Paper - Review

DOI: 10.1038/s41590-019-0556-1

Abstract

Pathogen-derived nucleic acids
→ are (crucial signals)
→ for (innate immunity)

Mammalian cells
→ have been able → to evolve (powerful innate immune signaling pathways)
← that originate ← from the detection of (cytosolic nucleic acid species)
← the most prominent being → 1⃣ the cGAS-STING pathway 2⃣ the RLR-MAVS pathways

A plethora of (regulatory mechanisms)
← that are crucial → for balancing (the activity ← of nucleic acid sensors)
→ for the maintenance of (overall cellular homeostasis)

Elucidation of (the various mechanisms)
← which enable cells → to maintain control → over (the activity ← of cytosolic nucleic acid sensors)
→ has 1⃣ provided (new insight) → into the pathology of (human diseases) 2⃣ offers → a (rich & largely) unexplored source → for (new therapeutic targets)

Introduction

Every organism
→ is assaulted ← by pathogens
← that must be counter-acted → for the maintenance of (1⃣ host integrity 2⃣ survival)

Elaborate (cellular surveillance mechanisms)
→ have evolved → that enable (individual cells)
→ to recognized microbe-associated (nucleic acid species)
❗: a powerful signal of infection

Activation (← of these nucleic acid sensors)
→ initiates (sophisticated signaling cascades)
← involves (the 1⃣ generation 2⃣ transmission) ← of an infection signal
→ to elicit a multitude of (cellular defense responses)

The usage (← nucleic acid-sensing systems)
← as "pathogen recognition"
→ is universal

The detection (← of pathogen-derived nucleic acids)
→ is carried out ← by a limited number of (nucleic acid receptor systems)
→ which triggers (diverse types) ←of effector response

1⃣ The identity of these systems 2⃣ their mode of activation 3⃣ their signal transduction
→ have been delineated
← over the pst decades

Sensors of (cytosolic nucleic acids)
→ act in synergy
→ to mediate (innate control) ←of (pathogen infection)
→ e.g. 1⃣ guiding → distinct cellular behaviors 2⃣ acting ← on selective cellular subsets 3⃣ reacting ← with differing activation threshold

2 sensing systems
→ seem to be important
→ for the generation of 1⃣ anti-viral 2⃣ pro-inflammatory transcriptional programs
→ induced within various 1⃣ cells 2⃣ species

∴ 1⃣ the cCAS-STING pathways → for the DNA recognition 2⃣ the RLRs (← RIG-I-like receptors) → for the RNA recognition

❓: How (nucleic acid-sensing mechanisms)
← in 1⃣ general 2⃣ cGAS pathways 3⃣ RLR pathways
→ affect → a variety of (biological processes)
← in the absence of infection

∴ Studies of (these mechanisms)
→ have revealed (diverse roles) → for nucleic acid sensors
→ e.g. 1⃣ in promoting → anti-tumor immunity 2⃣ in regulating responses → to (cellular & tissue) derangement 3⃣ in (driving & aggravating) auto-inflammatory conditions

❓: how sensors of (cytosolic nucleic acids)
→ can operate safety
← in the context of (a healthy cells)
← that is replete with (endogenous nucleic acid species)

❗: Regulatory mechanisms
← that act at different levels of (1⃣ the cGAS-STING 2⃣ RLR sensing systems)
❗: their importance
← in mediating (1⃣ cellular 2⃣ organismal) homeostasis

The molecular mechanism by which cGAS is activated by dsDNA

cGAS
→ is (a central receptor ← of cytosolic DNA)
← that mediates the up-regulation of (1⃣ type I interferons 2⃣ other inflammatory cytokines 3⃣ other inflammatory chemokines)

cGAS
→ is a dsDNA-activated enzyme
→ that initiates (signal transduction)
→ through the synthesis of (the second-messenger molecule cGAMP)

The enzymatic function
→ is achieved
← by the C-terminal part of cGAS
← which has 1⃣ nucleotidyltransferase activity 2⃣ MB21 domains ← along with 2 DNA-binding sites

Profound conformational changes
← that affect the catalytic pocket
∵ the binding of dsDNA

