Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1.

Wesley Tucker,Lewis Fraser,Yee-Wai Cheung,Julian Tanner,Andrew Kinghorn

doi:10.3390/ijms19030763

Wesley Tucker, Lewis Fraser + Show 3 more

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https://doi.org/10.3390/ijms19030763

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Abstract

Nucleic acid aptamers hold promise as therapeutic tools for specific, tailored inhibition of protein targets with several advantages when compared to small molecules or antibodies. Nuclear WW domain containing E3 ubiquitin ligase 1 (WWP1) ubiquitin ligase poly-ubiquitinates Runt-related transcription factor 2 (Runx2), a key transcription factor associated with osteoblast differentiation. Since WWP1 and an adapter known as Schnurri-3 are negative regulators of osteoblast function, the disruption of this complex has the potential to increase bone deposition for osteoporosis therapy. Here, we develop new DNA aptamers that bind and inhibit WWP1 then investigate efficacy in an osteoblastic cell culture. DNA aptamers were selected against three different truncations of the HECT domain of WWP1. Aptamers which bind specifically to a C-lobe HECT domain truncation were observed to enrich during the selection procedure. One particular DNA aptamer termed C3A was further evaluated for its ability to bind WWP1 and inhibit its ubiquitination activity. C3A showed a low µM binding affinity to WWP1 and was observed to be a non-competitive inhibitor of WWP1 HECT ubiquitin ligase activity. When SaOS-2 osteoblastic cells were treated with C3A, partial localization to the nucleus was observed. The C3A aptamer was also demonstrated to specifically promote extracellular mineralization in cell culture experiments. The C3A aptamer has potential for further development as a novel osteoporosis therapeutic strategy. Our results demonstrate that aptamer-mediated inhibition of protein ubiquitination can be a novel therapeutic strategy.

Highlights

In mammalian bone, the osteoblast is exclusively responsible for the deposition of bone matrix in the form of hydroxyapatite crystals together with various structural proteins [1]
As it is not possible to express the full length WWP1 protein in E. coli, truncations of the smaller yet active HECT domain were used as the targets for aptamer selection
The HECT domain has two lobes: the N-lobe is involved in E2 binding and the C-lobe is important in ubiquitination [8]

Summary

Introduction

The osteoblast is exclusively responsible for the deposition of bone matrix in the form of hydroxyapatite crystals together with various structural proteins [1]. The Nedd family HECT (‘Homologous to the E6-AP Carboxyl Terminus’) domain E3 ligase WWP1, in complex with an adapter Schnurri-3 (Shn3), ubiquitinates Runx leading to its degradation and reduction of osteoblast-mediated bone matrix synthesis [2]. WWP1 and Shn proteins negatively regulate Runx at the protein level, are promising targets to stimulate osteoblast differentiation, and by extension, higher bone mass in osteoporosis patients [4,5]. WWP1 is structurally and functionally characterized, is readily “expressible” in E. coli, and inhibition of WWP1 to maintain Runx levels is a promising therapeutic strategy to stimulate osteoblast function

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