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Cytosolic DNA sensing via cGAS: long, U-shaped and other DNA ligands and cellular co-factors
Cytosolic DNA sensing via cGAS: long, U-shaped and other DNA ligands and cellular co-factors
Innate immune sensing of cytosolic nuclear acids is executed by pattern recognition receptors and is a powerful tool to counteract viral and bacterial infection. Nucleic acids with their essential function as genetic information carriers serve as a very general pathogen-derived pattern and therefore trigger a powerful immune response. The innate immune system evolved to distinguish between pathogen-specific patterns such as secondary structures or modifications of RNA and host nucleic acids. In case of DNA, however, such strategies as DNA compartmentalization are used to prevent self-DNA recognition. Indeed, cellular DNA normally present in nuclei and mitochondria is immunosilent, whereas accumulation of cytosolic DNA triggers an inflammatory response. Such DNA can emerge due to viral infection, mitochondrial and nuclear stress or due to dysfunction of key proteins responsible for elimination of excessive DNA amounts in the cytosol and lysosomes. cGAS is a central sensor of cytosolic DNA expressed in almost all cell types. cGAS recognizes cytosolic dsDNA in a broad sequence-indiscriminatory manner and synthesizes the second messenger cyclic GMP-AMP (pG(2’-5’)pA(3’-5’), 2’3’-cGAMP) from ATP and GTP. Unlike bacterial cyclic dinucleotides (CDNs) with canonical 3’-5’ linkages, 2’3’-cGAMP is a unique metazoan CDN that comprises both 3’-5’ and a non-canonical 2’-5’ phosphodiester linkages connecting adenosine with guanosine and guanosine with adenosine, respectively. 2’3’-cGAMP produced upon infection binds and activates the downstream adaptor stimulator of interferon genes (STING). Residing on the endoplasmic reticulum, STING binds cGAS-generated cGAMP or bacterial CDNs and undergoes trafficking to the Golgi complex in perinuclear space where it recruits TANK-binding kinase 1 (TBK1) and transcription factor interferon regulatory factor 3 (IRF3). As a result of the pathway activation IRF3 gets phosphorylated, dimerizes and translocates into the nucleus resulting in type I interferons (IFNs) production. A range of cGAS structures and biochemical studies revealed activation and catalytic mechanisms of cGAS. cGAS was discovered to dimerize upon DNA binding in a way that two DNA molecules are sandwiched between two cGAS protomers and such dimerization was shown to be necessary for cGAS activation. The nature and physiological function of cGAS dimerization, however, remains elusive, since such conformation was not found for functionally and structurally similar 2'-5'-oligoadenylate synthetases (OAS) or other proteins suggesting it to be a unique feature of cGAS. Moreover, the proposed dimerization does not explain why short DNA constructs of 14-20 base pairs (bp) used for crystallization fail to fully activate cGAS in vivo, though they are capable of inducing all conformational changes known for cGAS activation in the crystal. Furthermore, the composition of such cGAS dimers would lead to steric clashes between two bound DNA molecules, if the length of DNA strands exceeds 18 bp present in the structure. The instability of such dimeric cGAS conformation is another enigma to be clarified, since cGAS2:DNA2 species could only be observed in non-physiological high concentrations of cGAS and DNA. In this work a mechanism of cGAS activation by biologically relevant ligands was studied. Furthermore, a model for cooperative sensing of long DNA by cGAS was established. Mab-21 domain of cGAS was found to have an intrinsic capability to measure DNA length, since its activity dramatically increased with DNA length by the same number of cGAS binding sites in vitro and in cell-based experiments. In order to investigate cGAS activity, a novel high-throughput fluorescence-based assay was developed. The first crystal structure of cGAS in complex with stimulatory DNA of 39 bp presented in this thesis provided an insight into cGAS activation by fibril formation. Such cGAS oligomers were found to make protein-DNA ladders with two nearly parallel DNA strands as “ladder sides” and cGAS dimers as “rungs” holding them together. cGAS-DNA oligomers, first observed in crystal packing, were confirmed by isothermal titration calorimetry (ITC) and by size exclusion chromatography coupled to right-angle light scattering (SEC-RALS). These methods revealed the stoichiometry and molecular weights of cGAS complexes with different DNA species that together determined an exact complex composition and confirmed the presence of (cGAS2)n:DNA2 complexes in solution. According to our model, the formation of the first dimer is highly unfavorable resulting in an unstable complex, however, it parallelizes two DNA strands and enables an effective binding