The mycosporine-like amino acids porphyra-334 and shinorine are antioxidants and direct antagonists of Keap1-Nrf2 binding

Ranko Gacesa,Karl P Lawrence,Nikolaos D Georgakopoulos,Kazuo Yabe,Walter C Dunlap,David J Barlow,Geoffrey Wells,Antony R Young,Paul F Long

doi:10.1016/j.biochi.2018.07.020

Abstract

Mycosporine-like amino acids (MAAs) are UVR-absorbing metabolites typically produced by cyanobacteria and marine algae, but their properties are not limited to direct sun screening protection. Herein, we examine the antioxidant activities of porphyra-334 and shinorine and demonstrate that these MAAs are prospective activators of the cytoprotective Keap1-Nrf2 pathway. The ability of porphyra-334 and shinorine to bind with Keap1 was determined using fluorescence polarization (FP) and thermal shift assays to detect Keap1 receptor antagonism. Concomitantly, the ability of porphyra-334 and shinorine to dissociate Nrf2 from Keap1 was confirmed also by measurement of increased mRNA expression of Nrf2 targeted genes encoding oxidative stress defense proteins in primary skin fibroblasts prior and post UVR exposure. Surprisingly, enhanced transcriptional regulation was only promoted by MAAs in cells after exposure to UVR-induced oxidative stress. Furthermore, the in-vitro antioxidant activities of porphyra-334 and shinorine determined by the DPPH free-radical quenching assay were low in comparison to ascorbic acid. However, their antioxidant capacity determined by the ORAC assay to quench free radicals via hydrogen atom transfer is substantial. Hence, the dual nature of MAAs to provide antioxidant protection may offer a prospective chemotherapeutic strategy to prevent or retard the progression of multiple degenerative disorders of ageing.

Highlights

The Kelch-like ECH-associated protein 1 (Keap1) is an actin bound homodimer that functions as a primary sensor of intracellular reduction-oxidation state regulation by controlling the activity of the master transcription nuclear factor erythroid 2erelated factor 2 protein (Nrf2), which regulates the transcription of a large number of genes under control of the cis-acting enhancer termed the antioxidant response element (ARE) [1,2]
Mycosporine-like amino acids (MAAs) Porphyra-334 and shinorine compete with Keap1-Nrf2 interaction in vitro
The MAAs porphyra-334 and shinorine together with eight selected antioxidants plus the known electrophilic Nrf2 activator sulforaphane were evaluated for their interaction with the Kelch-repeat domain of the Keap1 protein using both in vitro fluorescence polarization (FP) and thermal shift assays

Summary

Introduction

The Kelch-like ECH-associated protein 1 (Keap1) is an actin bound homodimer that functions as a primary sensor of intracellular reduction-oxidation (redox) state regulation by controlling the activity of the master transcription nuclear factor erythroid 2erelated factor 2 protein (Nrf2), which regulates the transcription of a large number of genes under control of the cis-acting enhancer termed the antioxidant response element (ARE) [1,2]. Nrf is targeted for ubiquitination and rapid 26S proteasomal degradation by Keap BTB domain bound Cullin3-Rbx E3 ubiquitin ligase (CRLKeap). During conditions of oxidative stress, the ubiquitination and degradation of Nrf by CRLKeap is disrupted by Nrf2-Keap dissociation. In the conformation cycling model, it is proposed that, in the presence of cellular oxidants and exogenous electrophiles, covalent modification of Cys151 in the BTB domain of Keap causes conformational changes that prevent ubiquitination of Nrf by the CRLKeap protein complex [6,7]. In the hinge and latch model, the Nrf2-Keap interaction is mediated by a high-affinity ETGE motif in the Neh domain of Nrf, which functions as a “hinge” by stabilising Nrf binding to the Kelch domain in the Keap dimer.

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