1⃣ ATP 2⃣ GTP
→ can then be accommodates ← within (the catalytic pocket)
→ serve as substrates → for synthesis of (the non canonical 2'-5'-linked cyclic dinucleotide)

The minimal (function unit) of (enzymatically active cGAS)
→ is a dimer
← the formation of which is (greatly facilitated) ← by dsDNA

Two (DNA helices)
→ engage → two separate DNA-binding sites
← e.g. 1⃣ A-site 2⃣ B-site
← on each cGAS molecule
∴ Efficiently cross-link → 2 individual cGAS units

This particular (mode of binding)
→ has important consequences
→ for a potent dsDNA agonist
❗: (the length of dsDNA) → is a key parameter

(Longer dsDNA templates)
→ provides multiple adjacent binding sites
← which enable cGAS to form (oligomeric & ladder-like) structures

dsDNA length
→ affects → the ensuring step of catalysis
∴ Longer dsDNA templates → achieve → higher overall enzymatic signaling outputs

Long dsDNA
→ is more potent
← in promoting cGAS liquid-liquid phase separation

The N-terminal region (← of cGAS)
→ facilitates → 1⃣ its phase-separation activity 2⃣ its enzymatic activity
← by increasing → the valence of the cGAS-DNA interaction

All mammalian cGAS homologes
→ have 1⃣ the basic 2⃣ un-structured N-terminal part

C-site
← the presence of a 3rd DNA-binding site
→ reinforces formation of (the DNA-cGAS complex)

∴ Multi-valent interactions of DNA (← with cGAS)
→ are critical
→ for regulation of (the overall enzymatic activity)

cGAS-cGAMP-STING signal transduction

STING
← an ER membrane protein
→ was the only reported downstream target of cGAMP ← inside mammalian cells
→ that converts → the activity of cGAS → into distinct cellular effector responses

The cGAS-STING signal transduction cascade
→ operate ← 1⃣ in a cell-intrinsic fashion 2⃣ across individual cells ← via gap-junction channels
→ incorporation → into 1⃣ cell-derived 2⃣ cellular secretion

These unconventional signaling routs
→ may facilitate → the rapid propagation of (inflammatory signals)
← throughout tissues

Amplificatory mechanisms
→ call → for a mean of (proper control)
→ to prevent its activation
← in response to spurious DNA signals

cGAMP
→ engages STING
← through a "butterfly-shaped" binding pocket

This pocket
→ is creates
← by the LBDs (← ligand-binding domains) ← of 2 STING molecules

An individual STING dimer
→ undergoes → a profound conformation transition
← within its LBDs
→ facilitates (side-by-side interaction) → for the creation of (larger oligomeric complexes)

1⃣ the mechanism ← of cGAMP-mediated activation of STING 2⃣ the consecutive initiation ← of signal transduction
→ is (a highly sophisticated process)
← which integrate (many distinct regulatory steps)

Activation of (the STING signal cascade)
→ manifest → itself cytologically
→ through → the formation of (relatively large STING-containing foci)
← that reside ← within (the Golgi compartment)
→ end up ← in the endolysosome → for degradation

The translocation of (activated STING) → to the Golgi compartment
← via ERGIC (← the ER-Golgi intermediate compartment)
→ relies ← on components of (the secretory pathway)
→ is mediated ← via (vesicle transport) ← by the vesicle coat protein COPII

STING's redistribution
→ to the ERGIC-Golgi compartment
→ is absolutely necessary
→ for 1⃣ the induced expression ← of STING-dependent target genes 2⃣ blocking ER-Golgi trafficking ← via (bacterial & chemical inhibitors)

Each STING unit
→ recruits → the kinase TBK1
← via a conserved binding motif
← which located in the C-terminal tail of STING

❗: Regulatory aspect (← of the STING signaling pathway)
→ is "self-limiting"

Degradation (← of STING)
→ is less efficient
∴ Apoptosis

∴ Modulation of (the degree ← of negative regulation)
→ guides → selective cell-fate decisions

