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Mol Cell Cui's team revealed a new mechanism by which protein pyrophosphorylation regulates antiviral innate immunity

 Last Update: 2023-02-03
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Protein is the material basis of life and the main bearer of life activities, but after the synthesis of proteins, it often needs to undergo a series of protein post-translational modifications (PTMs), through the addition or removal of functional groups, adjust the biochemical properties of proteins to change their activity
。 Common protein post-translational modifications include phosphorylation, ubiquitination, methylation, glycosylation, palmitoylation, etc
.
Among them, phosphorylation modification is the most widely distributed PTM
known.
Research on protein phosphorylation began in the early 20th century, and until the last decade, an average of 15,000 related studies were reported
each year.
It is estimated that nearly 1/3 of the proteins in eukaryotes can be phosphorylated
.
The human proteome contains more than 100,000 potential phosphorylation sites
.

Compared with phosphorylation modification, pyrophosphorylation modification of protein has a relatively short
research history as a newly discovered PTM.
Pyrophosphorylation modification was first discovered in
glycogen synthesis.
In 2007, Solomon H.
Snyder's group first determined that pyrophosphorylation modifications also exist on proteins [1].

At present, it is believed that protein pyrophosphorylation modification is mainly divided into two steps: (1) the serine or threonine site of the protein is phosphorylated by kinase; (2) On the basis of phosphorylation, IP₇ dissociates the β-phosphate group by hydrolysis, and releases a large amount of free energy to promote the transfer of the β-phosphate group to the phosphate group of the target protein through Mg2+ cofactors, and finally forms protein pyrophosphorylation
.
Since there are currently no reports of protein pyrophosphorylase, it is generally believed that protein pyrophosphorylation is an enzyme-free catalyzed modification
.

On January 4, 2023, Professor Cui Jun's research group from the School of Life Sciences of Sun Yat-sen University published a report entitled Metabolic enzyme UAP1 mediates IRF3 pyrophosphorylation to facilitate innate immune response in the journal Molecular cell In his paper, it was found that the pyrophosphorylase UAP1 can bind to the transcription factor IRF3 to directly positively regulate the type I interferon reaction, and also found that UAP1, as the first discovered protein pyrophosphorylase, enhances the body's innate immune response and reduces the new mechanism
of viral infection by catalyzing the pyrophosphorylation of IRF3.

In this study, through functional screening, it was found that knockout the key pyrophosphorylase UAP1 in the synthesis of glycosylated substrates can significantly inhibit the antiviral activity of cells, while overexpression of UAP1 enhances the expression
of type I interferon and interferon-stimulated genes 。 For further investigation, the researchers tried to construct Uap1 knockout mice and found that the mice with Uap1 knockout were homozygous and lethal, suggesting that UAP1 has important functions
in the body.
Fortunately, they found that Uap1 heterozygous mice were able to survive smoothly, and the protein expression of UAP1 in cells was reduced by more than half, which could be used as knockdown mice, and found that Uap1 knockdown mice were more sensitive to various viral infections, and their antiviral ability was significantly reduced, revealing its important role
in the body's antiviral immunity.

