Alpha-helical destabilization of the Bcl-2-BH4-domain peptide abolishes its ability to inhibit the IP3 receptor.

Giovanni Monaco,Tomas Luyten,Ludwig Missiaen,Luc Leybaert,Elke Decrock,Wim M De Borggraeve,David I Yule,Sergei V Strelkov,Humbert De Smedt,Koen Nuyts,Larry E Wagner Ii,Geert Bultynck,Jan B Parys,Alexander G Obukhov

doi:10.1371/journal.pone.0073386

Giovanni Monaco, Tomas Luyten + Show 12 more

Open Access

https://doi.org/10.1371/journal.pone.0073386

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Abstract

The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the primary Ca2+-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP3R-mediated Ca2+ release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4) has been identified as essential and sufficient for this IP3R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP 3R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine “hinges” replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG). By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP 3R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP3R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP3R with significant pharmacological implications.

Highlights

Intracellular Ca2+ homeostasis requires a tight cross-talk between the endoplasmic reticulum (ER) and the mitochondria
Using SDSPAGE, we showed that the Bcl-2-BH4–peptide strongly interacted with GST-Domain 3 while Bcl-2-BH4 IV/GG lost most of its IP3R-binding properties (Figure 2A)
Our findings indicate that the α-helicity of Bcl-2-BH4 peptide is a key determinant for its ability to directly suppress IP3R signaling and Ca2+-mediated apoptosis

Summary

Introduction

Intracellular Ca2+ homeostasis requires a tight cross-talk between the endoplasmic reticulum (ER) and the mitochondria. We used a modified version of the Bcl-2-BH4 peptide that carries a change in two hydrophobic residues, which are part of the N-terminal α -helix of the native Bcl-2 but display a poor surface accessibility in the full-length protein [13,22,23] In this peptide, residues I14 and V15 have been replaced with two glycines to introduce high flexibility in the structure and destabilize helical conformation of the peptide. Residues I14 and V15 have been replaced with two glycines to introduce high flexibility in the structure and destabilize helical conformation of the peptide We applied this modified peptide in a series of functional experiments, addressing its ability to bind IP 3R1, inhibit single-channel activity, curb IICR and protect against Ca2+-dependent apoptosis. We further examined the relevance of the α-helical domain organization of the BH4 domain by introducing other mutations that reduce the α-helical content

Materials and Methods

Findings

Discussion