Mechanisms for sensing cytosolic RNA

The sensing of (cytosolic RNA)
→ is mediated ← by RLRs
← e.g. 1⃣ RIG-I 2⃣ MDA5 3⃣ LGP2

Both 1⃣ RIG-I 2⃣ MDA5
→ activate → anti-viral signaling pathways
← through MAVS

Activation of MAVS
→ results in stimulation of (the kinase 1⃣ TBK1 2⃣ IKK)
∴ Stimulation of (the transcription factors 1⃣ IRF3 2⃣ IRF7 3⃣ NK-κB)
∴ Induction of (gene encoding 1⃣ type I & type III interferon 2⃣ pro-inflammatory cytokines)

1⃣ RIG-I 2⃣ MDA5
→ have been shown → to activate (interferon-independent apoptosis)

LGP2
→ does ❌ directly interact
← with MAVS
∴ LGP2 → lacks → the anti-viral signaling activity

LGP2
→ down-regulate → RIG-I's signaling activity
→ up-regulate → MDA5's signaling activity

∴ LGP2
→ is essential
→ for MDA5-mediated signaling activity

∴ LGP2
→ can promote → the recognition of dsRNA
← by MDA5 → through 2 independent mechanisms
← 1⃣ by protecting dsRNA 2⃣ by assisting recruitment of MDA5

❗: the mechanism
→ (RIG-I & MDA5) → recognize RNA
→ (RIG-I & MDA5) → activate MAVS
∵ Biochemical & structural studies

(RIG-I & MDA5)
→ have
→ 1⃣ N-terminal tanden CARDs (← caspase-recruitment domains) 2⃣ a central helicase domain 3⃣ CTD (← C-terminal domain)

CARDs
→ interact ← with MAVS
(1⃣ the helicase domain 2⃣ CTD)
→ recognize → cognate dsDNA

CARD is auto-suppressed
← in the absence of (dsRNA binding)
∴ The binding (← of dsRNA)
→ 1⃣ releases → the auto-suppression 2⃣ anti-viral signaling

(1⃣ binding of cognate RNA 2⃣ release of auto-repression)
→ is necessary
→ but not ❌ sufficient
(Homo-tetramerization of CARD)
→ is required → for (signal activation)
← by both 1⃣ RIG-I 2⃣ MDA5

The RNA-binding domain (← of 1⃣ RIG-I 2⃣ MDA5)
→ can independently form (filamentous oligomers)
← along the dsRNA length
∴ This can promote
→ 1⃣ CARD tetramerization 2⃣ anti-viral signaling

The formation (← of RIG-I filaments)
→ facilitates
→ 1⃣ the tetramerization of CARD 2⃣ the recruitment of RIPLET ← (the essential E3 ligase ← conjugates Lys63-linked ubiquitin chains to RIG-I)

(conjugated ubiquitin chains)
→ bind & stabilize → 1⃣ the RLR CARDs tetramer 2⃣ potentiate anti-viral signaling

Filamentous MAVS
→ serves as (signaling platform)
→ for the (recruitment & activation) ← of (members of the TRAF family)
← 1⃣ TBK1 2⃣ IRF3
→ for the transcriptional activation of (type I & type III) inteferons

Regulatory factors that filter noise from danger

(Nucleic acids)
→ are the most conserved molecules
← in the evolution of life

Pathogen-derived nucleic acids
→ can bear (certain structural features)
← which NOT present on cytosolic nucleic acid species

Nucleic acid sensors
→ are unable ❌
→ to molecularly distinguish ← self 🆚 non-self nucleic acids

Mammalian cells
→ are able → to mount adequate responses
∵ Multiple molecular safeguard strategies
← which restrict (the activity ← of nucleic acid sensors) → at steady state

Side-by-side mechanisms
→ interfere ← with 1⃣ DNA signal-transduction pathways 2⃣ RNA signal-transduction pathways
→ guide appropriate responses → as 1⃣ cGAS 2⃣ RLRs

The nucleic acid signaling cascades
← include 1⃣ spatio-temporal organization 2⃣ PTM (← post-translational modification) 3⃣ antagonizing of (protein binders) 4⃣ polymerization & oligomerization

