Structure and dynamics of Smoothened (SMO)– a potential target for regulation of the Hedgehog (HH) signaling pathway

Abstract

Hedgehog (HH) signaling pathway is the cellular pathway for patterning and morphogenesis. It is controlled by two membrane proteins, the HH receptor Patched1 (PTCH1), a 12-pass transmembrane (TM) protein, and Smoothened (SMO), a 7-pass transmembrane (TM) signal transducer. SMO transduces the signal from the extracellular (EC) region to the cytoplasmic (CP) region. Binding of HH ligand to PTCH1 receptor allows SMO entry to the primary cilium (PC) and activates the downstream signaling process. The malfunctioning of SMO results in misregulation of the HH signaling pathway, which contributes to birth defects and various cancers. The story has evolved from initial conjectures involving direct communication of PTCH1 with SMO to indirect communication between the two TM proteins. In the process, some of the old theories have been discarded in recent literature. However, the underlying mechanism of how PTCH1 controls SMO is still not very clear, and thus constitutes an important area of research. Based on available evidence, the consensus appears to be limited to the assertion that, driven by HH signaling, PTCH1 regulates the activity of SMO activity through the intermediacy of some small molecules (such as cholesterol). In addition, it appears that the lipid bilayer composition affects SMO orientation and function. In this context, several earlier hypotheses involving the primary role of cholesterol are being disputed. At the same time, several HH pathway inhibitors viz., cyclopamine, taladegib, and vismodegib, which target SMO have been reported. Notwithstanding their potential utility in the treatment of cancer, long-term administration of these drugs was reported to lead to the development of resistance. The broad aim of my research is to build upon the existing molecular-level understanding of the functioning of the HH pathway, with a view to facilitate the formulation of newer approaches towards therapeutic intervention. The focus of my research is to investigate the structure and domain organization of SMO, which is a class F GPCR. This thesis attempts to provide some insights into the functioning of the SMO receptor, using molecular dynamics-based approaches. It is shown that SMO is activated by cholesterol molecules and that its dynamics is influenced by other membrane lipids such as PI4P. The specific objectives include 1. To identify important motifs, especially including, but not limited to, the lipid-binding sites/motifs. 2. To study the conformational dynamics of SMO, to help understand its molecular behavior and also the role of the composition of the primary cilia. 3. To study the regulation mechanisms in SMO by exploring the different cholesterol-bound structures of SMO.