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Selection of mutant µplasmin for amyloid-? cleavage in vivo.

ABSTRACT: One of the main culprits of Alzheimer's disease (AD) is the formation of toxic amyloid-? (A?) peptide polymers and the aggregation of A? to form plaques in the brain. We have developed techniques to purify the catalytic domain of plasmin, micro-plasmin (µPlm), which can be used for an A?-clearance based AD therapy. However, in serum, µPlm is irreversibly inhibited by its principal inhibitor ?2-antiplasmin (?2-AP). In this study, we engineered and selected mutant forms of µPlm that are both catalytically active and insensitive to ?2-AP inhibition. We identified surface residues of ?Plm that might interact and bind ?2-AP, and used an alanine-scanning mutagenesis method to select residues having higher activity but lower ?2-AP inhibition. Then we employed saturation mutagenesis for further optimize both properties. Modeled complex structure of µPlm/?2-AP shows that F587 is a critical contact residue, which can be used as a starting position for further investigation.

SUBMITTER: Yang D

PROVIDER: S-EPMC7374754 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Selection of mutant µplasmin for amyloid-β cleavage in vivo.

Yang Dongying D Zhu Wei W Wang Yingjie Y Tan Fangmei F Ma Zhiping Z Gao Jiali J Lin Xinli X

Scientific reports 20200721 1

One of the main culprits of Alzheimer's disease (AD) is the formation of toxic amyloid-β (Aβ) peptide polymers and the aggregation of Aβ to form plaques in the brain. We have developed techniques to purify the catalytic domain of plasmin, micro-plasmin (µPlm), which can be used for an Aβ-clearance based AD therapy. However, in serum, µPlm is irreversibly inhibited by its principal inhibitor α2-antiplasmin (α2-AP). In this study, we engineered and selected mutant forms of µPlm that are both catal ...[more]

PMID: 32694536

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Project description:AimsThe accumulation and deposition of β-amyloid (Aβ) has always been considered a major pathological feature of Alzheimer's disease (AD). The latest and mainstream amyloid cascade hypothesis indicates that all the main pathological changes in AD are attributed to the accumulation of soluble Aβ. However, the exploration of therapeutic drugs for Aβ toxicity has progressed slowly. This study aims to investigate the protective effects of Icaritin on the Aβ-induced Drosophila AD model and its possible mechanism.MethodsTo identify the effects of Icaritin on AD, we constructed an excellent Drosophila AD model named Aβarc (arctic mutant Aβ42) Drosophila. Climbing ability, flight ability, and longevity were used to evaluate the effects of Icaritin on AD phenotypes. Aβarc was determined by immunostaining and ELISA. To identify the effects of Icaritin on oxidative stress, we performed the detection of ROS, hydrogen peroxide, MDA, SOD, catalase, GST, and Caspase-3. To identify the effects of Icaritin on energy metabolism, we performed the detection of ATP and lactate. Transcriptome analysis and qRT-PCR verifications were used to detect the genes directly involved in oxidative stress and energy metabolism. Mitochondrial structure and function were detected by an electron microscopy assay, a mitochondrial membrane potential assay, and a mitochondrial respiration assay.ResultsWe discovered that Icaritin almost completely rescues the climbing ability, flight ability, and longevity of Aβarc Drosophila. Aβarc was dramatically reduced by Icaritin treatment. We also found that Icaritin significantly reduces oxidative stress and greatly improves impaired energy metabolism. Importantly, transcriptome analysis and qRT-PCR verifications showed that many key genes, directly involved in oxidative stress and energy metabolism, are restored by Icaritin. Next, we found that Icaritin perfectly restores the integrity of mitochondrial structure and function damaged by Aβarc toxicity.ConclusionThis study suggested that Icaritin is a potential drug to deal with the toxicity of Aβarc, at least partially realized by restoring the mitochondria/oxidative stress/energy metabolism axis, and holds potential for translation to human AD.

Dataset Information

Selection of mutant µplasmin for amyloid-? cleavage in vivo.

Publications

Selection of mutant µplasmin for amyloid-β cleavage in vivo.

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