Regulatory determinants at the level of DNA

The ability of cGAS
→ is critically determined ← by (structural features) of (the DNA duplex)
→ is affected ← 1⃣ by auxiliary factors 2⃣ by antagonizing factors ← that 1⃣ bind 2⃣ modify dsDNA

The activity of cGAS
→ is regulated ← by dsDNA
← in a strictly length-dependent manner

The presence of (sufficiently long stretches) ← of cytosolic dsDNA
→ signals → 1⃣ serious damage 2⃣ serious infections
← that can no longer be counter-acted ← by the action of (cytosolic nucleases)

Long dsDNA
→ can be tuned ← through modifications
← that render it (more resistant) → to nuclease cleavage

Additional (PTM of DNA)
← e.g. methylation
→ affect (its capacity) → 1⃣ to bind 2⃣ to activate cGAS

RNA → does NOT ❌ activate cGAS
∵ RNA fails → to rearrange (the enzymatic pocket)

Possibly (DNA modification)
→ may exist ← which act in a similar manner
→ may serve ← as a dominant (negative ligand) ← on cGAS

A synthetic oligonucleotide
← comprising 1⃣ repetitive telomeric sequences 2⃣ phosphorothioate backbone modifications
→ inhibited (dsDNA-triggered cGAS activity)

Chromatininzed DNA
→ is less active as a ligand
← than (naked dsDNA)

DNA-binding proteins
→ facilitates → the activity of cGAS

The link
← between 1⃣ dysfunctional DNA metabolism 2⃣ innate DNA immunity
→ was crucially advanced ← by the molecular pathogenesis of (1⃣ AGS ← Aicardi-Goutié syndrome 2⃣ an early-onset autoimmune syndrome)
→ is biochemically characterized ← by (an increase ← in type I interferons)

The first genes
← to be mutated in patients ←with AGS
→ were those that encode enzymes
← with critical functions ← in the turn-over of (endogenous DNA)

TREX1
→ is a major 3' DNA exonuclease
← which located on the membranes of the ER
→ that exerts activity → against 1⃣ ssDNA 2⃣ dsDNA

SAMHD1
→ has dNTP phosphohydrolase activity
← that controls (the levels of dNTP) ← in a cell-cycle-dependent manner
→ influences DNA replications

RNaseH2
→ cleaves → DNA-RNA hybrids
→ functions ← in (the removal of ribonucleotides)
← that had been erroneously incorporated → into the genome

❓: What are the substrates of the enzymes above?

Treatment
← with (an inhibitor ←of human immuno-deficiency virus type I reverse transcriptase)
→ can diminish typ I inteferone signaling ←in patients with AGS
∵ a pilot open-label clinical trial

Activity of (AGS-related genes)
← can activate cGAS
→ is through interference ← with the disposal of (aberrant DNA species)
← that originate from errors ← during 1⃣ DNA replication 2⃣ DNA damage 3⃣ DNA repair

Trex1 cells
→ display → signs of (spontaneous DNA damage)
← in conjunction with (accrual of ssDNA)

Mutations
← in the gene ← encoding BLM RecQ-like helicase
→ compromise (genome integrity)
→ was associated ← with (cGAS-dependent recognition of DNA)

The gene products
→ balance → the amount of DNA ← that originated from intracellular sources
Malfunction
← in the turnover of (exogenous DNA)
→ provide (powerful ligands) → for cGAS

Counter-acting (the accumulation of DNA)
← from cell-extrinsic sources
→ is "DNase II"
← resides inside 1⃣ the lysosomes of macrophages 2⃣ non-phagocytic cells

Macrophages
← in Dnase2a-deficient mice
→ show intra-cellular accumulation of DNA
→ that drives (lethal & cGAS-STING-mediated anemia)

People
← carrying loss-of-function alleles of DNASE2
→ exhibit (severe neonatal anemia)
∴ This resemble → a milder version of the phenotype (← observed in mice)

The importance of (DNase II)
← in the homeostasis of (DNA-mediated inflammation)

DNase II
→ has been shown → to participate
← in the degradation of (cell-intrinsic DNA-damage products)
← within non-phagocytic cells ❗

