The antioxidant function of Sco proteins depends on a critical surface-exposed residue

https://doi.org/10.1016/j.bbagen.2020.129781Get rights and content

Highlights

  • The antioxidant function of eukaryotic Sco proteins is conserved irrespective of the extent of sequence homology.

  • The charge of the residue located 15 amino acid downstream of the conserved CxxxC domain affects oxidative stress defense.

  • This site is surface-exposed and required for binding of proteins that are connected to known antioxidant pathways.

Abstract

Background

Besides their role in copper metabolism, Sco proteins from different organisms have been shown to play a defensive role against oxidative stress. In the present study, we set out to identify crucial amino acid residues for the antioxidant activity.

Methods

Native and mutated Sco proteins from human, Arabidopsis thaliana and the yeast Kluyveromyces lactis were expressed in the model organism Saccharomyces cerevisiae. The oxidative stress resistance of the respective transformants was determined by growth and lipid peroxidation assays.

Results

A functionally important site, located 15 amino acids downstream of the well-conserved copper binding CxxxC motif, was identified. Mutational analysis revealed that a positive charge at this position has a detrimental effect on the antioxidant capacity. Bioinformatic analysis predicts that this site is surface-exposed, and according to Co-IP data it is required for binding of proteins that are connected to known antioxidant pathways.

Conclusion

This study shows that the antioxidant capacity of eukaryotic Sco proteins is conserved and depends on the presence of functional site(s) rather than the extent of overall sequence homology.

General significance

These findings provide an insight into the conserved functional sites of eukaryotic Sco proteins that are crucial for combating oxidative stress. This capacity is probably not due to an enzymatic activity but rather is indirectly mediated by interaction with other proteins.

Introduction

The evolutionary conserved Sco (synthesis of cytochrome c oxidase) proteins exist in all kingdoms of life. The number of SCO genes and their function may vary across organisms [1], but most eukaryotic Sco proteins share the following features: an N-terminal mitochondrial targeting sequence, a single transmembrane domain, a highly conserved CxxxC motif and histidine residue – crucial for copper binding – and a thioredoxin fold [2].

The first Sco protein (Sco1) was identified in the yeast Saccharomyces (S.) cerevisiae as an essential component for the assembly of the cytochrome c oxidase (COX) [3]. Subsequent studies in different organisms have shown divergent roles of Sco proteins including copper homeostasis [[4], [5], [6], [7]] and redox signaling [[8], [9], [10]]. Most recently, a role of Sco proteins in the defense against oxidative stress was reported [[11], [12], [13], [14]]. The presence of the thioredoxin fold in Sco proteins has long been pointed out as a structural homology to antioxidant enzymes [10,[15], [16], [17]]. However, the investigation of the antioxidant activity of Sco proteins is hampered by their concomitant role in the assembly of the respiratory chain. This difficulty can be overcome by using the facultative aerobic organism S. cerevisiae that can gain energy exclusively by glycolysis. Complementation studies in this model organism nicely figured out the conservation of the antioxidant role of eukaryotic Sco homologs among distant species independent of the extent of their sequence homology [14].

Structural characterization of human Sco proteins and the analysis of pathogenic mutations of human Sco2 led to the identification of several amino acid(s) (aa) with special importance for conformational stability as well as for physical and binding properties [10,18]. Subsequent analyses revealed the negative impact of the respective mutations on copper homeostasis [6], oxidoreductase activity [19] and antioxidant function [14].

In addition, several studies have emphasized the role of charged residues on both structural and functional features of Sco proteins and pointed out the contribution of salt bridges to the structure of human Sco proteins [15,16]. For instance, S. cerevisiae Sco1 interacts with the copper chaperone Cox17 and the Cox2 subunit of mitochondrial cytochrome c oxidase through complementary electrostatic surfaces [20].

In the present work, we aimed at investigating functionally important residues for the antioxidant role of eukaryotic Sco proteins. In the light of previous findings [14], we included the Sco homologs from Arabidopsis (A.) thaliana, human and Kluyveromyces (K.) lactis and analyzed the conserved site(s) by putting special emphasis on charged aa. Our results point out a novel functional site – 15 aa downstream of the CxxxC domain – which is crucial for oxidative stress defense. Mutagenesis data demonstrate charge-driven changes in the antioxidant function and propose a role to this conserved site in mediating electrostatic interactions.

Section snippets

Bioinformatic analysis

The protein sequences were retrieved from the UniProt database [21] and alignments were done by Clustal Omega [22]. The MitoFates tool [23] was used to predict mitochondrial targeting sequences. The transmembrane domain was predicted with TMpred [24] for SpSco and K07152; for the other Sco homologs this information was retrieved from the UniProt database.

