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Brazilin Inhibits Prostatic Acidic Phosphatase Fibrillogenesis and Decreases its Cytotoxicity
Ming Li1, Xiaoyan Dong1, Yang Liu2
1Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300354, P. R. China.
Abstract:
A 39-amino acid peptide fragment that is derived from prostatic acidic phosphatase (PAP), PAP248-286 , is secreted in large amounts in human semen and forms amyloid fibrils. These fibrils can capture HIV virions and increase the attachment of virions to target cells; as such, they are called a "semen-derived enhancer of virus infection" (SEVI). Therefore, the inhibition of the formation of PAP248-286 amyloid fibrils is of great significance. Herein, we demonstrate that brazilin effectively inhibits PAP248-286 aggregation. The inhibitory effect increases with increasing brazilin concentration. Thioflavin T fluorescence assays and TEM observations confirmed that a few fibrils formed when brazilin was present with PAP248-286 in an equimolar concentration. Circular dichroism spectroscopy indicated that brazilin inhibited the secondary structural transitions from α-helices and random coils into β-sheets. Cytotoxicity assays showed that brazilin significantly decreased the cytotoxicity of the fibrils at 0.01 mmol L-1 . Isothermal titration calorimetry revealed that hydrophobic interactions were the main driving force for the binding of brazilin to the PAP248-286 monomer (dissociation constant, 4.03 μmol L-1 ), and that the binding affinity of brazilin for the fibrils was at least three orders of magnitude lower than that for the monomer. These results indicate that brazilin holds great potential as a small-molecule agent against SEVIs.
Insights
Brazilin effectively inhibits the formation of semen-derived enhancer of virus infection (SEVI) amyloid fibrils, which enhance HIV attachment. This small molecule shows potential as an agent against SEVI, reducing its cytotoxicity.
Area of Science:
- Biochemistry
- Virology
- Drug Discovery
Background:
- Prostatic acidic phosphatase (PAP) fragment PAP248-286 forms amyloid fibrils known as SEVI.
- SEVI enhances HIV capture and attachment to target cells, posing a public health concern.
- Inhibiting SEVI fibril formation is crucial for developing antiviral strategies.
Purpose of the Study:
- To investigate the potential of brazilin as an inhibitor of PAP248-286 amyloid fibril formation.
- To elucidate the mechanism by which brazilin affects SEVI aggregation and cytotoxicity.
Main Methods:
- Thioflavin T fluorescence assays and Transmission Electron Microscopy (TEM) to observe fibril formation.
- Circular Dichroism (CD) spectroscopy to analyze secondary structural changes.
- Cytotoxicity assays to evaluate the effect of brazilin on SEVI-induced toxicity.
- Isothermal Titration Calorimetry (ITC) to determine binding interactions between brazilin and PAP248-286.
Main Results:
- Brazilin demonstrated dose-dependent inhibition of PAP248-286 aggregation.
- Equimolar concentrations of brazilin and PAP248-286 resulted in minimal fibril formation.
- CD spectroscopy revealed that brazilin prevented the transition to β-sheets, stabilizing α-helices and random coils.
- Brazilin significantly reduced SEVI fibril cytotoxicity at 0.01 mmol/L.
- ITC showed strong binding of brazilin to PAP248-286 monomers via hydrophobic interactions (Kd = 4.03 μmol/L), with much lower affinity for fibrils.
Conclusions:
- Brazilin effectively inhibits the formation of SEVI amyloid fibrils.
- Brazilin acts by preventing the structural transitions necessary for fibrillogenesis.
- Brazilin reduces the infectivity-enhancing and cytotoxic effects of SEVI.
- Brazilin shows significant promise as a small-molecule therapeutic agent against SEVI.