The promotion of (paracrine activation)
← of cGAS-STING signaling
→ is the propagation of (microbial DNA)
→ through (vesicle transport)

Disruption of proper subcellular compartmentalization driving cGAS-mediated inflammation

Loss (← of mitochondrial membrane integrity)
→ can lead → to 1⃣ the release of mtDNA (← mitochondrial DNA) 2⃣ powerful activation ← of cytosolic cGAS

Compromise ← (1⃣ mitochondrial homeostasis 2⃣ ranging ← from defects)
∵ Absence of TFAM
→ to pathogen-inflicted mitochondrial damage → to compromised mitophagy

mtDNA
→ can end up
← in the course of (normal physiological processes)
❗: Apoptosis
→ release mtDNA → into the cytosol

Cells
→ challenged → to undergo apoptosis typically
∵ fail ❌ → to mount → a cGAS-dependent inflammatory response

The inability of (apoptotic cells)
← to react to mtDNA
→ was (an active process)
← that relies on the action of (apoptotic caspases)

❓: How caspases contribute → to (1⃣ the silencing of cGAS 2⃣ its downstream effectors)
❓: Whether caspases exert → their effect
❗: (Cellular factors) exist → that actively suppress (mtDNA-induced cytokine responses)
❗: (Cellular factors) → limit inflammatory off-target effects
← under homeostatic conditions

Mitochondrial damage
→ is (a prominent side effect) ← of (pathogen invasion)
∴ Viruses → trigger inflammatory response
← through (the recognition of mtDNA) by cGAS

Mitochondrial dysfunctions
→ contributes → to a variety of (1⃣ metabolic 2⃣ neurological diseases)
∵ (cGAS-mediate inflammatory response) → is found
→ to (cause & aggravate) these pathologies

An absence of STING
→ prevents → (1⃣ the movement defects 2⃣ neuronal losses)
← that usually occur ← in (old mutant mice ← that lack Parkin)

∴ (A possible role) → for (self DNA-mediated inflammation)
← in contributing → to the molecular pathologies
← e.g. neuro-degenerative disorders ← e.g. Parkinson's disease

Genomic DNA
→ is (a powerful cell-intrinsic source)
→ for the activation of cGAS

Fragment of (genomic DNA)
→ can gain access → to the cytosol
→ trigger cGAS-dependent (inflammatory signaling)

(Single & multiple) chromosome
→ can be captured
← in aberrant micro-nuclei structures
← that are physically separated ←from the main nucleus

Mis-segregated chromosomes
→ are frequently exposed → to the cytosol
← as a consequence of (spontaneous rupture ← of micro-nuclei)

"Non-core" nuclear
→ envelope proteins
← that render them (1⃣ highly fragile 2⃣ likely to collapse)

cGAS
→ accumulates ← on exposed chromosomal fragments
← is correlated ← with the up-regulation of cytokines

Various conditions
← 1⃣ Prominently genotoxic treatment 2⃣ Defects ← in (DNA replication & DNA repair)
→ can cause → chromosome mis-segregation
→ lead → to the formation of micro-nuclei

Micro-nuclei
→ are often found
← 1⃣ inside cancer cells 2⃣ cells en route → to transformtion

Chromosomes
← that originate from micro-nuclei
→ can be heavily mutagenic
∵ 1⃣ Faulty replication 2⃣ Detrimental

The cGAS-stimulatory "bystander" effect
→ may serve ← as (an essential surveillance mechanism)
→ through (the generation of 1⃣ and anti-proliferative 2⃣ inflammatory response)
← which provides protection → against (the further propagation) of (cells ← that accumulate micro-nuclei)

Genomic DNA
→ escape ←from the main nucleus
→ elicit (a cGAS-dependent response) ← in non-dividing cells

A meshwork
→ formed ← by polymers of (intermediate-filament proteins)
← e.g. 1⃣ LaminA/C 2⃣ LaminB
→ as well as → inner-nuclear-membrane proteins
← which is essential → for the maintenance of (the physical intactness)

