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Published on: October 27, 2014
Paclitaxel binding to human and murine MD-2
Shanta M Zimmer1, Jin Liu2, Jaime L Clayton2
1Department of Chemistry, Emory University, Atlanta, Georgia 30322.
Abstract:
Paclitaxel (PTX) is an important cancer chemotherapeutic agent that binds to beta-tubulin and prevents mitosis through microtubule overstabilization. Recent evidence also implicates PTX in the induction of apoptosis of cancer cells via the TLR4 innate immune pathway. The TLR4 accessory protein, MD-2, is an essential component for the species-specific proinflammatory activity of PTX on murine cells. However, whether PTX binds to human MD-2 and how MD-2 and TLR4 interact with PTX are not well defined. Recombinant human MD-2 (rhMD-2) was produced in a Pichia pastoris expression system, and the interaction between rhMD-2 and PTX was assessed by an enzyme-linked immunosorbent assay to show that PTX binds rhMD-2. Formation of the latter complex was found to be dose-dependent and inhibited by anti-MD-2 antibody but not by an isotype control antibody. As measured by human tumor necrosis factor alpha production, human THP-1 monocytes expressing TLR4 and MD-2 were poorly responsive to the addition of PTX, but murine macrophages expressing TLR4 and MD-2 responded in a dose-dependent manner. Human embryonic kidney (HEK293) cells transfected with both human TLR4 and human MD-2 or human MD-2 and murine TLR4 were also poorly responsive to PTX (10 microm). However, HEK293 cells transfected with murine MD-2 and human TLR4 or murine MD-2 and murine TLR4 were highly responsive to PTX (10 microm), indicating that the murine MD-2/PTX interaction is required for TLR4 activation. To further define the structural differences for MD-2/TLR4 activation, crystal structures of both murine and human MD-2 were subjected to PTX docking by computational methods. These models indicate that PTX binds in the pocket of both human and mouse MD-2 structures. The species-specific difference between human and murine MD-2 activation of TLR4 by PTX can be explained by alterations of surface charge distribution (i.e. electrostatic potential), binding pocket size, and the locus of PTX binding within the MD-2 pocket, which results in reorganization of the 123-130 amino acid loop. In particular, Phe(126) appears to operate as a bridge for TLR4.MD-2 dimerization in the mouse but not the human protein.
Insights
Paclitaxel (PTX) activates the Toll-like receptor 4 (TLR4) innate immune pathway through binding to MD-2, but this interaction is species-specific. Murine MD-2 is required for PTX-induced TLR4 activation in humans, unlike in mice.
Area of Science:
- Immunology
- Molecular Biology
- Pharmacology
Background:
- Paclitaxel (PTX) is a chemotherapy drug that targets beta-tubulin and induces apoptosis via the Toll-like receptor 4 (TLR4) pathway.
- MD-2 is a crucial accessory protein for TLR4 activation, and its interaction with PTX is known to be species-specific in murine cells.
- The precise binding of PTX to human MD-2 and the subsequent TLR4 interaction remain incompletely understood.
Purpose of the Study:
- To investigate whether Paclitaxel (PTX) binds to human MD-2.
- To elucidate the interaction mechanisms between MD-2, TLR4, and PTX.
- To identify the structural basis for the species-specific activation of TLR4 by PTX.
Main Methods:
- Produced recombinant human MD-2 (rhMD-2) using a Pichia pastoris expression system.
- Assessed PTX-rhMD-2 binding using enzyme-linked immunosorbent assay (ELISA).
- Evaluated TLR4 activation by measuring human tumor necrosis factor alpha production in THP-1 monocytes and HEK293 cells transfected with various combinations of human and murine TLR4 and MD-2.
- Performed computational docking of PTX onto crystal structures of murine and human MD-2.
Main Results:
- Paclitaxel (PTX) directly binds to recombinant human MD-2 (rhMD-2) in a dose-dependent manner, inhibited by anti-MD-2 antibodies.
- Human monocytes and HEK293 cells expressing human MD-2 showed poor responsiveness to PTX, while murine cells responded robustly.
- HEK293 cells transfected with murine MD-2 and human TLR4 were highly responsive to PTX, indicating murine MD-2 is essential for PTX-induced TLR4 activation.
- Computational models revealed PTX binds within the MD-2 pocket in both species, but differences in electrostatic potential, pocket size, and Phe(126) location explain species-specific TLR4 activation.
Conclusions:
- Paclitaxel (PTX) binds to both human and murine MD-2, but the interaction with murine MD-2 is critical for activating the TLR4 pathway.
- Species-specific differences in MD-2 structure, particularly involving Phe(126), dictate the ability of PTX to bridge MD-2 and TLR4 for dimerization and activation.
- Understanding these molecular interactions provides insights into the differential immune responses to PTX across species.
