Stepwise Strategy to Identify Thrombin as a Hydrolytic Substrate for Nattokinase

Area of Science:

• Biochemistry
• Molecular Biology
• Cardiovascular Research

Background:

• Nattokinase (NK) is a serine protease with significant thrombolytic and cardiovascular benefits.
• Understanding NK's molecular mechanisms, particularly its substrates, is crucial for developing novel cardiovascular disease (CVD) therapies.
• NK's role in mitigating inflammation, oxidative stress, and thrombosis highlights the importance of identifying its targets, like thrombin.

Purpose of the Study:

• To computationally identify and experimentally validate novel biological substrates of Nattokinase (NK).
• To elucidate the molecular mechanism underlying NK's thrombolytic and anticoagulant activities.
• To develop a robust computational strategy for predicting protease-cleavage sites and substrate interactions.

Main Methods:

• A stepwise computational approach combining protein-protein complex modeling for substrate recognition and protease-peptide complex modeling for cleavage site identification.
• Mass spectrometry (MS)-based N-terminal sequencing to verify predicted cleavage sites.
• Functional assays to confirm the biological activity and substrate interaction.

Main Results:

• Thrombin was computationally predicted and experimentally identified as a novel biological substrate of Nattokinase (NK).
• This finding expands the known antithrombotic mechanisms of NK, particularly its role in disrupting the inflammation-oxidative stress-thrombosis cycle.
• The study provides detailed insights into the interaction between NK and its substrate, thrombin.

Conclusions:

• Nattokinase (NK) directly targets and cleaves thrombin, offering a new therapeutic avenue for cardiovascular diseases.
• The developed computational strategy is effective for identifying protease substrates and can be applied to discover other NK targets.
• This research enhances our understanding of NK's catalytic mechanism and its comprehensive antithrombotic effects.