∴ Senescent cells
→ display → (marked down-regulation ← of LMNB1 expression)
← which causes a profound alteration (← of the nuclear architecture)
∴ This facilitates → (the release ← chromatin fragments)

cGAS
→ undergo enrichment ← on cytosolic chromatin fragments
→ accumulate ← in senescent cells
∴ cGAS → establish → (the senescence-associated secretory phenotype)

Genomic DNA species
→ escape the nucleus
→ loosely accumulate ← in context of (1⃣ DNA damage 2⃣ replication stress)

SAMHD1-deficient cells
→ accumulate (cytosolic ssDNA)
→ depleted → the DNA damage-repair proteins (1⃣ Rad51 2⃣ RPA)

❓: How DNA
← whether 1⃣ dsDNA 2⃣ ssDNA
→ would leave → an intact nucleus
← in these circumstances

Regulatory determinants at the level of RNA

RLRs
→ utilize (multiple criteria)
→ to ensure 1⃣ selective recognition ← of non-self RNA 2⃣ robust discrimination → against cellular RNA

Duplex RNA structure
→ is (necessary & not sufficient) → for (the sensing of foreign RNA)

5'ppp
← 5' triphosphate group
← in 1⃣ all nascent transcripts 2⃣ unprocessed viral RNAs
→ is additionally required

∴ 5' processing
← during (the maturation ← of many cellular RNAs)
→ is the chief mechanism
← which (cellular RNAs) → are prevented ← from activating RIG-I

1⃣ a dsRNA (← length of >50 bp) 2⃣ a blunt-end dsRNA structure
→ promote → the appropriate formation of (RIG-I filaments)
← along the dsRNA length
∴ This process → ensures (robust discrimination) → against cellular RNAs

MDA5
→ has more-stringent criteria
→ for 1⃣ dsRNA length 2⃣ dsRNA structural integrity
← in 1⃣ the selective recognition ← of foreign dsRNA 2⃣ the discrimination → against (shorter & imperfect) cellular dsRNAs

❗: how (RIG-I & MDA5)
→ utilize (different mechanisms)
→ for 1⃣ RNA binding 2⃣ filament assembly & diassembly

RLRs
→ can mis-recognize (cellular RNAs)
← under (various physiological conditions)

∴ (Multiple regulatory mechanisms)
→ are needed
→ to prevent (the recognition ← of self-RNA by RLRs)

❗: RNA modification
→ can selective mark (cellular RNAs)
→ block recognition ← by (the innate immune system)

(A 7-methyl guanosine → attached to 5'ppp)
→ is NOT sufficient
→ to suppress (the stimulation of RIG-I)
❗: 2'-O methylation

Host circular RNAs
← formed by back-splicing
→ can also stimulate RIG-I
∴ Host circular RNAs
→ evade (recognition by RIG-I)
→ through N6-methyladenosine modification

Adenosine-to-inosine modification
→ block (the recognition of dsRNA ← by MDA5)

❗: the importance ← of (1⃣ co-transcriptional modification 2⃣ post-transcriptional modification)

∴ Some viruses
→ utilize (virus-encoded methyltransferases)
→ to 1⃣ mimic → cellular RNAs 2⃣ evade detection ← by the innate immune system

Self-RNA
→ are protected ← from being sensed (← by the innate immune system)
→ through interaction (← with cellular functional partners)

Cellular RNAs
→ contain (5'ppp) ← with secondary structures
∴ Cellular RNAs
→ have a potential → to stimulate RIG-I

7SL RNA
→ is bound ← by (signal-recognition particle protein partners)
→ is shielded ← from being detected by RIG-I

Unprotected 7SL RNA
→ (accumulates ← in the cytosol) & (activates → RIG-I)
← when (RNA is mis-regulated) & (RNA occurs ← in excess over the protein partners)

(Un-shielding & cytosolic localization)
← of (1⃣ otherwise nuclear 2⃣ protected RNAs)
→ were accompanied ← by (activation of RIG-I)

Perturbed localization (← of host RNAs)
→ may represent → (a damage ← associated molecular pattern)
→ that alerts cells of (1⃣ pathogen infection 2⃣ cellular stress)