of the subsequent cGAS dimer. This results in cooperative binding of cGAS on long stimulatory DNA with cooperativity emerging already from cGAS-DNA interactions. cGAS dimers are mutually stabilized within a fibril resulting in higher cGAS activity. Furthermore, potential cGAS co-factors were proposed. Based on similarities in DNA conformations and a recent research revealing mitochondrial DNA to be a cGAS activator, mitochondrial transcription factor A (TFAM), as well as high-mobility group box 1 (HMGB1) and bacterial nucleoid HU proteins were discovered to enhance cGAS activity. According to our hypothesis, TFAM and other DNA-bending proteins introduce a U-turn in long DNA, prearrange DNA in a manner favorable for cGAS dimerization and thus serve as nucleation points for cGAS-DNA ladder formation. Another part of this thesis was dedicated to discover other physiological nucleic acid ligands of cGAS. Cell line experiments with different synthetic constructs revealed RNA:DNA hybrids to be potent cGAS activators. Furthermore, ssDNA hairpins formed by HIV-1 reverse-transcribed ssDNA were found to stimulate cGAS during HIV-1 infection. Substitution of guanines flanking the double-stranded hairpin regions abolished cGAS activity and specific short Y-shaped DNA with G-overhangs (YSD) were found to be potent cGAS ligands. Intriguingly, such YSDs were capable of cGAS activation despite their short length (< 20 bp) leaving the question open, whether the cGAS-DNA ladder model is applicable in this case. Taken together, this work presents a range of cGAS activators that include RNA:DNA hybrids, HIV-1 ssDNA hairpins and partly unfolded mitochondrial and bacterial nucleoids rather than naked dsDNA as specific cGAS ligands. The oligomerization mechanism of cGAS described in this thesis provides a link between a peculiar cGAS dimerization, on the one hand, and high stimulatory activity of long dsDNA in vivo, on the other hand. Proposed cGAS co-factors further clarify the complexity of DNA recognition by cGAS in the living cell suggesting that analogically to RNA sensing by RIG-I, cGAS recognizes specific structures of dsDNA rather than just mislocalized DNA fragments. However, further studies are needed to evaluate the relevance of the oligomerization mechanism for cGAS sensing of other ligands and to discover other cellular co-factors facilitating cGAS activity in vivo.
innate immunity, pattern recognition receptors, cGAS, HIV-1, interferon, x-ray crystallography, nucleic acid sensing
Andreeva, Liudmila
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Andreeva, Liudmila (2018): Cytosolic DNA sensing via cGAS: long, U-shaped and other DNA ligands and cellular co-factors. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Innate immune sensing of cytosolic nuclear acids is executed by pattern recognition receptors and is a powerful tool to counteract viral and bacterial infection. Nucleic acids with their essential function as genetic information carriers serve as a very general pathogen-derived pattern and therefore trigger a powerful immune response. The innate immune system evolved to distinguish between pathogen-specific patterns such as secondary structures or modifications of RNA and host nucleic acids. In case of DNA, however, such strategies as DNA compartmentalization are used to prevent self-DNA recognition. Indeed, cellular DNA normally present in nuclei and mitochondria is immunosilent, whereas accumulation of cytosolic DNA triggers an inflammatory response. Such DNA can emerge due to viral infection, mitochondrial and nuclear stress or due to dysfunction of key proteins responsible for elimination of excessive DNA amounts in the cytosol and lysosomes. cGAS is a central sensor of cytosolic DNA expressed in almost all cell types. cGAS recognizes cytosolic dsDNA in a broad sequence-indiscriminatory manner and synthesizes the second messenger cyclic GMP-AMP (pG(2’-5’)pA(3’-5’), 2’3’-cGAMP) from ATP and GTP. Unlike bacterial cyclic dinucleotides (CDNs) with canonical 3’-5’ linkages, 2’3’-cGAMP is a unique metazoan CDN that comprises both 3’-5’ and a non-canonical 2’-5’ phosphodiester linkages connecting adenosine with guanosine and guanosine with adenosine, respectively. 