Signal transduction in innate immunity:

the case of Toll-like, NOD and RIG

receptors
CINVESTAV

Claudia González Espinosa, PhD

Pharmacobiology Department
Cinvestav
 Contents
1.  General concepts in signal transduction

2.  Signal transduction through TLR receptors

3.  Signaling through RIG-like receptors

4.  Signal transduction through NOD-like receptors

5.  Recent advances on innate immune signaling

Fernando de Szyszlo, Sin título, 2010.

 Immune System is a Distributed Autonomous System

We can define a system as an interacting collection of entities or agents, such as cells or ants.
If a system is termed distributed, it implied that the system is composed by many entities
(such as the trillions of cells in the immune system) and that activity of the system is
accomplished by the combined action of many of these entities. Immune system is autonomous
because there is no a special entity that directs its actions.

Major design principles for the immune system are:

Layering (new processes are built on top of older, less-effective processess).

Scaffolding (early steps in the immune system provide the conditions
needed for later steps)
Parallel processing
Dynamic engagement (cells act briefly and then are replaced by other cells).
Variable network connectivity

Segel, L.A. and Cohen, I.R., Design principles for the immune system and other distributed autonomous systems. Oxford University
Press, 2001. ISSBN 0-19-513699-3
 Concept of Signal Transduction

Transduction. Conversion of energy from one form into another. 1930.

Transduction is the mechanism of changing light into chemical signals

inside a cell in photoreception phenomena. Hildebrand, E. 1977.

Signal Transduction is the process of translate extracellular chemical

signals in a coherent group of metabolic changes in bacteria. Adler, J. 1979.

Signal Transduction refers to the molecular changes during the process

of recognition of a ligand by a given receptor, the formation of active
intermediates and the final response of any cell. Rodbell, M., 1980.

Signal Transduction is the mechanism by which extracellular

physicochemical stimuli are transmitted via a signaling cascade into
intracellular chemical changes that orchestrate appropriate responses.

Signal Transduction refers to the metabolism of the information that is

received by a cell and is converted into physiological changes.

Gomperts, B.D. et al. Signal Transduction, Second Edition, 2009. Academic Press.
 Is signal transduction really important?

At least 15% of our genome codifies

for proteins known to participate in
the process of signal transduction.

Together with transcription factors

and histone-modifying proteins,
Around 23.2% of the human genome is
involved in to translate signals
from the environment into a
coherent group of chemical
reactions inside cells.

1543 receptors
868 kinases
988 regulatory proteins
376 peptide ligands
902 regulators of cell division

Venter, JC., et al., Science, 291:1304-1351, 2001; International Human Genome Sequencing Consortium, Nature, 409:860-921, 2001
 Panther Categories of Human Genes

Venter, JC et al. Science 291:1304- 51, 2001

Main types of signaling cascades
Receptors
Enzymes
Transcription factors
 Spatial organization of cell signaling

Good, M.C., et al. Science 332: 680-686, 2011

 Complex formation and signal amplification

Protein complex
formation adds
velocity and
specificity on
signal
transduction
Negative
feedback
loop
Scaffold
proteins are
formed by
interaction
domains

Alberts, B. et al. Molecular Biology of the Cell, Garland Press, 2017.

 Concept of protein domain

A domain is a part of a protein sequence that:

1)  Can evolve and function independently of the rest of

the protein chain.

2) Forms a 3D structure and often can be independently

stable and folded (motif).

3) Confers specific interaction/catalytic characteristics to the

proteins they belong.

4) Can be found combined with others in to create proteins with

different functions.

5) Vary in lenght from 25 to 500 aminoacids.

 Signaling proteins contain multiple domains

Waksman, G., et al. Cell 72(5):779-790, 1993

 Examples of domain- specific interactions

Bedrrige, M. Cell Signaling

 Common interaction domains in signal transduction

Pawson, T., and Nash, P., Science 18:445-452, 2003

Common post-translational protein modifications

Enzimatic Activity Protein Modification

Tyrosine kinase Tyrosine
Phosphorylation
Serine-Threonine Serine-Threonine
kinase Phosphorylation
Ubiquitin ligase Ubiquitin transfer (K63,
K48, etc)
Protease Proteolysis
 Phosphorylation-based signaling cascade

Example:

The MAP kinase

cascade
The MAPK signaling cascade
 Ubiquitination

Ubiquitin
(76 aa, 8.5 kDa)

Glycine
Structure of Lys48 and Lys 63 ubiquitination
The ubiquitination code
 Proteolysis

External Stimuli
Distinct post-translational modifications in a single pathway

The NFkB signaling cascade

 Controlling intensity and specificity

Pathway
signaling
dynamics
Isoforms,
Expression of
specific elements
of the pathway

