WTAP and BIRC3 are involved in the posttranscriptional mechanisms that impact on the expression and activity of the human lactonase PON2

Teresa Maria Carusone,Giovanna Cardiero,Giuseppina Lacerra,Giuliana Catara,Luigi Mandrich,Oscar Moran,Elena Porzio,Giuseppe Manco,Mariangela Cerreta

doi:10.1038/s41419-020-2504-2

Abstract

The activity of human paraoxonase 2 (PON2) is rapidly reduced in cells incubated with the bacterial quorormone 3-Oxo-dodecanoyl Homoserine Lactone (3OC12HSL), an observation that led to hypothesize a fast PON2 post-translational modification (PTM). Recently, we detected a 3OC12HSL-induced PTM in a cell-free system in which a crude extract from 3OC12HSL-treated HeLa cells was able to inactivate and ubiquitinate at position 144 a recombinant PON2. Here we show the occurrence of this and new PTMs on PON2 in HeLa cells. PTMs were found to gather nearby the two SNPs, A148G, and S311C, that are related to type-2 diabetes and its complications. Furthermore, we detected a PTM nearby a 12 amino acids region that is deleted in PON2 Isoform 2. An in vitro mutation analysis showed that the SNPs and the deletion are involved in PON2 activity and suggested a role of PTMs on its modulation, while a SAXS analysis pointed to Isoform 2 as being largely unstructured, compared to the wild type. Besides, we discovered a control of PON2 expression via a putative mRNA operon involving the Wilms tumor 1 associated protein (WTAP) and the E3 ubiquitin ligase (E3UbL) baculoviral IAP repeat-containing 3 (BIRC3).

Highlights

The human paraoxonase (PON) family comprises three highly conserved lactonases: PON1, paraoxonase 2 (PON2), and PON31,2
Based on the analysis of the regulatory function of the 3′ untranslated (3′ UTRs) sequences[21], we identified a cluster of 21 human genes comprising PON2 characterized by the sharing at the 3′ UTR leader of the conserved sequence: UUUCCUUAAAAU21,26 (Supplementary Table 6)
PON2 expression in HeLa and A549 cell lines Aiming at studying posttranslational modification (PTM) of PON2 we first analyzed the

Summary

Introduction

The human paraoxonase (PON) family comprises three highly conserved lactonases: PON1, PON2, and PON31,2. For the mutant rPON2 S311C we have mutagenized the residue 311 into C and used as mutagenic primers the following complementary pairs of oligonucleotides: PON2-S311C forward 5′-gcatccagaacattctatgtgagaagccta cagtga-3′; Fig. 1 PON2 presents different isoforms (RNA and proteins) in HeLa cells. B Western Blot analysis with anti-PON2 antibody of HeLa cells detergents-treated soluble extracts.

Results

Conclusion