Formation of FADD amyloid fiber and its role in immune signaling in Drosophila melanogaster / 生物工程学报

In this research, we studied the formation of Drosophila melanogaster FADD (Fas-associated death domain-containing protein) amyloid fiber and its influence on signal transduction in IMD (Immune deficiency) signaling pathway to better understand the regulation mechanism of Drosophila innate immune signaling pathway, which will provide reference for the immune regulation in other species. First, we purified dFADD protein expressed in Escherichia coli and performed Sulfur flavin T binding and transmission electron microscopy to identify the dFADD amyloid fibers formed in vitro. Then we investigated the formation of dFADD polymers in S2 cells using SDD-AGE and confocal microscope. We also constructed dFADD mutants to find out which domain is essential to fiber formation and its effect on IMD signal transduction. Our results revealed that dFADD could be polymerized to form amyloid fiber polymers in vitro and inside the cells. Formation of fibers relies on DED (Death-effector domain) domain of dFADD, since DED domain-deleted mutant existed as a monomer. Dual luciferase reporter assay showed that intact DED domain was required for the induction of downstream antimicrobial peptides, indicating that fiber formation was the key to IMD signal transduction. Our study revealed the role of dFADD in mediating the cascade between IMD and Dredd in the IMD signaling pathway by forming amyloid fibers, suggesting an evolutionarily conserved regulatory mechanism of innate immune signaling pathway.

Prediction of fibril formation by early-stage amyloid peptide aggregation / 药物分析学报

Amyloid fibrils are found in systemic amyloidosis diseases such as Alzheimer's disease, Parkinson's disease, and type II diabetes. Currently, these diseases are diagnosed by observation of fibrils or plaques, which is an ineffective method for early diagnosis and treatment of disease. The goal of this study was to develop a simple and quick method to predict the possibility and speed of fibril formation before its occurrence. Oligomers generated from seven representative peptide segments were first isolated and detected by ion-mobility mass spectrometry (IM-MS). Then, their assemblies were disrupted using formic acid (FA). Interestingly, oligomers that showed small ion intensity changes upon FA addition had rapid fibril formation. By contrast, oligomers that had large ion intensity changes generated fibrils slowly. Two control peptides (aggregation/no fibrils and no aggregation/no fibrils) did not show changes in their ion intensities, which confirmed the ability of this method to predict amyloid formation. In summary, the developed method correlated MS intensity ratio changes of peptide oligomers on FA addition with their amyloid propensities. This method will be useful for monitoring peptide/protein aggregation behavior and essential for their mechanism studies.