RESUMEN
Human Raf1 kinase inhibitory protein (hRKIP) is an important modulator of the Ras/Raf1/MEK/ERK signaling pathway. Here, we demonstrated that anti-leprosy drug Clofazimine can bind to hRKIP with a significantly stronger affinity than the endogenous substrate phosphatidylethanolamine (PE) by using Biolayer interference technology. Moreover, we identified that residues P74, S75, K80, P111, P112, V177, and P178 play crucial roles in the binding of hRKIP to Clofazimine by using a combination of Nuclear Magnetic Resonance spectroscopy and molecular docking approach. These residues are located at the conserved ligand-binding pocket of hRKIP. Furthermore, we found that 3.2 µM Clofazimine could significantly increase the ERK phosphorylation level by about 37%. Our results indicate that Clofazimine can enhance Ras/Raf1/MEK/ERK signaling transduction pathway via binding to hRKIP. This work provides valuable hints for exploiting Clofazimine as a potential lead compound to efficiently treat the diseases related to RKIP or the Ras/Raf/MEK/ERK pathway.
Asunto(s)
Clofazimina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Unión Competitiva , Clofazimina/química , Clofazimina/farmacología , Células HEK293 , Humanos , Leprostáticos/química , Leprostáticos/metabolismo , Leprostáticos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Estructura Molecular , Proteínas de Unión a Fosfatidiletanolamina/química , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Dominios ProteicosRESUMEN
BACKGROUND: Advanced stages of leprosy show T cell unresponsiveness and lipids of mycobacterial origin are speculated to modulate immune responses in these patients. Present study elucidates the role of phenolicglycolipid (PGL-1) and Mannose-capped lipoarabinomannan (Man-LAM) on TCR- and TCR/CD28- mediated signalling. RESULTS: We observed that lipid antigens significantly inhibit proximal early signalling events like Zap-70 phosphorylation and calcium mobilization. Interestingly, these antigens preferentially curtailed TCR-triggered early downstream signalling events like p38 phosphorylation whereas potentiated that of Erk1/2. Further, at later stages inhibition of NFAT binding, IL-2 message, CD25 expression and T-cell blastogenesis by PGL-1 and Man-LAM was noted. CONCLUSION: Altogether, we report that Man-LAM and PGL-1 preferentially interfere with TCR/CD28-triggered upstream cell signalling events, leading to reduced IL-2 secretion and T-cell blastogenesis which potentially could lead to immunosupression and thus, disease exacerbation, as noted in disease spectrum.