Helicases as Powerful Tools in Innate Immunity

Helicases are among the largest and most highly conserved families of enzymatic proteins in eukaryotic organisms. These proteins utilize NTP hydrolysis (usually ATP) to drive their recognition, remodeling, and response to target DNA or RNA.¹

Nearly every aspect of nucleic acid metabolism is mediated by helicases. DNA helicases function in replication, repair, recombination, transcription, chromosome segregation, telomere maintenance, and unwinding alternative DNA structures, like G-quadruplexes. RNA helicases affect nearly all crucial RNA metabolic activities, like transcription, translation, pre-mRNA splicing, RNA export, mRNA decay, ribosome biogenesis, miRNA processing, and organelle RNA processing. A few helicases can recognize both DNA and RNA. Recent work suggests that some helicases can actually rewind or anneal nucleic acid strands.¹

All helicases share great similarity in their core regions. Their cores possess conserved amino acid residues, specifying NTP binding and hydrolysis, and an arginine residue that enables the coupling of released NTP energy with nucleic acid interaction. They also contain varying accessory protein motifs, particular to their specific function.  Accordingly, a sequence-based classification system was devised to establish 6 superfamilies of helicase. Further distinction between groups of families is based upon their structure. Superfamilies 3 to 6 assemble as hexameric toroids. Superfamilies 1 and 2 are present as monomers or dimers. Superfamilies 3 to 5 are endemic to bacteria and viruses. Superfamilies 1, 2, and 6 are found in eukaryotes.³

Helicases in Health and Disease

Both DNA and RNA helicases have been found to be vital elements in innate immunity. When such helicases cease to function normally and generate inappropriate, long term, defensive inflammation, autoimmune diseases and cancer can arise. DDX41 and DHX36 are two examples of helicases currently under investigation for their roles in innate immunity and disease. They are both classified as DEAD (Asp-Glu-Ala-Asp)/DEAH (Asp-Glu-Ala-His)-box helicases that are essential for RNA metabolism.²

DDX41

DDX41 [DEAD-Box Polypeptide 41] is most known as a cytosolic helicase sensor for dsDNA, DNA/RNA complexes, and cyclic dinucleotides (CDNs). It is a multi-faceted helicase, also playing parts in translocation and unwinding double-stranded nucleic acid structures. Recent work has additionally found that DDX41 selectively regulates the alternative splicing of many genes involved in the innate immune response.²

Structurally, DDX41 consists of an N-terminal domain, DEAD domain, and C-terminal domain. The N-terminal domain of DDX41 is responsible for the translocation from the cytoplasm to the nucleus. Its DEAD domain is important for ATP powered DNA/CDN detection and signaling. The remaining C-terminal domain functions as the helicase, unwinding the targeted nucleic acid structures.²

DDX41 not only acts as a sensor to invading bacteria or viruses, but also initiates downstream innate immune responses following detection. Prior to bacterial/viral invasion, DDX41 remains inactivated, bound to TRIM21 (an E3 ubiquitin ligase). Upon release of dsDNA or c-di-GMP by the virus or bacteria, DDX41 activates via phosphorylation by Bruton’s Tyrosine Kinase (BTK). This stimulates downstream activation of STING (stimulator of interferon genes). STING signaling activates transcription factors via TBK1, inducing the production of type 1 interferons and pro-inflammatory cytokines, such as TNF and IL-6.⁴

If DDX41 is overactivated, the autoimmune pathologies associated with excessive type 1 IFN expression, such as lupus erythematosus, occur.⁴

DHX36

DEAH-box polypeptide 36 (DHX36) is a 1000 amino acid RNA helicase protein with a central helicase core that is capable of binding single stranded DNA or RNA. Specifically, DHX36 binds both DNA and RNA G-quadruplexes (G4s) and plays an important role in unwinding these G4s.⁵

Structurally, DHX36 consists of N terminal and C terminal domains.  The N-terminal domain recognizes the 5’ most G-quadruplex quartet, specifically in parallel G4 DNA structures. The C terminal of the core (the OB fold domain) recognizes the 3’ most G tract sugar phosphate backbone.⁵

G4s aid viral replication and neurological diseases through gene expression, replication, recombination, translation, and telomere activity. Due to damaging structural changes to G4 when DHX36’s binds, DHX36’s roles in the innate antiviral immune response and neurological diseases are under investigation in hopes of finding new treatments.⁶

DHX36 is an important detector of viral nucleic acids in the cytoplasm.  It also is an important component of initiating downstream innate cellular immune responses. When infection occurs, DHX36 induces double-stranded RNA-dependent protein kinase, leading to the formation of stress granules, and downstream cytokine production. Without sufficient DX36 activity, infection by RNA viruses is much more severe.⁶

The implication of DHX36 in neurodegenerative disease and brain aging has also gained significant traction. Altered G4 DNA, RNA, and DNA-RNA hybrids are now seen as significant contributors to diseases, such as frontotemporal dementia, amyotrophic lateral sclerosis, and fragile X syndrome. DHX36 is now believed to be an important agent for autophagy in neurons, essential for removing the accumulation of stress-inducing, aberrant G4 complexes.⁷

The essential roles of helicases in innate immunity and other diseases open tremendous opportunities for advancements in antiviral treatments and therapeutic interventions for other serious diseases.

Learn more about how you can screen for helicase activity with the Transcreener ADP² Assay.

Learn More About The Transcreener ADP² Assay

References

1. Wu, Yuliang. (2012) Unwinding and Rewinding: Double Faces of Helicase? Journal of Nucleic Acids, 2012, 140601. Review. https://doi.org/10.1155/2012/140601 
2. Perculija, V. and Ouyang, S. (2018) Diverse Roles of DEAD/DEAH-Box Helicases in Innate Immunity and Diseases. Helicases from All Domains of Life, 141-171. https://www.sciencedirect.com/science/article/pii/B9780128146859000099
3. 3. Sami, A. A. et al. (2021) Deciphering the role of helicases and translocases: A multifunctional gene family safeguarding plants from diverse environmental adversities. Current Plant Biology, 26, 100204. Review.      https://doi.org/10.1016/j.cpb.2021.100204
4. Jiang, Y. et al. (2017) The Emerging Roles of DDX41 Protein in Immunity and Diseases. Protein and Cell, 8, 83-89. Review. https://doi.org/10.1007/s13238-016-0303-4
5. Chen, M.C. et al. (2018) Structural Basis of G-Quadruplex Unfolding by DEAH/RHA Helicase DHX36. Nature, 558(7710), 465-469. https://doi.org/10.1038/s41586-018-0209-9
6. Antcliff, A. et al. (2021) G-Quadruplexes and the DNA/RNA Helicase DHX36 in Health, Disease, and Aging. Aging, 13(23), 25578-25587. Review. https://doi.org/10.18632/aging.203738
7. Moruno-Manchon, J. F. et al (2020) Small Molecule G-Quadruplex Stabilizers Reveal a Novel Pathway of Autophagy Regulation in Neurons. eLife, 9, e52283. https://doi.org/10.7554/eLife.52283