Human VDAC isoforms differ in their capability to interact with minocycline and to contribute to its cytoprotective activity

Highlights

• HVDAC3 isolated from yeast cells displays canonical channel activity.

• Minocycline interacts differently with human VDAC isoforms.

• Only HVDAC3 mediates cytoprotective effect of minocycline against H₂O₂.

Abstract

It has been previously demonstrated that cytoprotective activity displayed by minocycline in the case of the yeast Saccharomyces cerevisiae cells pretreated with H₂O₂ requires the presence of functional VDAC (YVDAC1). Thus, we decided to transform YVDAC1-depleted yeast cells (Δpor1 cells) with plasmids expressing human VDAC isoforms (HVDAC1, HVDAC2, HVDAC3) to estimate their involvement in the minocycline cytoprotective effect. We observed that only expression of HVDAC3 in Δpor1 cells provided minocycline-mediated cytoprotection against H₂O₂ although all human isoforms are functional in Δpor1 cells. The observation appears to be important for on-going discussion concerning VDAC isoform roles in mitochondria and cell functioning.

Introduction

Studies concerning minocycline (a derivative of tetracycline; 7-dimethylamino-6-dimethyl-6-deoxytetracycline) indicate that the antibiotic has multi-faced effects on cell functions and consequently a number of clinical properties that are useful and/or could be useful for treatment of different diseases, including bacterial infections, cancer, autoimmune disorders, ischemia as well as neurodegenerative and psychiatric diseases (for review, see for example, Garrido-Mesa et al., 2013, Karachitos et al., 2013, Stock et al., 2013 and Fond et al., 2014). Accordingly, it is becoming evident that minocycline-mediated cytoprotection is an important aspect of its clinical application. However, till now the mechanism(s) of minocycline-mediated cytoprotection has(ve) not been clearly defined. Moreover, there are also data pointing at detrimental effects of minocycline including hepatotoxicity (e.g. Ferrajolo et al., 2010) and dysfunction of liver and brain mitochondria (e.g. Månsson et al., 2007 and Schönfeld et al., 2013, respectively). Correspondingly, minocycline is regarded as an anticancer and a proapoptotic factor (e.g. Teicher, 1998, Niu et al., 2008, Lokeshwar, 2011 and Pourgholami et al., 2012). Thus, detailed description of cellular and molecular mechanism(s) triggered by minocycline appears to be current and important topic of study.

As it is commonly accepted that mitochondrial dysfunction contributes to cell death and mitochondria are important target of cytoprotective drugs, the effect of minocycline on isolated mitochondria has been studied, but yet the published data do not support consistent explanation of minocycline effect (e.g. Fernandez-Gomez et al., 2005, Gieseler et al., 2009, Kupsch et al., 2009, Antonenko et al., 2010, Garcia-Martinez et al., 2010, Månsson et al., 2010, Cuenca-Lopez et al., 2012; and Schönfeld et al., 2013). However, despite the differences, the data point at changes at the level of the inner membrane permeability as a direct or indirect consequence of minocycline interaction with mitochondria. Undoubtedly, the changes affect the mitochondrial respiratory chain whose activity requires, among others, an efficient metabolite exchange between mitochondria and cytoplasm. The latter is supported by VDAC (voltage-dependent anion-selective channel), also known as mitochondrial porin and regarded as crucial for mitochondrial functioning (for review, see for example, Colombini, 2004, Lemasters and Holmuhamedov, 2006, Mannella and Kinnally, 2008, Rostovtseva and Bezrukov, 2008, Gałgańska et al., 2010a, Shoshan-Barmatz et al., 2010, Homblé et al., 2012 and Mertins et al., 2014).

Interestingly, we have shown that minocycline is able to affect channel properties of reconstituted VDAC (Garcia-Martinez et al., 2010). Moreover, we have shown that cytoprotective effect of minocycline on the yeast Saccharomyces cerevisiae cells treated with proapoptotic factor (i.e. H₂O₂) requires the presence of functional VDAC (Gałgańska et al., 2010b) and the effect correlates with improvement of the status of energy coupling within the cell (Karachitos et al., 2012). It should be, however, remembered that in many organisms VDAC isoforms encoded by distinct genes may be present in mitochondria and these isoforms have been proposed to play different functional roles (e.g. Blachly-Dyson and Forte, 2001, De Pinto et al., 2010, Anflous-Pharayra et al., 2011, Brahimi-Horn et al., 2012, Messina et al., 2012; and Amodeo et al., 2014). S. cerevisiae genome contains two genes for VDAC isoforms (YVDAC1 and YVDAC2) although until now only YVDAC1 has been proved to form a channel with functional properties highly conserved for VDAC1 from other species (Blachly-Dyson et al., 1997; Lee et al., 1998). In human mitochondria, as in the case of other vertebrates, three VDAC isoforms (VDAC1, VDAC2 and VDAC3) have been identified. As summarized in (Messina et al., 2012), VDAC1 and VDAC2 form channels with conserved properties when reconstituted in lipid bilayers, whereas VDAC3 has no evident channel-forming ability or is unable to insert properly in the artificial membranes although discrepant data concerning mammalian VDAC3 channel activity also have been shown (Xu et al., 1999, Checchetto et al., 2014 and Okazaki et al., 2015). Moreover, VDAC1 is the most abundant isoform, VDAC2 has similar activity that VDAC1 and both are involved in apoptosis regulation, whereas VDAC3 is specifically expressed in mammalian testis and its function is the least known, although it is suggested to be other than bioenergetics (De Pinto et al., 2010). However, molecular dynamics simulations performed recently for human VDAC isoforms suggest that human VDAC1 and VDAC3 are similar in terms of both electrostatics and dynamics of the channel pore and differ in this respect from VDAC2 (Amodeo et al., 2014).

It is well known that S. cerevisiae complementation assay is a useful method to assess the ability of an exogenous protein to recover the physiological growth phenotype of the mutated strain. Especially, the presence of only one channel-forming VDAC isoform in S. cerevisiae mitochondria enables to study a given VDAC isoform in the cellular context of YVDAC1-depleted (Δpor1) mutant (Xu et al., 1999 and De Pinto et al., 2010). Therefore, we decided to transform Δpor1 cells with plasmids expressing human VDAC isoforms (HVDAC1, HVDAC2, HVDAC3) to estimate their significance for the occurrence of the cytoprotective effect of minocycline against H₂O₂ treatment. The results indicate that among human VDAC isoforms only HVDAC3 is able to contribute to the cytoprotective effect of minocycline and it may ensue from differences in their channel properties as well as changes of the properties due to direct interaction with minocycline.