Protein facility users reveal the molecular mechanism of autophagy receptor protein recruitment to ULK complexes related to autophagy initiation

Autophagy is a highly regulated and lysosome-dependent cellular metabolic process in eukaryotic cells, which plays a vital role in maintaining cell homeostasis and promoting cell growth, development and survival
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Selective autophagy uses autophagy receptor proteins to specifically recognize and degrade specific substrates, including protein aggregates, damaged mitochondria, and exogenous invading pathogens
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Selective autophagy plays an important role in many physiological processes, and its dysfunction is also associated with a large number of human diseases, such as neurodegenerative diseases and microbial infections
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In mammalian cells, the autophagy initiating ULK complex consists of four subunits: ULK1, ATG13, ATG101, and FIP200, which play a key role in initiating the initial stage of autophagosome formation
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In some selective autophagy processes, autophagy receptor proteins can recruit and activate ULK complexes by binding to the FIP200 subunit in the ULK complex, and mediate the formation of autophagosomes in situ near the autophagy substrate
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However, at present, the molecular mechanism and related regulation mechanism of autophagy receptor protein to recruit ULK complex by binding to FIP200 are still unknown
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On March 10, 2021, a user of the National Protein Science Research (Shanghai) facility, Pan Lifeng's group from the State Key Laboratory of Bioorganic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, published a titled "Phosphorylation regulates the binding of autophagy receptors to FIP200" in Nature Communications.
The article "Claw domain for selective autophagy initiation" reveals the molecular mechanism by which a universal, phosphorylation-regulated autophagy receptor protein binds to the Claw domain of FIP200 to recruit the autophagy initiation ULK complex
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The team used a series of biochemical characterization methods such as nuclear magnetic resonance technology, rapid protein liquid chromatography, analytical ultracentrifugation, and fluorescence polarization experiments to discover for the first time the FIR2 motif interval of the autophagy receptor protein CCPG1, which is involved in the selective autophagy of the endoplasmic reticulum.
The phosphorylation modification of the 104-position serine will significantly enhance the interaction between CCPG1 FIR2 and FIP200 Claw domain
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At the same time, the team further research found that the TBK1 kinase-mediated autophagy receptor protein Optineurin (involved in the selective autophagy process of mediating protein aggregates, invading pathogens, and damaged mitochondria) of the 177-position serine phosphate of the LIR motif Chemical will regulate and enhance the interaction between Optineurin LIR and FIP200 Claw
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Subsequently, through the X-ray single crystal diffraction method, the team analyzed for the first time the high-resolution complex structure of FIP200 Claw combined with phosphorylated CCPG1 FIR2 (p-CCPG1 FIR2) and phosphorylated Optineurin LIR (p-Optineurin LIR).

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Consistent with the biochemical results, the relevant structural analysis showed that FIP200 Claw exists as a stable dimer and symmetrically combines with two phosphorylated CCPG1 FIR2 or Optineurin LIR to form a heterotetramer.
The subsequent detailed structural analysis not only revealed for the first time The specific molecular mechanism of p-CCPG1 FIR2 and p-Optineurin LIR binding to FIP200 Claw, and it was found that the mechanism of phosphorylation modification regulating the interaction between CCPG1 FIR2 and Optineurin LIR and FIP200 Claw is different
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Figure 1: Biochemical and structural data of FIP200 Claw combined with phosphorylated CCPG1 FIR2 and Optineurin LIR

 

Based on the resolved complex structure and related biochemical studies, the team defined the sequence pattern of the FIR motif that can bind to the FIP200 Claw domain for the first time, and through bioinformatics analysis, it was found that the classical LIR interval of a large number of autophagy receptor proteins contained Potential FIR motifs, and then the researchers summarized the currently known and potential FIR and FIP200 Claw interaction systems into two different modes
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At the same time, further structural analysis and biochemical experiments showed that CCPG1 FIR2 or Optineurin LIR can compete with FIP200 Claw and ATG8 family proteins through the same amino acid residues, indicating that autophagy receptor proteins should first be recruited by binding FIP200 after recognizing autophagy substrates.
The ULK complex then mediates the generation of autophagy precursors in situ, and subsequently promotes the extension and closure of the autophagy precursors by binding to ATG8 family proteins
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Finally, the researchers proposed a model diagram of the autophagy receptor protein in the selective autophagy process by binding to the FIP200 subunit to recruit the autophagy initiation ULK complex to mediate the process of autophagosome formation
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Figure 2.
A cartoon schematic diagram of the recruitment of ULK complexes and ATG8 family proteins to mediate autophagosome formation by FIR/LIR-containing autophagy receptor proteins during selective autophagy

In summary, this research work for the first time systematically defines the sequence types of FIR motifs that can bind to the FIP200 Claw domain, and reveals a universal, phosphorylation-regulated autophagy receptor protein that binds to the FIP200 Claw domain.
The new mechanism of recruiting autophagy initiating ULK complex has important guiding significance for further understanding of the selective autophagy process mediated by autophagy receptor protein
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The National Protein Science Research (Shanghai) Facility Mass Spectrometry System used mass spectrometry to identify that the phosphorylation of Serine 177 of Optineurin, the key protein of the subject, was regulated by TBK1 kinase, which provided important support for subsequent mechanism research
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