A Redox-Sensitive Cysteine Is Required for PIN1At Function.

Benjamin Selles,Jérémy Couturier,Nicolas Rouhier,Alexis Boutilliat,Tiphaine Dhalleine

doi:10.3389/fpls.2021.735423

Abstract

Parvulins are ubiquitous peptidyl-prolyl isomerases (PPIases) required for protein folding and regulation. Among parvulin members, Arabidopsis PIN1At, human PIN1, and yeast ESS1 share a conserved cysteine residue but differ by the presence of an N-terminal WW domain, absent in PIN1At. In this study, we have explored whether the cysteine residue of Arabidopsis PIN1At is involved in catalysis and subject to oxidative modifications. From the functional complementation of yeast ess1 mutant, we concluded that the cysteine at position 69 is mandatory for PIN1At function in vivo, unless being replaced by an Asp which is found in a few parvulin members. This result correlates with a decrease of the in vitro PPIase activity of non-functional PIN1At cysteinic variants. A decrease of PIN1At activity was observed upon H2O2 treatment. The in vitro oxidation of cysteine 69, which has an acidic pKa value of 4.9, leads to the formation of covalent dimers that are reduced by thioredoxins, or to sulfinic or sulfonic acid forms at higher H2O2 excess. These investigations highlight the importance of the sole cysteine residue of PIN1At for activity. The reversible formation of an intermolecular disulfide bond might constitute a protective or regulatory mechanism under oxidizing conditions.

Highlights

The cis-trans isomerization of the peptide bond between a prolyl residue and the preceding amino acid (Xaa-Pro) is considered as a molecular switch involved notably in the regulation of protein function (Lu et al, 2007)
It was previously reported that plant parvulins were able to complement the yeast ess1 strains even though they lack the N-terminal WW domain (Metzner et al, 2001; Yao et al, 2001)
The Cys69 was substituted to an aspartate, mimicking the residue present in a few isoforms, and to serine and asparagine to compare with the work conducted on human PIN1 (Behrsin et al, 2007)

Summary

Introduction

The cis-trans isomerization of the peptide bond between a prolyl residue and the preceding amino acid (Xaa-Pro) is considered as a molecular switch involved notably in the regulation of protein function (Lu et al, 2007). Parvulins are widespread proteins that were initially identified in Escherichia coli as PAR10 (or PpiC) protein (Rahfeld et al, 1994). An alternative initiation transcription site for the PIN4 gene leads to the synthesis of a second isoform referred to as PAR17 protein, which is targeted to mitochondria due to the presence of an N-terminal matrix targeting sequence (Figure 1; Mueller et al, 2006; Kessler et al, 2007). Three genes encode proteins with a parvulin domain (PAR1/PIN1At, PAR2/PIN2 and PAR3/STR12; Kouri et al, 2009; He, 2012; Selles et al, 2019; Moseler et al, 2020)

Methods

Results

Discussion

Conclusion