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▎WuXi AppTec content team editor
, MARCH8 attenuates cGAS-mediated innate immune responses through ubiquitylation.
Science Signaling.
2022 May 3; 15(732)】
Natural immunity is the body's "first line of defense"
against pathogen invasion.
When a pathogen invades, the molecular patterns associated with the pathogen are recognized by the body's pattern recognition receptors and initiate an innate immune response
.
In 2013, circular guanylate-adenylate synthase (cGAS) was discovered by Professor Chen Zhijian of Southwestern Medical Center in the United States, as an important double-stranded DNA recognition receptor, cGAS is a research hotspot
in the field of natural immunity in recent years.
cGAS-mediated natural immune signaling pathways play an important role
in the occurrence of diseases such as anti-viral infection, tumor immunity and autoinflammation.
Therefore, it is of great significance
to explore the mechanism of cGAS function regulation.
Yin Hang's research group at the School of Pharmacy of Tsinghua University has long focused on the exploration
of the regulation of natural immune signaling pathways.
In 2022, taking protein post-translational modification as the starting point, the research group focused on the regulatory mechanism of cGAS and achieved a series of research results
.
Based on the discovery of novel protein post-translational modifications during cGAS activation, enzymes responsible for post-translational modifications of related proteins, including palmitoylation modifier ZDHHC18, ubiquitination ligase MARCH8 and deubiquitinase USP15, were identified, providing new targets
for the specific regulation of cGAS functions 。 In particular, in the autoimmune disease model caused by blisty simplex virus type I (HSV-1) infection and exonuclease deletion (Trex1 knockout), the research team revealed the biological functions of post-translational modifications of several proteins, which provided application prospects
for the treatment of herismic simplex virus infection and autoimmune diseases.
In April, Yin Hang's research group reported for the first time that cGAS can undergo palmitoyl modification and negatively regulate the activation of cGAS, and the cysteine at cGAS site 474 was identified as a palmitoylation modification site
by protein mass spectrometry.
Yin Hang's research group combined shRNA-mediated gene knockdown and functional screening to find that the palmitoylation modification of cGAS was mainly catalyzed
by ZDHHC18.
In terms of molecular mechanism, the binding of palmitoylated cGAS to DNA and its own dimerization are inhibited
.
In human and animal macrophages knocked down by ZDHHC18, DNA-initiated innate immune signals were significantly enhanced, indicating the negative regulatory effect
of palmitoylation on cGAS activation in vivo.
At the animal level, mouse strains knocked out by the ZDHHC18 gene exhibited greater resistance to DNA virus infection, revealing the important biological significance
of cGAS palmitoylation modifications.
The results were published in The EMBO Journal (DOI: 10.
15252/embj.
2021109272).
ZDHHC18-mediated palmitoylation modification negatively regulated cGAS activation [Image source: Shi et al.
, ZDHHC18 negatively regulates cGAS-mediated innate immunity through palmitoylation.
The EMBO Journal.
2022 Jun 1; 41(11)】
In May of the same year, in order to explore the ubiquitination modification of cGAS, Yin Hang's research group screened and identified the ubiquitination ligase MARCH8 as the interacting protein
of cGAS.
In cell lines, overexpressing MARCH8 weakens the natural immune response induced by dsDNA, while knocking down or knocking out MARCH8 has the opposite effect
.
In terms of molecular mechanism, MARCH8 catalyzes the polyubiquitination of K63-linked type at the Lys411 site of cGAS, which inhibits the binding of cGAS to its ligand DNA, and ultimately weakens the production
of downstream interferon factors.
Using a mouse model of MARCH8 knockout, the authors further confirmed the negative regulatory role
of MARCH8 on cGAS-mediated antiviral innate immune signaling.
The results were published as a cover story in the biochemistry journal Science Signaling (DOI: 10.
1126/scisignal.
abk3067).
MARCH8-mediated ubiquitination inhibits cGAS activation [Image source: Yang et al.
, MARCH8 attenuates cGAS-mediated innate immune responses through ubiquitylation.
Science Signaling.
2022 May 3; 15(732)】
On October 16, 2022, Yin Hang's research group published a research paper entitled "USP15 promotes cGAS activation through deubiquitylation and liquid condensation" (DOI: 10.
1093/nar/gkac823)
in Nucleic Acids Research.
In this study, from the perspective of deubiquitination modification, the research group identified the deubiquitinase USP15 as an interacting protein of cGAS for the first time through co-immunoprecipitation and protein mass spectrometry, and USP15 can promote cGAS recognition of DNA and activate downstream signaling pathways
.
The double-stranded DNA in the cytoplasm increases the expression of USP15, and the activation of the natural immune signaling pathway caused by the loss of DNA in USP15 gene deletion macrophages indicates that USP15 promotes the activation
of DNA-induced natural immune signaling pathway in a positive feedback manner.
In terms of molecular mechanisms, USP15 promotes the activation
of cGAS through two mechanisms.
First, USP15 can enhance cGAS activity by hydrolyzing multiple types of ubiquitination modifications of cGAS.
Second, USP15 can promote the phase separation structure formed by cGAS and DNA, and promote the binding
of cGAS to DNA.
The above two mechanisms are parallel to each other and jointly promote the activation
of cGAS.
USP15-mediated cGAS deubiquitination modification and enhanced phase separation promote DNA-induced native immune signaling, revealing novel microregulatory mechanisms
in native immune pathways.
USP15 promotes cGAS activation through deubiquitylation and liquid condensation.
Nucleic Acids Research.
DOI: 10.
1093/nar/gkac823）
In addition, Yin Hang's research group also paid attention to the regulation mechanism of cell pyrotic signaling pathway closely related to natural immunity and achieved research results
.
On September 30, 2022, Yin Hang's research group of Tsinghua University was invited by Chemical Communications to publish a special issue entitled "Protein phosphatase 1 regulates phosphorylation of gasdermin D and pyroptosis" in the special issue of Emerging Investigator's tenth anniversary review.
(Phosphatase PP1 regulates phosphorylation and pyroptosis of gasdermin D protein) (DOI: 10.
1039/d2cc03590a).
This work found that phosphorylation modification has an outstanding contribution to the regulation of gasdermin D protein (GSDMD) in immune cells, and phosphatase PP1 regulates GSDMD-NT through dephosphorylation, affecting GSDMD-NT oligomerization, and then regulating cell pyrosis
.
Using protein profiling, Yin Hang's research group identified multiple phosphorylation modification sites of GSDMD
.
By constructing mutants for phenotypic verification, phosphorylation modifications, including T213, were found to significantly inhibit cell pyrosis
.
Using the solved crystal structure of GSDMD-NT, phosphorylation modifications were analyzed to regulate their function, and it was found that these sites could affect the binding to membrane phospholipids and the formation
of protein oligomers.
In vitro experiments were used to confirm that phosphorylation at the T213 site did not affect its binding to membrane phospholipids but could inhibit protein oligomerization, and the increase of steric hindrance caused by phosphorylation modification may be the main reason for inhibiting the formation of
protein oligomers.
The mechanistic study found that the phosphatase PP1 interacted with GSDMD, and through the construction and phenotypic verification of cell lines such as knockdown and overexpression, it was found that PP1 dephosphorylated GSDMD after it was cleaved, so that GSDMD-NT oligomerization induced pyrosis
.
▲ Phosphatase PP1 regulates the function of GSDMD (Image source: Li et al.
, Protein phosphatase 1 regulates phosphorylation of gasdermin D and pyroptosis.
Chemical Communications.
doi: 10.
1039/d2cc03590a）
