Highly Conserved C-Terminal Region of Indian Hedgehog N-Fragment Contributes to Its Auto-Processing and Multimer Formation.

Xiaoqing Wang,Lin He,Hao Liu,Yushu Wang,Gang Ma,Haifeng Chen,Gefei Han,Yanfang Liu

doi:10.3390/biom11060792

Abstract

Hedgehog (HH) is a highly conserved secretory signalling protein family mainly involved in embryonic development, homeostasis, and tumorigenesis. HH is generally synthesised as a precursor, which subsequently undergoes autoproteolytic cleavage to generate an amino-terminal fragment (HH-N), mediating signalling, and a carboxyl-terminal fragment (HH-C), catalysing the auto-processing reaction. The N-terminal region of HH-N is required for HH multimer formation to promote signal transduction, whilst the functions of the C-terminal region of HH-N remain ambiguous. This study focused on Indian Hedgehog (IHH), a member of the HH family, to explore the functions of the C-terminal region of the amino-terminal fragment of IHH (IHH-N) via protein truncation, cell-based assays, and 3D structure prediction. The results revealed that three amino acids, including S195, A196, and A197, were crucial for the multimer formation by inserting the mutual binding of IHH-N proteins. K191, S192, E193, and H194 had an extremely remarkable effect on IHH self-cleavage. In addition, A198, K199, and T200 evidently affected the stability of IHH-N. This work suggested that the C-terminus of IHH-N played an important role in the physiological function of IHH at multiple levels, thus deepening the understanding of HH biochemical properties.

Highlights

Introduction published maps and institutional affilThe Hedgehog (HH) family is well known for its essential role in the normal embryonic development of invertebrates and vertebrates and the maintenance of adult tissue homeostasis [1,2]
Human Indian Hedgehog (IHH) cDNA was cloned and inserted into the pIND (Sp1)-based inducible expression vector (Invitrogen, Carlsbad, CA, USA) with double FLAG-tags were introduced into the IHH-N as described previously [15], or the pCMV-based expression vector (Promega, Madison, WI, USA) with double FLAG-tags were introduced into the IHH-C
S195, A196, and A197 Are Involved in Multimerization of IHH-N Protein by Maintaining

Summary

Introduction

The Hedgehog (HH) family is well known for its essential role in the normal embryonic development of invertebrates and vertebrates and the maintenance of adult tissue homeostasis [1,2]. Hedgehog (SHH), Indian Hedgehog (IHH), and Desert Hedgehog (DHH) [5]. Their expression patterns and physiological functions are different, their protein processing and signal transduction exhibit high similarities. HH is regularly synthesised as a precursor in the endoplasmic reticulum of the producing cells, and it undergoes autoproteolysis to generate a functional HH-N, mediating signalling, and a HH-C, catalysing the auto-processing reaction [6,7]. A cholesterol molecule attacks the self-cleavage site to promote the self-cleavage reaction, and binds iations

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Conclusion