RNA degradation
→ has (an important role)
← in preventing → the accumulation of (1⃣ potentially immunogenic aberrant RNA transcripts 2⃣ RNA metabolic byproducts)

A defect ← in SKIV2L
← a component of (the cytosolic RNA-degrading machinery)
→ increase → (the basal signaling activity ← of RIG-I)

IRE-1
← RNA endonuclease ← that is activated ← during (the unfolded protein response)
→ generates as-yet-unknown RIG-I ligands
← which may be normally degraded ← by the SKIV2L exosome

A defect (← in the mitochondrial nuclease PNPase)
→ result ← in the accumulation (← of 1⃣ mitochondrial dsRNA 2⃣ its leakage)

Mitochondrial genomes
→ are circular & transcribed bi-directionally
∴ Mitochondrial genomes
→ can lead → to (the formation of dsRNA)

Epigenetic regulation
→ has (additional function)
← in restring (the synthesis ←of immunostimulatory RNAs)

(1⃣ Genetic & chemical ablation ← of DNA methyltransferases 2⃣ the histone methyltransferase → SETDB1 3⃣ the histone demethylase → LSD1)
→ results ← in (aberrant activation ← of MDA5)

Endogenous retro-viruses
→ were responsible
→ for (the activation of MDA5)
∵ These reagents → up-regulate (expression ← of endogenous retroviruses)

The activities (← of RLRs)
→ are further regulated
← by (inhibitory cellular RNAs)

TRIM25
→ is not involved ← in RIG-I-mediated anti-viral signaling

❗: diversity (← in sequence & structure)
← in both 1⃣ host RNAs 2⃣ viral RNAs
→ result in (substantial overlap) ← between (the physicochemical properties)

∴ A variety ← of (1⃣ transcriptional 2⃣ post-transcriptional) regulatory mechanisms
→ have (key roles) ← in keeping (the level of virus-like host RNAs)
← under the self-tolerance threshold

Regulation at the level of cGAS

Various mechanisms
→ control → 1⃣ the enzymatic output 2⃣ the immuno-stimulatory activity
← by controlling cGAS

Human cGAS
→ is more selective ← in detecting long dsDNA
→ involves (species-specific substitutions) ← at distinct DNA-binding sites
∵ Comparison between species

A host of PTMs
→ linked → to regulation of (the responsiveness of cGAS → to DNA)
→ through 1⃣ diminishing DNA binding 2⃣ cGAS dimerization

Reversible PTMs
→ are useful → for (rapid adjustment ← of the responsiveness of cGAS)
→ to the environmental context of the cells

Adaptation process
→ is based ← on regulation of (the local concentration ← of 1⃣ cGAS 2⃣ its substrates)

❗: the importance of (1⃣ dimerization 2⃣ cooperation 3⃣ phase separation)
← in (the achievement ← of full activity by cGAS)

Intra-cellular positioning of cGAS
→ represent → a crucial determinant

A considerable fraction of cGAS
→ is (inside the nucleus)
∵ the intra-cellular distribution of cGAS

cGAS
→ rapidly localizes to chromatin ← during mitosis
This interaction (← between cGAS 🆚 chromatin)
→ is responsible → for carrying the protein → to the nucleus interior in dividing cells
∵ Live-cell imaging studies

❓: how cell regulate (cGAS activity)
← in the face of (immense amounts of self-DNA)

Forced expression
← of 1⃣ a cGAS deletion mutant → that lacks the N-terminal domain 2⃣ a version of cGAS ← with nuclear-localization signal
→ triggers (spontaneous activation ← of cGAS activity)
→ accompanied ← by (the induction of type I interferons)

∴ Intra-nuclear cGAS
→ is in (principal capable) of (responding to self DNA)
∴ The interior of the nucleus
→ contains (regulatory components) ← which suppress overt (cGAS activity)

Regulation at the level of RLRs

RLRs
→ are subject → to (multiple layer of regulation)
→ to further limit mis-recognition of cellular RNAs

RIG-I
→ is in tis auto-repressed conformation ← in the absence of RNA
→ is released from this conformation ← after RNA binding

CARD
← the signaling domain
→ is bound ← by the helicase domain
→ is prevented ← from forming (the active conformation)
∵ the structure of RNA-free RIG-I

