Archaea. 2016 Sep 26;2016:7424870

Discovering antioxidant molecules in the Archaea domain: peroxiredoxin Bcp1 from Sufolobus solfaricus protects H9c2 cardiomyoblasts against oxidative stress.

 

Carmen Sarcinelli, Gabriella Fiorentino, Elio Pizzo, Simonetta Bartolucci, Danila Limauro

Dipartimento di Biologia, Università di Napoli “Federico II”, Via Cinthia, 80126 Naples, Italy.

Correspondence to: Danila Limauro, Dipartimento di Biologia, Università di Napoli “Federico II”, Via Cinthia, 80126 Naples, Italy. E-mail: limauro@unina.it

 

Abstract

Peroxiredoxins (Prxs) are ubiquitous thiol peroxidases that are involved in the reduction of peroxides. It has been reported that prokaryotic Prxs generally show greater structural robustness than their eukaryotic counterparts, making them less prone to inactivation by overoxidation. This difference has inspired the search for new antioxidants from prokaryotic sources that can be used as possible therapeutic biodrugs. Bacterioferritin comigratory proteins (Bcps) of the hyperthermophilic archaeon Sulfolobus solfataricus that belong to the Prx family have recently been characterized. One of these proteins, Bcp1, was chosen to determine its antioxidant effects in H9c2 rat cardiomyoblast cells. Bcp1 activity was measured in vitro under physiological temperature and pH conditions that are typical of mammalian cells; the yeast thioredoxin reductase (yTrxR)/thioredoxin (yTrx) reducing system was used to evaluate enzyme activity. A TAT-Bcp1 fusion protein was constructed to allow its internalization and verify the effect of Bcp1 on H9c2 rat cardiomyoblasts subjected to oxidative stress. The results reveal that TAT-Bcp1 is not cytotoxic and inhibits H2O2-induced apoptosis in H9c2 cells by reducing the H2O2 content inside these cells

PMID: 27752237; DOI: 10.1155/2016/7424870

 

Hightlights

  • Prokaryotic Prxs are more robust to over oxidation than eukaryotic counterparts
  • The archaeal Prx, Bcp1 can work at same physiological condition of eukaryotic Prxs
  • TAT mediates the intracellular delivery of Bcp1 resulting not cytotoxic for H9c2cells
  • Delivery of TAT-Bcp1 in H2O2-stressed H9c2rat cardiomyoblasts protects against cell apoptosis

 

Supplement

H2O2 is involved in the development and pathogenesis of many diseases such as cancer, diabetes, chronic inflammation, cardiovascular disorders and the antioxidant enzymes can play an important function in their prevention. Among these, Prxs, are ubiquitous thiol peroxidases, recently characterized in different organisms, that catalyze the reduction of peroxides generally coupled with Thioredoxin reductase (TrxR) /Thioredoxin (Trx) regenerating system [Fig 1]. These enzymes, usually expressed in different isoforms, are evolutionarily conserved from the Archaea, such as S. solfataricus, in which four enzymes have been characterized, to man in which six isoforms have been identified [1-3].

In eukaryotic organisms, Prxs, although have retained fold and catalytic mechanism, have diversified their function, in fact they play also a regulatory role in the peroxide-mediated signaling pathways. Many evidences suggest both the strong involvement of Prxs in the oxidative stress response and their role in the regulation of intracellular levels of H2O2. It was widely reported that H2O2 contributes to the initiation and the progression of cardiovascular diseases including the atherosclerosis. In Apolipoprotein E knockout mice, fed with high-cholesterol diet, the overexpression of Prx4 attenuates atherosclerosis progression, reducing apoptosis of macrophages and Smooth Muscular Cells (SMCs), protecting aorta from oxidative stress [4]. Therefore, Prxs could be a potential therapeutic bio-tool for ROS-related cardiovascular dysfunctions, helping in the prevention of atherosclerotic vascular diseases and contributing to the clinical effectiveness. Unfortunately, in conditions of high ROS levels, such as hypoxia/reperfusion and acute inflammation, mammalian 2-Cys Prxs can be over-oxidized losing their antioxidant activity.

In order to develop new antioxidant enzyme less prone to the inactivation by over-oxidation during the increase of peroxide, we have chosen a previously characterized prokaryotic Prx, Bcp1 from S. solfataricus, (Figure 1) that lacking of sensitivity motifs, has a higher robustness compared to eukaryotic counterparts.

 

 

Figure 1. An Antioxidant defense system from hot spring habitat. Solfataric environment near Naples (Pisciarelli, Naples, Italy) where Sulfolobus solfataricus was isolated. Bcp1 (in green 3D structure) isolated from S. solfataricus and the general action mechanism of Prxs (on the top).

 

Starting from this structural difference, the manuscript “Discovering antioxidant molecules in the Archaea domain: peroxiredoxin Bcp1 from Sufolobus solfaricus protects H9c2 cardiomyoblasts against oxidative stress” [3] shows that: 1) an archaeal Prx, Bcp1, has antioxidant effect on H9c2 cardiomyoblasts undergone to oxidative stress 2) a non-recombinant system for the delivery of BioDrugs could be used instead of virus-mediated DNA transfection.