The protein structures were retrieved from Protein Data Bank (PDB) and model structures were built by SWISS-MODEL [25] using the top-ranked

Identification of a critical aa site for antioxidant function by multiple sequence alignment of Sco proteins

In our previous study, we described the oxidative stress sensitivity of a double mutant yeast strain lacking SCO2 and SOD1 – encoding superoxide dismutase 1. We used this strain, ∆sco2sod1, to investigate the ability of different eukaryotic Sco proteins to rescue the oxidative stress sensitivity [14]. Based on these results, the analyzed Sco homologs were grouped into functional (ySco2, SpSco, hSco1, hSco2, Scox, HCC2) and non-functional (HCC1, K07152) homologs. The finding that the K. lactis

Discussion

The defensive role of eukaryotic Sco proteins against oxidative stress has been shown previously in a model system with a yeast strain harboring concomitantly a deletion of SCO2 and SOD1, Δsco2Δsod1 [14]. The similar phenotype of this mutant and of the Δtrx3Δsod1 mutant strain under oxidative stress has supported the idea of a possible thioredoxin-like activity of Sco proteins as predicted by structural analyses [49] and shown before for both prokaryotic Sco proteins [[50], [51], [52]] and

Author contributions

AEK and UG conceptualized the study; AEK designed and performed experiments; AEK, GR and UG validated and analyzed data; GR and UG supervised the project; AEK wrote the manuscript; GR and UG made manuscript revisions; GR acquired the funding.

Funding

This work was supported by the Dresden International Graduate School for Biomedicine and Bioengineering (DIGS-BB) funded by the German Research Foundation (DFG grant GSC 97) to AEK.

Declaration of Competing Interest

The authors state no conflict of interest.

Acknowledgements

We are grateful to Prof. Michael Schroeder and Dr. Sebastian Salentin for their assistance in bioinformatic analysis. We are thankful to MS facility of the Max Planck Institute of Molecular Biology and Genetics (MPI-CBG) for mass spectrometry and statistical analyses.

References (59)

  • G. Daum et al.

    Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria

    J. Biol. Chem.

    (1982)
  • A.J. McCoy et al.

    Electrostatic complementarity at protein/protein interfaces

    J. Mol. Biol.

    (1997)
  • C.S. Gibbs et al.

    Identification of functional residues in proteins by charged-to-alanine scanning mutagenesis

    Methods

    (1991)
  • J. Reinders et al.

    Profiling phosphoproteins of yeast mitochondria reveals a role of phosphorylation in assembly of the ATP synthase

    Mol. Cell. Proteomics

    (2007)
  • F. Willmund et al.

    The cotranslational function of ribosome-associated Hsp70 in eukaryotic protein homeostasis

    Cell

    (2013)
  • K. von der Malsburg et al.

    Dual role of mitofilin in mitochondrial membrane organization and protein biogenesis

    Dev. Cell

    (2011)
  • A.C. Badrick et al.

    PrrC, a Sco homologue from Rhodobacter sphaeroides, possesses thiol-disulfide oxidoreductase activity

    FEBS Lett.

    (2007)
  • G. Capitani et al.

    Crystal structures of two functionally different thioredoxins in spinach chloroplasts

    J. Mol. Biol.

    (2000)
  • S. Mora-Garcia et al.

    Role of electrostatic interactions on the affinity of thioredoxin for target proteins. Recognition of chloroplast fructose-1, 6-bisphosphatase by mutant Escherichia coli thioredoxins

    J. Biol. Chem.

    (1998)
  • M.E. Palm-Espling et al.

    Role of metal in folding and stability of copper proteins in vitro

    Biochim. Biophys. Acta

    (2012)
  • L.E. Netto et al.

    The roles of Peroxiredoxin and Thioredoxin in hydrogen peroxide sensing and in signal transduction

    Mol. Cell

    (2016)
  • L. Banci et al.

    The functions of Sco proteins from genome-based analysis

    J. Proteome Res.

    (2007)
  • L. Banci et al.

    Seeking the determinants of the elusive functions of Sco proteins

    FEBS J.

    (2011)
  • M. Schulze et al.

    SCO1, a yeast nuclear gene essential for accumulation of mitochondrial cytochrome c oxidase subunit II

    Mol. Gen. Genet.

    (1988)
  • S.C. Leary et al.

    Human SCO1 and SCO2 have independent, cooperative functions in copper delivery to cytochrome c oxidase

    Hum. Mol. Genet.

    (2004)
  • S.C. Leary et al.

    COX19 mediates the transduction of a mitochondrial redox signal from SCO1 that regulates ATP7A-mediated cellular copper efflux

    Mol. Biol. Cell

    (2013)
  • C.V. Attallah et al.

    Plants contain two SCO proteins that are differentially involved in cytochrome c oxidase function and copper and redox homeostasis

    J. Exp. Bot.

    (2011)
  • S.C. Leary

    Redox regulation of SCO protein function: controlling copper at a mitochondrial crossroad

    Antioxid. Redox Signal.

    (2010)
  • P. Saenkham et al.

    Mutation in sco affects cytochrome c assembly and alters oxidative stress resistance in agrobacterium tumefaciens

    FEMS Microbiol. Lett.

    (2009)
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    Current address: Department of Biology, University of Fribourg, Chemin du Musée 10, 1700 Fribourg, Switzerland.

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