In order to explore the specific mechanism of UAP1 regulating antiviral immunity, the researchers found that UAP1 can specifically bind to the transcription factor IRF3
through co-immunoprecipitation experiments.
The enzymatic active mutant of UAP1 lost its ability to promote activation of the type I interferon pathway, indicating that the antiviral function of UAP1 depends on its enzymatic activity
.
The researchers further determined that UAP1 affects the activity
of IRF3 primarily by catalyzing the pyrophosphorylation modification of IRF3, rather than glycosylation modification.
Through inhibitor experiments, the researchers found that O-glycosylation inhibitor (OSMI-1) or N-glycosylation inhibitor (TM) treated cells did not affect the promoting effect of UAP1 on type I interferon, while pyrophosphorylation modification inhibitor (TNP).
Treatment significantly inhibits the antiviral function
of UAP1.
Supplementing UDP-N-acetylglucosamine, an important product of UAP1 in the hexosamine pathway, could not restore the regulatory effect of UAP1 on IRF3 and its antiviral immunity, revealing that UAP1 may directly exert its function
through pyrophosphorylation of catalytic proteins.
Further studies proved that UAP1 can specifically catalyze the pyrophosphorylation modification of the Ser386 site of IRF3 through different experimental methods such as isotope tracing method, pyrophosphate kit detection and mass spectrometry experiment
.
The researchers constructed an S386A mutant inactivated at the Ser386 site and found that IRF3 could not be further activated after the S386 point mutation, nor could the 5ST active site (Ser396) at the C-terminal of IRF3 be phosphorylated, and the dimerization and nucleation capacity of IRF3 was also significantly reduced, which showed that UAP1-mediated pyrophosphorylation modification is essential for the regulation of antiviral innate immunity
。
Figure UAP1 regulates IRF3 activity and the working model of type I interferon signaling pathway by catalyzing IRF3 pyrophosphorylation
The study not only reveals a new mechanism by which UAP1 regulates antiviral signaling pathways as a pyrophosphorylase by catalyzing pyrophosphate of IRF3, but also raises an interesting question: How common is protein pyrophosphorylation at the cell signaling level? Since pyrophosphorylation modification is an extension of phosphorylation modification, how much of the previous research on phosphorylation regulation function is actually caused by pyrophosphorylation? Follow-up research on these issues may lead to a series of breakthroughs
in the future.

Professor Cui Jun from the School of Life Sciences of Sun Yat-sen University is the corresponding author
of the work.
Dr.
Yang Shuai from the School of Life Sciences, Sun Yat-sen University is the first author
of the research paper.

Attached Cui Jun's recent work is as follows: 1 Mol Cell | Cui Jun's team reveals a new mechanism by which protein palmitation mediates autophagy and NLRP3 inflammasome interaction to regulate inflammation and related diseases (2022)2.
Nat CommunCui Jun/Li Chunwei collaborated to reveal that organic pollutants induce innate immune receptor AIM2 activation (2021)3.
Adv Sci | Cui Jun's team reveals a new mechanism by which USP38-KDM5B complex inhibits inflammation by regulating histone ubiquitination/methylation chain reaction (2020)4.
Cui Jun/Zhang Yuxia/Geng Lanlan et al.
collaborated to reveal a new mechanism of early onset inflammatory bowel disease (VEOIBD) caused by NLRP3 inflammasome with abnormal ubiquitination modification (2020)5.
Adv Sci | Cui Jun's team at Sun Yat-sen University reveals a new mechanism of autophagy regulating non-classical NF-kB pathways and inflammatory responses (2019)6.
Cui Jun/Li Chunwei collaborated to reveal the function and mechanism of NLRP3 inflammasome in rhinovirus-induced airway mucosal remodeling (2019)7.
Jiang Li/Jun Cui collaboration reveals the function and mechanism of natural immune signaling pathways in tumor microenvironment (2019).
8.
Mol Cell of Cui Jun Group, Sun Yat-sen University reported a new regulatory model of selective autophagy intervention against viral immunity (2017) 9.
【Academic Express】Three domestic "Molecular Cell" were published online at the same time (2016).

Original link:

https://doi.
org/10.
1016/j.
molcel.
2022.
12.
007

Platemaker: Eleven

References

1.
Bhandari R, Saiardi A, Ahmadibeni Y, Snowman AM, Resnick AC, Kristiansen TZ, Molina H, Pandey A, Werner JJ, Juluri KR, Xu Y, Prestwich GD, Parang K, Snyder SH.
Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event.
Proc Natl Acad Sci U S A 2007, 104(39): 15305-15310.

2.
Saiardi A.
Protein pyrophosphorylation: moving forward.
BIOCHEM J 2016, 473(21): 3765-3768.

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