Madsen, R. and Vanhaesebroeck, B., Science Signaling 13, eeay2940, 2020

 Cell Biology and Signaling

Empty
space?
Signaling on a crowded cytoplasm
Signaling is controlled by vesicle trafficking
 Main Innate Immunity Receptors

1.  Toll-like receptors (TLR)

2.  Retinoic acid-inducble gene-I (RIG-I)-like receptors

3.  Nucleotide-binding domain, leucine repeat-containing

protein receptors (NLRs)

4.  C-type lectin receptors (CLR)

5.  Others:

Absent in melanoma (AIM)-like receptors (ALRs)

Formyl-methionine receptors (fMLPs)
 Main Innate Immunity Receptors

Abbas, A., et al. Molecular Immunology 8th Edition, 2018.

 Toll-like Receptors (TLRs)

TLRs are integral polytopic

proteins
(transmembrane proteins)

TLRs are Type I

transmembrane
glycoproteins, because their
Carboxi-terminus is towards
cytosol.

10 in humans
13 in mice

Abbas, A., et al. Molecular Immunology 8th Edition, Elsevier, 2018.

Structure of LRR and TIR domains

LRR

TIR

135-160 aa. Five-stranded beta

sheet and five alpha helixes
 Canonical TLR
signaling cascade

O´Neil, L.A.J., et al.

Nat. Rev. Immunol.
13:453-460, 2013
 Receptors

O´Neil, L.A.J., et al.

Nat. Rev. Immunol.
13:453-460, 2013
 Adapters

O´Neil, L.A.J., et al.

Nat. Rev. Immunol.
13:453-460, 2013
 Ubiquitin
Ligases

O´Neil, L.A.J., et al.

Nat. Rev. Immunol.
13:453-460, 2013
 Ser-Thr
Kinases

O´Neil, L.A.J., et al.

Nat. Rev. Immunol.
13:453-460, 2013
 Ser-Thr-Tyr
Kinases

O´Neil, L.A.J., et al.

Nat. Rev. Immunol.
13:453-460, 2013
 Tyrosine
Kinases

Lyn/Fyn

O´Neil, L.A.J., et al.

Nat. Rev. Immunol.
13:453-460, 2013
 Transcription
Factors

O´Neil, L.A.J., et al.

Nat. Rev. Immunol.
13:453-460, 2013
 Gene products

O´Neil, L.A.J., et al.

Nat. Rev. Immunol.
13:453-460, 2013
 Desensitization of TLR signaling
(negative feedback loop, endotoxin tolerance)

Biswas, S., and López-Collazo, E. Trends Immunol, 2009. DOI: 10.1016/j.it.2009.07.009

 Pattern-recognition molecules

Abbas, A., et al. Molecular Immunology 8th Edition, Elsevier, 2018.

 Pattern-recognition molecules

Three members of the family:

1.  The retinoic acid-inducble gene I (RIG-1, discovered in 2004)

2.  The melanoma differentiation-associated gene 5 (MDA5)

3.  The laboratory of physiology and genetics 2 (LGP2)

Those molecules are part of the SF2 family of nucleic acid-

dependent nucleotidases (NTPases) because they use energy
to conduct functions at nucleic acids, and the family includes
helicases, motor proteins and translocases.
 Ligands of RIG-like receptors are RNAs

RIG-1

Viral genomic RNA; viral RNA replication intermediates, leader

RNAs, short dsRNAs; dsRNA ends (phosphorylated)

MDA5

Long dsRNAs (>2000 bp); replicative forms of viruses. MDA

does not bind ssRNA

LPG2

dsRNA ends (phosphorylated or not).

 Structure of RIG receptors Substrate
specificity

N-terminal
caspase
recruitment dsRNA
and activation Oligomerization
domain C-terminal Domain
(2-CARD) domain

NTPase
domain
(RecA1 and ATP
RecA2)

Lässig, C. J Biol Chem 292(22):9000-9009, 2017

Signaling through
RIG-I-like receptors

LPG2 contributes
to MDA5 signaling

MVAS, mitocondrial
antiviral signaling
adapter protein

Lässig, C. J Biol Chem 292(22):9000-9009, 2017

Signaling through
RIG-I-like receptors
induces IRF and
NFkB activation
Signaling through
RIG-I-like receptors Receptors
induces IRF and
NFkB activation

Ubiquitin
Adapters
ligase

Ser-Thr
kinases Caspases

Transcription
Factors Gene
Products
 Self RNAs are protected from RLR activation

miRNAs and siRNAs produced rRNAs are protected by Mature mRNAs are 2´-O-methiyated
by Dicer have a characteristic ribosomal proteins in the first nucleotide
3´overhang that prevents RIG-1
binding tRNAs are cleaveged to Immature mRNAs are located inside
Produce 5´monophosphate ends cell nucleus

Mature mRNAs present short dsRNA

segments that are prevented by
the conversion of A to I by specialized
enzymes

Lässig, C. J Biol Chem 292(22):9000-9009, 2017

 Pattern-recognition molecules

Abbas, A., et al. Molecular Immunology 8th Edition, Elsevier, 2018.

