The role sensing pathways play in our innate immune system
When cells are threatened, for example by viral infection, special sensors are activated to kick-start the immune system. Now, new genetic techniques are increasing our knowledge about how this response mechanism actually unfolds.
The genetic material of more biologically advanced organisms, such as humans, is normally to be found in the cell nucleus and the mitochondria (membrane-bounded organelles). Therefore, DNA found in the surrounding cytoplasm is likely to be due to nucleus or mitochondria damage or the result of bacterial or viral infection.
The cGAS enzyme has been described as a ‘cytosolic DNA sensor’ meaning that its job is to detect out-of-place DNA – which which may indicate infection – on behalf of the cell's innate immune system. A team led by Professor Karl-Peter Hopfner of the Ludwig-Maximilians-University, Munich, drawing on work developed as part of the EU-funded GENESIS project, discovered that the efficiency with which the cGAS enzyme can discover cytoplasmic DNA, is influenced by the length of the latter.
Why every rung of the ladder helps
The researchers, publishing in the journal ‘Nature’, discovered that cGAS binds to cytosolic DNA creating a ladder-like structure. The process triggers the formation of a molecule (cGAMP) which acts as a messenger to the immune system prompting the synthesis of interferons, immunostimulatory proteins which strengthen cellular defences.
The team demonstrated that this activation of the innate immune system required the ladder-like structure to exceed a certain length. A complex consisting of only a short strand of DNA lacks the robustness to be sustainable. As Prof. Hopfner explains, ‘The complex functions like a zipper. If only one of the projections interlocks it can be easily displaced, but when many are slotted into place, the central part cannot fall apart.’
To determine the complex’s structure, the team used X-ray diffraction after crystallising a complex consisting of longer DNA fragments and cGAS dimers (the enzyme’s functional twin subunits). The ladder-like structure is produced by either two separate DNA strands or a U-shaped single DNA molecule forming the vertical sides, with the cGAS dimers slotting between them like 'rungs'. This structure stabilises the active enzyme dimers enabling them to synthesize cGAMP, the signal molecule. As Liudmila Andreeva, lead author of the ‘Nature’ paper puts it, ‘The more rungs are inserted, the more stable the complex becomes, as neighbouring dimers stabilise one another (…) We were able to construct a mathematical model that accounts for this mechanism.’
It is known that various proteins can enable the formation of the ladder structure by triggering the DNA to form U-turns and so start the process of inserting cGAS dimers as rungs. The team demonstrated that certain stress related and DNA packaging proteins in the nucleus, bacteria and mitochondria were able to trigger this mechanism.
Earlier studies had already shown that longer DNA strands activated this protective process more readily than shorter strands, in cells containing equal amounts of cytosolic DNA. The working theory is that having a minimal length threshold for DNA strands avoids the innate immune system reacting unnecessarily, for example to short cytosolic DNA molecules that are the result of normal cellular processes.
Finding new answers
The GENESIS project has been able to take advantage of advances in genome engineering and the opportunities offered to generate and test hypotheses by knocking out genes or through the functional genetic screening of human cells. The project has developed a platform for genome targeting and validation, which allows large-scale, loss-of-function studies.
The project continues to systematically explore the role of known human DNA sensing pathways which detect potentially dangerous DNA and orchestrate protective measures. Through a large-scale perturbation study the project is looking specifically at the influence of the kinome in antiviral innate immune signalling pathways.