Nature Communications enables real-time monitoring of itaconic acid concentration changes at subcellular resolution

Itaconic acid is an intermediate metabolite
with anti-inflammatory function synthesized by activating macrophages.
Previous research evidence suggests that cis-aconitic acid in mitochondria is decarboxylated to produce itaconic acid under the catalysis of metabolic enzyme IRG1, which is subsequently transported to cytoplasm to exert immunomodulatory functions
.
Li Xinjian's team previously reported that itaconic acid can induce macrophage lysosomal biosynthesis to improve the body's natural immunity against bacterial invasion (Molecular Cell 2022; PMID: 35662396)
。 Itaconic acid as a cellular endogenous metabolite in the host natural immune response process plays an important function, the current quantitative detection of itaconic acid relies on liquid chromatography-mass spectrometry technology, this method requires cell lysis, can not obtain the concentration distribution information of metabolites in different regions of the cell, in order to promote further research on the biological function of itaconic acid, it is urgent to develop a method
with spatiotemporal resolution to detect itaconic acid concentration.

The binding of metabolic small molecules to proteins can induce conformational changes in protein molecules, such protein molecules are connected with fluorescent proteins, which can convert protein molecule conformational change signals into fluorescent signal output, and use this principle to engineer metabolic small molecule-binding proteins and develop bioreceptors
that monitor the concentration changes of target metabolic small molecules.

On November 4, 2022, Li Xinjian's team published an online publication in Nature Communications magazine entitled "A genetically encoded fluorescent biosensor for detecting itaconate with subcellular resolution in living macrophages" (https://doi.
org/10.
1038/s41467-022-34306-5), reporting the development of a genetically encoded itaconic acid fluorescent probe to achieve real-time monitoring of itaconic acid concentration changes
at subcellular resolution 。 The researchers fused the bacteria-derived itaconate-binding domain (IBD) with circularly permuted green fluorescent protein (cpGFP), and linked a human centromere protein B dimerization domain at the C-terminus Since IBD exists as a dimer at the N-terminus of the fusion protein, the fusion protein is eventually dimerized to form an internal cyclized molecule
by dimerization at the anterior and posterior ends.
Through a series of linkage peptide sequence optimizations, the researchers obtained bioreceptors that responded to changes in itaconic acid concentration within the physiological concentration range, named BioITA
。 Using BioITA to monitor the kinetic curve of changes in intracellular itaconic acid concentration in mouse macrophages, the researchers found that the concentration of itaconic acid in the mitochondrial matrix rose before the cytoplasm after LPS stimulation, and the concentration of itaconic acid in both the mitochondrial matrix and cytoplasm peaked about 12 hours after LPS stimulation, and unexpectedly found that the peak itaconic acid concentration in the cytoplasm (about 1757 M) was significantly higher than the peak itaconic acid concentration in the mitochondrial matrix (about 551 M).

In summary, this result provides innovative tool support
for further research on the anti-inflammatory, antibacterial and other biological functions of itaconic acid.

Figure: BioITA, as a dimer internal cyclizing molecule, monitors itaconic acid concentration changes
in real time at subcellular resolution.

Sun Pengkai, a doctoral student at the Institute of Biophysics, Chinese Academy of Sciences, and Zhang Zhenxing, a postdoctoral fellow, are the co-first authors of the paper, and researcher Li Xinjian is the corresponding author
of the paper.
The research was supported
by the National Natural Science Foundation of China, the Chinese Academy of Sciences, and the National Key Research and Development Program.

Link to the article: style="text-align:justify;"> 

(Contributed by: Zeng Yixin/Li Xinjian Research Group)