The MDA5 CARD
→ is phosphorylated ← in the resting state
∴ Such phosphorylation
→ may prevent (its tetramerization)
← in the absence of (an activating ligand)

❓: all MDA5 molecules
← in the resting state
→ are suppressed ← by phosphorylation

RLR
→ is activated → through (multiple steps ← of receptor oligomerization)
These process
→ function as checkpoints
← which restrict (aberrant activation ← of downstream signaling)

Only multi-merized RIG-I
→ interacts ←with (the essential E3 ligase RIPLET)
→ can be conjugated ← with K63-linked ubiquitin
← a requirement (for 1⃣ the formation ← of stable CARD tetramers 2⃣ the activation ← of MAVS)

RIPLET
→ can induce → (filament bridging)
← which creates a local environment
← that 1⃣ stabilizes → CARD tetramerization 2⃣ potentiates → the signaling activity of RIG-I

∴ Multiple steps ← of oligomerization
→ allows (fine tuning) ← of (the amplitude ← of the immune response)

1⃣ filament formation 2⃣ dsRNA length
→ are required → for efficient signaling by MDA5

❓: whether (similar multi-step signal amplification)
→ may occur ← with MDA5

Ubiquitin chains
← with other linkage types
← e.g. 1⃣ K48-linked 2⃣ linear chains
→ is (negatively regulate) → their signaling activities
← by inducing degradation

Various proteins
← 1⃣ RNF122 2⃣ RNF125 3⃣ RNF123 4⃣ TRIM13 5⃣ TRIM40 6⃣ CHIP 7⃣ LUBAC
→ conjugate (ubiquitin chains) → for 1⃣ proteasomal 2⃣ auto-phagosomal degradation ← of (RIG-I & MDA5)

Regulation mechanisms downstream of cGAS

The binding of (cGAMP → to STING)
→ initiates → (a complex series ← of events)

Targeting (the levels of cGAMP)
→ represent (an obvious point of action) → for tuning cellular responses to DNA

Poxins
→ is as a mean of (antagonizing DNA-induced anti-viral effector responses)

ENPP1
← the plasma membrane-bound pyrophosphatase
→ degrades cGAMP
∴ cGAMP need to be exported → for degradation by ENPP1

Cancer cells
→ secreted cGAMP
∵ in vitro experiments

Transport of cGAMP
← across (the plasma membrane)
→ controls (activity of STING)
← at the multi-cellular level

❓: whether (regulatory mechanisms) exist
← which affect 1⃣ the import 2⃣ the export 3⃣ the gap-junction transfer

Cellular activation
→ requires → the assembly of homo-oligomers
Oligomerization
→ facilitation of (the activation of STING)

Oligomerization
→ is important
→ for the exist of STING ← from the ER

Oligomerization
← at the ERGIC-Golgi compartment
→ is necessary → for the assembly of STING-TBK1-(IRF3)
← signaling-competent complexes

Binding of cGAMP
→ is sufficient → to induce (side-by-side oligomerization ← of STING dimers)
→ though (a cytosolic tetramer interface)
∵ in vitro

This oligomerization
→ is supported
← by the palmitoylation of (two cysteine residues)

❓: the enzymes
← responsible for STING (de)palmitoylation
→ are not identified

Activating mutations
← in the gene encoding STING
→ result in SAVI
← the auto-inflammatory syndrome

Some disease-causative (point mutation)
→ result in → changes to (the oligomerization interface ←of STING)
→ allow STING → to form oligomers
← which independently of (the presence of cGAMP)

∴ Ligand-independent oligomerization
→ offers (potential explanation)
→ how (these mutations) can trigger → (continued activation of the STING pathway)
← which leads → to chronic inflammation

Other (point mutations)
→ results in → changes at (the dimer interface ← of STING)
← which connects (the LBDs) ← with the transmembrane regions

These mutations
→ could promote (rotation of the LBDs)
∴ These mutations → initiate (spontaneous activation of STING)

❗: the relationship
← between (STING oligomerization) 🆚 (activation ← of downstream signaling)

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