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

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.

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Structural basis of human γ-secretase assembly

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1506242112 ◽

2015 ◽

Vol 112 (19) ◽

pp. 6003-6008 ◽

Cited By ~ 82

Author(s):

Linfeng Sun ◽

Lingyun Zhao ◽

Guanghui Yang ◽

Chuangye Yan ◽

Rui Zhou ◽

...

Keyword(s):

3D Structure ◽

Structural Basis ◽

Wild Type ◽

Transmembrane Region ◽

Electron Cryomicroscopy ◽

Amino Terminal ◽

Transmembrane Segments ◽

Terminal Fragment ◽

Carboxyl Terminal ◽

Amino Terminal Fragment

The four-component intramembrane protease γ-secretase is intricately linked to the development of Alzheimer’s disease. Despite recent structural advances, the transmembrane segments (TMs) of γ-secretase remain to be specifically assigned. Here we report a 3D structure of human γ-secretase at 4.32-Å resolution, determined by single-particle, electron cryomicroscopy in the presence of digitonin and with a T4 lysozyme fused to the amino terminus of presenilin 1 (PS1). The overall structure of this human γ-secretase is very similar to that of wild-type γ-secretase determined in the presence of amphipols. The 20 TMs are unambiguously assigned to the four components, revealing principles of subunit assembly. Within the transmembrane region, PS1 is centrally located, with its amino-terminal fragment (NTF) packing against Pen-2 and its carboxyl-terminal fragment (CTF) interacting with Aph-1. The only TM of nicastrin associates with Aph-1 at the thick end of the TM horseshoe, and the extracellular domain of nicastrin directly binds Pen-2 at the thin end. TM6 and TM7 in PS1, which harbor the catalytic aspartate residues, are located on the convex side of the TM horseshoe. This structure serves as an important framework for understanding the function and mechanism of γ-secretase.

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Evolution of the hedgehog Gene Family

Genetics ◽

10.1093/genetics/142.3.965 ◽

1996 ◽

Vol 142 (3) ◽

pp. 965-972 ◽

Cited By ~ 2

Author(s):

Sudhir Kumar ◽

Kristi A Balczarek ◽

Zhi-Chun Lai

Keyword(s):

Intercellular Communication ◽

Short Range ◽

Gene Duplications ◽

Future Directions ◽

Amino Terminal ◽

Terminal Fragment ◽

Carboxyl Terminal ◽

Multicellular Organisms ◽

Amino Terminal Fragment

Abstract Effective intercellular communication is an important feature in the development of multicellular organisms. Secreted hedgehog (hh) protein is essential for both long- and short-range cellular signaling required for body pattern formation in animals. In a molecular evolutionary study, we find that the vertebrate homologs of the Drosophila hh gene arose by two gene duplications: the first gave rise to Desert hh, whereas the second produced the Indian and Sonic hh genes. Both duplications occurred before the emergence of vertebrates and probably before the evolution of chordates. The amino-terminal fragment of the hh precursor, crucial in long- and short-range intercellular communication, evolves two to four times slower than the carboxyl-terminal fragment in both Drosophila hh and its vertebrate homologues, suggesting conservation of mechanism of hh action in animals. A majority of amino acid substitutions in the amino- and carboxyl-terminal fragments are conservative, but the carboxyl-terminal domain has undergone extensive insertion-deletion events while maintaining its autocleavage protease activity. Our results point to similarity of evolutionary constraints among sites of Drosophila and vertebrate hh homologs and suggest some future directions for understanding the role of hh genes in the evolution of developmental complexity in animals.

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