Cyclic GMP-AMP Synthase
cGAS (cyclic GMP-AMP synthase) has a dsDNA-binding domain that can sense cytosolic DNA and catalyze the production of second messenger, cGAMP (cyclic GMP-AMP dinucleotide). This further binds to and activates ER-localised adapter protein STING (stimulator of interferon genes) that induces type I IFN (IFN? or IFN?) via TBK1-IRF3 signalling. After their secretion, type I IFNs bind to the type I IFN receptor (IFNAR) in an autocrine as well as paracrine manner. This signal induces the expression of interferon-stimulated genes (ISGs) in the signalling cell and can also be transferred to neighbouring cells by gap junctions. Since cGAS does not have any specificity to bind to a particular type of dsDNA fragment, this allows recognition of viral dsDNA, retroviral reverse-transcribed cDNA, or dsDNA of intracellular bacteria.
However, the same property poses an obstacle since it allows detection of self-DNA in a variety of situations and activation of immune response. There are several mechanisms by which the immune system ensures its efficient activation upon exposure to pathogens, while avoiding improper activation under homeostatic conditions- cGAS has the intrinsic property of length-dependent DNA recognition, independent of accessory proteins. This is especially prominent at lower concentrations which are similar to the ones present during infection. Longer DNA fragments produce more cGAMP and induce type I IFN response more strongly than smaller or intermediate-sized fragments. This ensures that shorter DNAs that accumulate as a by-product of cell division and DNA repair do not evoke immune responses. Caspases are inflammatory or apoptotic cysteinyl aspartate-specific proteases that are known to mediate innate immune responses by cleaving precursors of proinflammatory cytokines such as IL-1b and IL-18, thereby facilitating their secretion. Intrinsic apoptotic pathway is controlled by effectors including Bak and Bax (in check in healthy cells).
Apoptotic signals trigger their activation. Bak- and Bax-mediated mitochondrial damage triggers the release of mitochondrial DNA (mtDNA) due to MOMP (mitochondrial outer membrane permeabilization), which is recognized by the cGAS/STING-mediated cytosolic DNA sensing pathway. But apoptotic caspases cleave one of the molecules involved in cGAS-STING signaling like IRF3 or mitochondrial DNA is degraded by nucleases which are activated by apoptotic caspases. In the absence of the apoptotic caspases, this leads to the induction of IFN-? transcription and subsequent secretion by the dying cell. Loss of the caspase-cascade leads to elevated IFN-? levels in vivo. This feeds back to, and has a profound impact on, the HSC (hematopoietic stem cells) compartment, which is highly sensitive to the effects of type I IFN. Thus, the apoptotic caspase-cascade regulates the immunological impact an apoptotic cell has on the host by preventing damage associated molecular pattern (DAMP) signaling induced by mtDNA. Caspase-deficient mice exhibit phenotypic abnormalities – even lethality.
Genomic DNA can be damaged by endogenous or environmental stresses. During repair, these short single-stranded DNAs are continuously generated and may leak out to the cytoplasm, where they are generally drawn back into the nucleus by DNA repair and replication factors (RPA and Rad51). Alongside, TREX1, cytoplasmic exonuclease, is anchored on the outer nuclear membrane to degrade leaking DNAs immediately. This prevents activation of the cGAS/STING pathway against self DNA. Inhibition of any of the above mentioned proteins leads to accumulation of cytoplasmic DNA and subsequent inflammatory cytokine production. During interphase, self DNA, existing as nucleosomes, does not trigger cGAS. However, during mitosis, when the nuclear envelope disassembles, there exists a possibility of self-DNA detection by cGAS and production of type I IFNs. cGAS is shown to bind to nucleosomes with much higher affinity than to naked DNA (even mechanisms of cGAS binding to naked DNA and to nucleosomes are distinct.). Nucleosomes also suppress the cGAMP synthase activity of cGAS and hence, do not allow production/activation of downstream molecules. So, at mitosis, nucleosomes competitively interfere with cGAS activation.
However, during mitotic arrest, when cGAS is bound for a prolonged time, it gets activated and serves as a signal for error in the cell cycle. It stimulates mitochondrial relocalisation of IRF3, which in turn activates Bax-mediated apoptosis. Apart from cGAS, other exogenous DNA sensors are also known – one of them being IFI16 (interferon-g inducible protein 16). IFI16 is shown (in human keratinocytes) to be required for the cGAMP-induced activation of STING, and its interaction with STING promotes STING phosphorylation and translocation. It can be said that these two cytoplasmic DNA sensors cooperate during DNA sensing, and both are required for the full activation of an innate immune response and also prevent spurious activation of the type I interferon response by adding another level of regulation. DNA binding to cGAS induces formation of liquid-like droplets, which function as micro reactors in which the enzyme (activated cGAS) and reactants (ATP, GTP) are concentrated to enhance the production of cGAMP. This mechanism allows cGAS to detect the presence of DNA in the cytoplasm only above a certain threshold. Such a switch-like response is made possible by the multivalent interactions between the DNA binding domains of cGAS and DNA in a manner that depends on the DNA length as well as on Zn2+ concentration.