Direct protein\u2013protein interaction between Npas4 and IPAS mutually inhibits their critical roles in neuronal cell survival and death

Shuya Kasai,Kazuhiro Sogawa,Xianyu Li,Satoru Torii,Ken-Ichi Yasumoto

doi:10.1038/s41420-021-00690-y

Abstract

Inhibitory PAS domain protein (IPAS) is a bifunctional protein that acts as a transcriptional repressor in hypoxia and as a pro-apoptotic protein involved in neuronal cell death. Npas4 (NXF or LE-PAS) is a transcriptional factor that protects nerve cells from endogenous and foreign neurotoxins. Here we show that IPAS and Npas4 antagonize each other through their direct interaction. Coimmunoprecipitation experiments revealed that multiple binding sites on each protein were involved in the interaction. CoCl2 treatment of PC12 cells that induces IPAS repressed the transactivation activity of Npas4, and IPAS siRNA treatment reduced the CoCl2-induced repression. CoCl2-induced apoptosis was suppressed by the addition of KCl that induces Npas4. The protective effect of KCl was attenuated by siRNA-mediated gene silencing of Npas4. Npas4 and IPAS proteins were induced and localized in the cytoplasm of the dopaminergic neurons in the substantia nigra pars compacta after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Npas4−/− mice exhibited greater sensitivity to MPTP in nigral dopaminergic neurons. Together, these results strongly suggest that neuroprotective activity of Npas4 was, at least partly, exerted by inhibiting the pro-apoptotic activity of IPAS through direct interaction.

Highlights

Inhibitory PAS domain protein (IPAS) was originally found as a negative regulator of HIF-1 [1], a master transcriptional regulator of numerous genes under hypoxic conditions [2]
Interaction between IPAS and Npas4 We investigated direct interaction between Myc-IPAS and FLAGNpas4 that were overexpressed in HEK293T cells
Further deletions of the N-terminal segment of Npas4 strongly suggested that basic helixloop-helix (bHLH), PAS A, and PAS B domains, which are involved in dimerization with Arnt2, were used for binding to IPAS (Fig. 1E)

Summary

Introduction

Inhibitory PAS domain protein (IPAS) was originally found as a negative regulator of HIF-1 [1], a master transcriptional regulator of numerous genes under hypoxic conditions [2]. HIF-1 is composed of an oxygen-dependent HIF-1α subunit and a constitutive Arnt (HIF-1β) subunit [3, 4]. Both HIF-1α and Arnt are protein family members that are characterized by a basic helixloop-helix (bHLH) motif and two PAS (PAS A and PAS B) domains, both of which are required for their dimerization. IPAS is a splicing variant of HIF-3α, which shows sequence similarity to HIF-1α in the bHLH and PAS domains. IPAS contains the same bHLH sequence as that of HIF-3α and one PAS A-like domain modified by alternative splicing, but lacks the PAS B domain [5]. IPAS represses HIF-1 transactivation activity by directly interacting with HIF-1α and abrogating its DNA binding activity

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Results

Conclusion