Comparison between
LRR domains found in
NLR and TLR receptors
 The Nucleotide-binding Oligomerization Domain receptors

Cytosolic sensors for microbes, endogenous danger signals

and exogenous insults.

The defining feature of NLR family members is the presence

of a nucleotide-binding domain (NATCH) and a second
domain harbouring a leucine-rich domain.

22 human and 34 murine NODR have been identified

Four families of NOD
receptors

Domain found in
NAIP, CIITA, HET-E
and TP1 proteins
(NATCH)

The NATCH domain

consists in seven
specific motifs, including
the ATP-GTPase loop,
a Mg2+ binding site and
five more.
 The Nucleotide-binding Oligomerization Domain receptors

Martinon,F., Annual Rev Immunol, 2009

 Signal transduction through NOD-like receptors

NOD-like receptors transduce signals by

1) Forming cytoplasmic marcomolecular complexes called

Inflammasomes.

2) Activating NFκB transcription factor

Inflammasomes are stabilized by homotypic interactions

between aminoacids located in the NATCH domain.

Inflammasomes are coupled to the activation of inflammatory caspases (1,

12,11,4 and 5), leading to the production of IL-1β and IL-18.
 Signal transduction through NOD-like receptors

Martinon,F., Annual Rev Immunol, 2009

 Signal transduction through NOD-like receptors

Adapter-dependent or independent aggregation of NLRs lead to the formation

of molecular aggregates named Inflammasomes.

Functions: Cytokine processing, atypical secretion, cell death

Martinon,F., Annual Rev Immunol, 2009

 Structure of NLRP3 inflammasome by cryo-EM

Shen, C., et al. Curr. Op. Struct. Biol. 58:18-25, 2019

 Mammalian inflammasome assembly

Xue, Y., et al. Trends Immunol, 40:1035-1040, 2019

 Inflammasomes lead to cell death by pyroptosis

Xue, Y., et al. Trends Immunol, 40:1035-1040, 2019

 Pore formation and structure of Gasdermin D

High resolution atomic force (≤ 2 nm)

microscopic view of Gasdermin D

Agiletti, F.A., et al., Trends Immunol. 38:261-271, 2017; Mulvihill, E., et al. EMBO J e98321, 2018
 Structure of Gasdermin D by cryo-EM

Shen, C., et al. Curr. Op. Struct. Biol. 58:18-25, 2019

 NLRs induce Apoptosis and Necroptosis

Necroptosome

Jorgensen, I., et al., Nat. Rev Immunol. 17: 151-164, 2017

 Membrane changes on immune cell death

Zhang, Y., et al., Cell Res. 28: 9-21, 2018

 Memory in innate immunity responses

Innate immune
memory:

acquisition of
long-term
phenotypes

Crisan, T. O., et al., Eur. J.Immunol. 46: 817-828, 2016

 Concepts behind
“transduction”

Shorter Oxford English Dictionary

3rd Edition, 1994, Oxford University Press.
 SNP in RIG-I-Like receptors and human diseases

T1D. Type I diabetes; AGS: Aaircardi-Goutières syndrome;

IgAD: immunoglobulin A deficiency; SLE: Systemic lupus erytematosus;
SMS: Singleton-Merten syndrome

Lässig, C. J Biol Chem 292(22):9000-9009, 2017

 Recent advances in innate immune signaling

1.  Co-receptor function

2.  Second messenger production (S1P, PIP3, Ca)
3.  Role of lipid rafts
4.  Role of tyrosine kinases (SFK)
5.  Role of calcium channels
6.  Cell-to-cell communication by exosomes
7.  Regulation of innate immunity by nervous system
8.  Innate immunity memory
 Exosomes

Kalluri, R., and LeBleu, V.S., Science 367:640-646, 2020.

 Exosomes

Kalluri, R., and LeBleu, V.S., Science 367:640-646, 2020.

 Exosomes

Kalluri, R., and LeBleu, V.S., Science 367:640-646, 2020.

TLR transfer by exosomes
 TLR transfer by exosomes

Zhang, Y., et al., Immunohorizons, 3:186-193, 2019

 TLR transfer by exosomes

Zhang, Y., et al., Immunohorizons, 3:186-193, 2019

Signal transduction is in your future!!!