria
What are NLR sensing?
Luciferase experiment used for transcription factors; luciferase gene and NFkB promoter, so
overexpress luciferase, how much NFkB activity detected, so luciferase dpenedent on NfkB activity
Peptidoglycan from different bacteria NOD1 could only be stimulated by some peptidoglycans, those
that could stimulate were gram-positive bacteria
Saw difference between types of bacteria
Knock downs can be done down to confirm the results done a decade ago
Nod1 fractionated peptidoglycan in different fractions analyed by mass spectrometry
Only some bacteria were detected by Nod1 something in peptidoglycan specifically was detected
Peptidoglycan is made up of repeating sugars, peptide links peptides are cross-linking chains
There is heterogeneity in peptidoglycan some chains made up of 2,3,4, 5, peptides some cross-
linked, creating heterogeneity
In mass spec, see different populations
When separate the peaks, repeat the experiments only 2 peaks activating Nod1
The two molecules that were in fraction were very similar they had TRIPEPTIDE that were not cross-
linked
The whole detection system dGlu and mesoDAP these are absent from petidostructures of the host;
no mesoDAP in mammals
Really specific to bacteria
It explains why gram-positive bacteria not detected
Similar work on NOD2
NDP worked right away see difference between NOD1 and 2
NOD2 detects muramic acid; nod1 detecs muramid acids plus mesoDAP
Nod 1 detects gram positive bacteria specifically
In cytoplasm of cells, have NOD1/2 that were evolved to detected specific bacterial structures i.e.
peptidoglycan and different peptidoglycan motifs
Once NOD1/2 activated some variability nod 1 has cap domain that interatcts with RIp2 which also
has CAL domain
TLR receptor also TIR domain in cytoplasm activation of pathway is TIR-TIR hemophilic interaction
Here is CAL-CAL hemophilic interaction
NOD2 and Chron’s disease
Two papers identified the first gene for Crohn’s disease
Most commonly associated locus in CD
CD
Chronic inflammatory disorder of gastrointestinal system can affect any part of GI tract
Usually have flares of inflammation and periods of remission there is no cure for Crohn’s disease – but
if look at incidence of Crohn’s disease in the world, it is dramatically increasing – reasons unclear
Hygiene hypothesis balance between infectious disease vs inflammatory disorders
NLR protein associated with disease of the intestinal mucosa?
Pathogens, commensals the intestine is not any type of mucosal surface because of the nuber of
bacteria resident to intestine there is affinity for immune system in intestine, because have 1) tons of
bacteria, so must be silent to microbiota, which si already a challenge to the host; 2) pathogens also
present in intestine. Major issue silent to microbiota, which is made up of bacteria but also aggressive
to pathogens that could enter by the intestine; one of the ways the immune system has challenged the
problem is by location of immune system sensors NLRs in cells, where it is supposed to be sterile
Expression of functional NOD2 in eptheillial cells
Probably CD has something to do with bacteria simple idea that CD was associated with mishandling of
bacteria only recently due to knowing that NOD2 sensing peptidoglycan, that is known that CD due to
mishandling of peptidoglycan
NOD2 expressed macropahges cells in sub-mucosal not in lumen
Begue et al. when already have inflammation cytokines released such as TNF; the epithelium
changes, and one of the major effects is TLR induces NOD2 expression in epithelial cells ONLY IN
INFLAMED STATE
Mutations in NOD2 associated with CD most common one is frame-shift mutation
One feature is that the mutated form of NOD2 in CD cannot detect bacteria products suggesting that
there is some misdetection of bacteria in case of CD
Other CD associated with NOD2 mutations
All the different mutations found in NOD2 associated with CD; most common is SNP13
Took all the different forms of NOD2 and recreate in vitro all the different mutations even the rare
ones
Compare the capacity of every mutant to detect peptidoglycan with the wild-type
If at 100%, mutant detects as well as wild-type
If at 1%, detect 99% less than wild-type
Read out is looking at NF kappa B
Most of the mutations lead to detrimental function
How can you infer causality? What can you conclude of the link between NOD2 and CD??
Maybe NF kappa B is not the only read out; looking from the narrow angle of NF kappa B maybe the
mutants have different functions at different effectiveness
Current models to explain the paradoxical link between NOD2 loss of function and CD
The mutations associated with NOD2 causes decreased NFkB activity yet inflammation is increased
Something is missing… NOD2 is a way to try to dive into those questions
1. Loss of antimicrobial activity
Over expressed NOD2 in Kappa 2 cells less Salmonella so NOD2 is antimicrobial
Overexpress the frameshift mutation interesting observation even though mechanism was not known
Paneth cells important for defense produce antimicrobial peptides stem cells in general at base of
crypt stem cells for renewing epithelium
NOD2 is strongly expressed in paneth cells antimicrobial peptides might serve as link between
detection of bacteria and production of antimicrobial peptides
Those stains maybe artifacts however, because Paneth cells are so electron dense, so non-specific
staining
More recent experiments with Nod2 knockout mice NOD2 can kill bacteria assay done from isolated
crypts in intestine; cell from intestine can kill bacteria if add NDP NOD2 konckouts NDP inducing
NOD2 would induce antimicrobial ability
Flavell showed that if take intestine of NOD2 knockout mice vs WT mice observe that there was
defect in secretion of specific antimicrobial peptides alpha defensins seemed to be defective in
knock out argues for antimicroibial function has not been reproduced
Not well controlled