2’3’-cGAMP produced upon infection binds and activates the downstream adaptor stimulator of interferon genes (STING). Residing on the endoplasmic reticulum, STING binds cGAS-generated cGAMP or bacterial CDNs and undergoes trafficking to the Golgi complex in perinuclear space where it recruits TANK-binding kinase 1 (TBK1) and transcription factor interferon regulatory factor 3 (IRF3). As a result of the pathway activation IRF3 gets phosphorylated, dimerizes and translocates into the nucleus resulting in type I interferons (IFNs) production. A range of cGAS structures and biochemical studies revealed activation and catalytic mechanisms of cGAS. cGAS was discovered to dimerize upon DNA binding in a way that two DNA molecules are sandwiched between two cGAS protomers and such dimerization was shown to be necessary for cGAS activation. The nature and physiological function of cGAS dimerization, however, remains elusive, since such conformation was not found for functionally and structurally similar 2'-5'-oligoadenylate synthetases (OAS) or other proteins suggesting it to be a unique feature of cGAS. Moreover, the proposed dimerization does not explain why short DNA constructs of 14-20 base pairs (bp) used for crystallization fail to fully activate cGAS in vivo, though they are capable of inducing all conformational changes known for cGAS activation in the crystal. Furthermore, the composition of such cGAS dimers would lead to steric clashes between two bound DNA molecules, if the length of DNA strands exceeds 18 bp present in the structure. The instability of such dimeric cGAS conformation is another enigma to be clarified, since cGAS2:DNA2 species could only be observed in non-physiological high concentrations of cGAS and DNA. In this work a mechanism of cGAS activation by biologically relevant ligands was studied. Furthermore, a model for cooperative sensing of long DNA by cGAS was established. Mab-21 domain of cGAS was found to have an intrinsic capability to measure DNA length, since its activity dramatically increased with DNA length by the same number of cGAS binding sites in vitro and in cell-based experiments. In order to investigate cGAS activity, a novel high-throughput fluorescence-based assay was developed. The first crystal structure of cGAS in complex with stimulatory DNA of 39 bp presented in this thesis provided an insight into cGAS activation by fibril formation. Such cGAS oligomers were found to make protein-DNA ladders with two nearly parallel DNA strands as “ladder sides” and cGAS dimers as “rungs” holding them together. cGAS-DNA oligomers, first observed in crystal packing, were confirmed by isothermal titration calorimetry (ITC) and by size exclusion chromatography coupled to right-angle light scattering (SEC-RALS). These methods revealed the stoichiometry and molecular weights of cGAS complexes with different DNA species that together determined an exact complex composition and confirmed the presence of (cGAS2)n:DNA2 complexes in solution. According to our model, the formation of the first dimer is highly unfavorable resulting in an unstable complex, however, it parallelizes two DNA strands and enables an effective binding of the subsequent cGAS dimer. This results in cooperative binding of cGAS on long stimulatory DNA with cooperativity emerging already from cGAS-DNA interactions. cGAS dimers are mutually stabilized within a fibril resulting in higher cGAS activity. Furthermore, potential cGAS co-factors were proposed. Based on similarities in DNA conformations and a recent research revealing mitochondrial DNA to be a cGAS activator, mitochondrial transcription factor A (TFAM), as well as high-mobility group box 1 (HMGB1) and bacterial nucleoid HU proteins were discovered to enhance cGAS activity. According to our hypothesis, TFAM and other DNA-bending proteins introduce a U-turn in long DNA, prearrange DNA in a manner favorable for cGAS dimerization and thus serve as nucleation points for cGAS-DNA ladder formation. Another part of this thesis was dedicated to discover other physiological nucleic acid ligands of cGAS. Cell line experiments with different synthetic constructs revealed RNA:DNA hybrids to be potent cGAS activators. Furthermore, ssDNA hairpins formed by HIV-1 reverse-transcribed ssDNA were found to stimulate cGAS during HIV-1 infection. Substitution of guanines flanking the double-stranded hairpin regions abolished cGAS activity and specific short Y-shaped DNA with G-overhangs (YSD) were found to be potent cGAS ligands. Intriguingly, such YSDs were capable of cGAS activation despite their short length (< 20 bp) leaving the question open, whether the cGAS-DNA ladder model is applicable in this case. Taken together, this work presents a range of cGAS activators that include RNA:DNA hybrids, HIV-1 ssDNA hairpins and partly unfolded mitochondrial and bacterial nucleoids rather than naked dsDNA as specific cGAS ligands. The oligomerization mechanism of cGAS described in this thesis provides a link between a peculiar cGAS dimerization, on the one hand, and high stimulatory activity of long dsDNA in vivo, on the other hand. Proposed cGAS co-factors further clarify the complexity of DNA recognition by cGAS in the living cell suggesting that analogically to RNA sensing by RIG-I, cGAS recognizes specific structures of dsDNA rather than just mislocalized DNA fragments. However, further studies are needed to evaluate the relevance of the oligomerization mechanism for cGAS sensing of other ligands and to discover other cellular co-factors facilitating cGAS activity in vivo.