Firstly, we showed that Bcp1, homologous to human Prxs (hPrx1 and hPrx2), is active in physiological conditions typical of mammalian cell (pH 7.00 and 37°C); in addition the yeast reducing system yTrxR/yTrx recycles Bcp1, similarly to hPrxs, suggesting the possibility of Bcp1 to act in eukaryotic cells.

With the aim to show the possible use the archaeal Prx as antioxidant, we analyzed the effect of Bcp1 on H2O2-induced apoptosis into H9c2 cells [3].

Previous studies showed that HIV-Trans-Activating Transduction (TAT) domain, a short basic polypeptide of 11 amino acids residues, fused with a target protein confers it the capability to penetrate the cells across the lipid bilayer, thanks to the strong binding of positive TAT domain to negative charges of the cell surface. To allow Bcp1 cell internalization, we generated the fusion protein TAT-Bcp1. We chose as model system H9c2 rat cardiomyoblasts, non-malignant cardiac-like cells, commonly used to study the molecular response to oxidative damage, to analyse the entrance of TAT-Bcp1 inside the cells, its possible cytotoxicity and the antioxidant effect.

The results can be summarized in three main points:

1) after 1 h by the administration TAT-Bcp1 was effectively detected into the cells (Figure 2A) and gradually decreased until 70% after 24 h, in agreement with the protein turnover. Furthermore, the protein never induced any cytotoxic effect in the cells, but rather it could have a cytoprotective effect, as demonstrated by a slight but significant increase of cell viability at 16h and 24h.

2) TAT-Bcp1 efficiently decreased H2O2 levels in H9c2 cells, both in physiological and stressed conditions, so showing that the enzyme can use endogenous recycling system.

3) TAT-Bcp1 is able to reduce apoptosis (Figure 2 B-C).

All together these results suggest that the antioxidant activity of TAT-Bcp1 could play a role in protecting cells against apoptosis.

This work opens a new scenario regarding the possibility of using alternative and safe sources of antioxidant enzymes such as thermophilic microorganisms, to prevent oxidative damage. This is the first report regarding an archaeal enzyme delivered into mammalian cultured cells and able to protect against oxidative stress-induced apoptosis. The intrinsic stability of Bcp1 and its modification, that provides the ability to penetrate cells, makes TAT-Bcp1 an effective tool for the treatment in vascular oxidative injuries. Studies on the pathways induced by Bcp1 in the H9c2 cardiomyoblasts during the oxidative stress and in vivo studies are in progress, nevertheless these encouraging results suggest that Bcp1 could be a promising antioxidant for the treatment of cardiovascular disorder.

 

 

Figure 2. Effect of TAT-Bcp1 administration to H9c2 cells in normal growth and undergone to oxidative stress. (A) TAT-Bcp1 (in green) enters inside the cells and  is not cytotoxic. (B)  Effect of oxidative stress on cellular growth without TAT-Bcp1 pretreatment. (C) Effect of TAT-Bcp1 pretreatment on cells undergone to oxidative stress.

 

Acknowledgements.

The authors thank Dr. Sang Won Kang, Center for Cell Signaling Research and Division of Molecular Life Sciences, Ewha Woman’s University, Seoul, Korea, for generously providing the plasmids: pET17yTrx and pET17yTrxR. The work was supported by grants from POR Campania FSE 2007/2013, Progetto CARINA CUP B25B09000080007, and SostegnoTerritoriale alle Attività di Ricerca (STAR) 2014, University of Naples Federico II, CUP E68C13000020003.

 

References

[1] D’Ambrosio K, Limauro D, Pedone E, Galdi I, Pedone C, Bartolucci S, De Simone G (2009) Insights into the catalytic mechanism of the Bcp family: functional and structural analysis of Bcp1 from Sulfolobus solfataricus. Proteins 76:995-1006. doi: 10.1002/prot.22408

[2] Limauro D, D’Ambrosio K, Langella E, De Simone G, Galdi, I, Pedone, C, Pedone E, Bartolucci S (2010) Exploring the catalytic mechanism of the first dimeric Bcp: Functional, structural and docking analyses of Bcp4 from Sulfolobus solfataricus. Biochimie 92:1435-44 doi: 10.1016/j.biochi.2010.07.006

[3] Sarcinelli C., Fiorentino G, Pizzo E, Bartolucci S, Limauro D (2016) Discovering antioxidant molecules in the Archaea domain: the peroxiredoxin Bcp1 from Sufolobus solfaricus protects H9c2 cardiomyoblasts against oxidative stress. Archaea 2016:7424870. doi: 10.1155/2016/7424870

[4] Guo X, Yamada S, Tanimoto A, Ding Y, Wang K Y, Shimajiri S, Murata Y, Kimura S, Tasaki T, Nabeshima A, Watanabe T, Kohno K, Sasaguri Y(2012) Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice. Antioxid Redox Signal. 17:1362-75 doi: 10.1089/ars.2012.4549

 

 

 

 

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