Extended Data Fig. 6: Site-specific lactylation of cGAS abrogates their Liquid-Liquid Phase Separation (LLPS) and sensing of DNA.
From: AARS1 and AARS2 sense l-lactate to regulate cGAS as global lysine lactyltransferases
a, A schematic diagram describing the generation of site-specifically-lactylated recombinant h- or m- cGAS proteins with an lactyl-lysine Methanosarcina barkeri (Mb) pyrrolysyl-tRNA synthetase/tRNACUA pair in pSupAR backbone vector (see Materials and Methods). b, Immunoblot (IB) of the purified non-lactylated and site-specifically-lactylated h-cGAS (left) or m-cGAS (right) proteins with specific antibodies. The anti-Strep blots indicate loading of lanes. c, MST binding affinity between Cy3-45-bp ISD and h-cGASNon-lac, h-cGASK131-lac (left), m-cGASNon-lac or m-cGASK156-lac (right) proteins purified in a. d, Representative images (left) and quantification (right) of droplet formation by mixing 45-bp ISD (1 μM, 5% labeled with Cy3) with cGAS proteins (2 μM) purified in a. e, Representative image (left) and quantification (right) of FRAP over a 120 s time course of 45-bp ISD (1 μM, 5% labeled with Cy3) with h-cGAS (upper, 2 μM) or m-cGAS (lower, 2 μM) proteins purified in a. f, Fluorescence of GFP-fused m-cGAS (10 μM) proteins mixed with 100-bp DNA (1 μM, 5% labeled with AF647) at room temperature with 150 mM NaCl at pH 7.0. Quantitative line profile of co-localization along the indicated arrow of the left image (right). n = 3. g, Fluorescence of m-cGAS (20 μM) proteins purified in a after incubated with HT-DNA (0.05 μg/μl) and fluorescent tetramethulrhodamine dextran (10 kDa, 40 kDa or 155 kDa as indicated). h, FRAP over a 150-s time course of droplet formation by mixing 45-bp ISD (1 μM), 100-bp DNA (1 μM), HT-DNA (0.05 μg/μl) with AF488-labeled h-cGASNon-Lac (upper-left), h-cGASK131-Lac (upper-right), m-cGASNon-Lac (lower-left) or m-cGAS-K156-Lac (lower-right) proteins purified in a. i, Working model on low-diffusive nature of the DNA-repellent cGASLac condensate. cGASNon-Lac condensates with DNA and forms liquid-like droplets that are highly diffusive and permeable by 10 kDa, 40 kDa and 155 kDa tetramethylrhodamine dextran molecules. After N-terminal lactylation, cGASLac tend to repel DNA and self-condensate into DNA-free, small gel-like droplets with high-viscosity, low-fluorescence recovery and low-permeability, resulting in the exclusion of 155 kDa tetramethylrhodamine dextran molecules (Rh ≈ 9 nm). cGAS and dextran molecules were depicted in blue and red respectively. cGAS lactylation (Lac) was highlighted with the orange circle. j, MST binding affinity between 100-bp DNA and m-cGAS proteins purified in a. Data are representative of at least three independent experiments (b-h, j). Scale bar, 5 μm (d-g). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (d) or two-way ANOVA (e, h). Supplementary Fig. 1 shows full gels.
