Identification new potential multidrug resistance proteins of Saccharomyces cerevisiae
Introduction
Members of the ATP binding cassette (ABC) protein superfamily, including human and yeast ABC transporters, transfer a variety of substances including ions, anticancer drugs, antibiotics, peptides, and phospholipids across biological membranes (Linton, 2007). ABC proteins fulfill a stunning variety of functions, ranging from ATP-driven transmembrane transport of great many different molecules, to the regulation of important cellular processes. In particular, ABC proteins can function as ion channels, channel regulators, receptors, proteases, as well as environmental sensors (Higgins, 1995; Dean and Allikmets, 1995; Kuchler and Thorner, 1992).
Most members of the ABC protein family share a similar molecular architecture and domain organization. But the typical ABC transporter contains two transmembrane domains (TMD1, TMD2) and two nucleotide-binding domains (NBD1, NBD2) (Bauer et al., 1999), (Dassa, 2003). The domain architecture of MDR proteins of both species includes full-size transporters (TMS6 − NBD)2 or (NBD − TMS6)2, half-size transporters (TMS6 − NBD) and members lacking obvious TMDs or NBDs (Gaur et al., 2005), (Tusnády et al., 2006). All ABC-transporters contain conserved regions (Hollenstein et al., 2007). NBD is approximately 200 residues in length and possesses six highly conserved motifs: the Walker A, Q-loop, ABC-signature, Walker B, D-loop and H-loop (Saurin et al., 1999). The main function of the NBDs is to bind and hydrolyze ATP or other NTPs, thereby fueling transport processes (Hollenstein et al., 2007). The most conserved features found in any given NBD are the Walker A and B motifs (Higgins, 1992). The Walker A motif has the sequence “GxxGxGKS/T,” where x is any amino acid. Walker B reported the consensus sequence of this motif to be “xxxxD, where D denotes aspartic acid residues respectively, and x represents any of the 20 standard amino acids (Higgins, 1995), (Dean et al., 2001). Walker A and Walker B motifs are involved in the ATP binding (Kerr, 2002). ABC-signature is found only in ABC transporters (Kim and Chen, 2018). In general, particular positions of these motifs are invariantly conserved and occupied by different amino acids in each subfamily (Mishra et al., 2014). Another important domains in ABC-transporters are TMDs. These domains are the primary determinants of substrate specificity through specific substrate-binding sites (Fig. 1) (Hollenstein et al., 2007), (Lewis et al., 2012). Each TMD usually contains six predicted α-helical transmembrane-spanning segments (TMSs), although in some cases four to eight predicted TMSs per TMD are also known (Biemans-Oldehinkel et al., 2006).
Despite their main functions in cell metabolism, ABC transporters can be harmful to humans. For instance, human diseases such as cystic fibrosis, adrenoleukodystrophy, Dubin-Johnson syndrome, Tangier disease are associated with mutations in human genes encoding ABC transporters. But one of the biggest problems with ABC proteins is their overexpression confers the resistance to hundreds of chemically unrelated drugs, including anticancer drugs and many others. Mainly these ABC-transporters localize in the plasma membrane and are called the multidrug resistant (MDR) proteins (Glavinas et al., 2005). For successful chemotherapy, it is necessary to search new inhibitors of MDR proteins in human cells. But the most important requirements are the low toxicity of inhibitors to the human cells. One of the main limitations of using human cells is different cell types need different expensive media to grow and survive, and most of the primary cells in culture have limited number of passages.
According to literature, Saccharomyces cerevisiae is an ideal model organism for the functional dissection of disease-related genes such as those of the ABC superfamily, because they grow easily, rapidly, cheaply, and have well understood genome (Karathia et al., 2011), (Wolters et al., 2015). Also, for example, up to 30% of genes implicated in human disease may have orthologs in the yeast proteome.
Genome of S. cerevisiae is shown to contain 30 ABC proteins which consists of six subfamilies: MDR (ABCB), MRP/CFTR (ABCC), ALDP (ABCD), RLI (ABCE), YEF3 (ABCF) and PDR5 (ABCG) (Bauer et al., 1999), (Piecuch and Obłak, 2014). Among these subfamilies, there is a network of genes involved in the multiple drug resistance phenotype, called the pleiotropic drug resistance (PDR) proteins in yeast (Piecuch and Obłak, 2014). Currently, yeast PDR network is shown to consist of PDR5 (ABCG), SNQ2 (ABCG), and YOR1 (ABCC) genes (Yibmantasiri et al., 2014). Likely, other ABC subfamily proteins of yeast cells can also perform the function of MDR under certain conditions (Bauer et al., 1999). The function of many ABC-transporters of S. cerevisiae has not been established yet. In the literature is shown that attempts to identify multidrug resistance functions of yeast ABC-proteins are being made by using quantitative reverse transcription PCR (RT-qPCR) analysis, GFP accumulation assay and flow cytometry (Galkina et al., 2018).
Besides the fact that multidrug resistance proteins of ABC superfamily are actively studied in clinical area, some of them are widespread in nature and have agricultural or biotechnological implications including agricultural fungicides, azoles, mycotoxins, herbicides and many others. For example, PDR18 of S. cerevisiae features the resistance to chemical stress agents, including herbicides, agricultural fungicides, and some metals (Teixeira et al., 2012). Also based on a genome-wide screening, PDR18 expression was also found to confer the resistance to the anticancer drugs cisplatin and carboplatin and the antifungal drug nocodazol (Teixeira et al., 2012). Pdr18 was found to play a role in plasma membrane sterol incorporation, and this physiological trait is proposed to contribute to its action as a multidrug resistance determinant (Cabrito et al., 2011). In the same time, PDR18 gene overexpression increases yeast ethanol tolerance and fermentation performance at more highly inhibitory concentrations of ethanol (Cabrito et al., 2011). PDR18 overexpressing in industrial yeast strains appears to be a promising approach to increase the bio-ethanol production (Godinho et al., 2018).
Despite the biological importance of the MDR phenotype, the identification of MDR functions of ABC proteins is carried out either by using time-consuming and expensive methods, or by establishing homology between proteins of different organisms using BLAST algorithms. Conventionally, only one specific part of the polypeptide chain is used for BLAST analysis (for example, the full protein sequence). By this way, the high level of homology of ABC-transporters of S. cerevisiae with Candida albicans, Schizosaccharomyces pombe, Drosophila melanogaster, Mycobacterium tuberculosis, Staphylococcus aureus has shown in previous researches (Shukla et al., 2003), (Christensen et al., 1997), (Braibant et al., 2000), (Burnie et al., 2000). The disadvantage of this method is that the established homology does not allow to assume the functional similarity of the studied proteins, since it relies on one parameter.
In our article, we proposed novel approach for identification potential MDR proteins using cluster analysis. For our analysis we use a number of parameters, namely cell localization and several homology parameters in the functionally important regions of ABC proteins, which are localized in the substrate-binding, ATP-binding and conserved sites of the protein molecules. In addition, to reveal functional similarity among ABC-proteins we use not only yeast ABC proteins that having proven PDR phenotype, but also human ABC proteins with a well-established MDR function, which increases the probability of establishing the MDR activity and functional analogs of yeast ABC proteins.
As a result of applying this approach, we have revealed new functional analogues of human MDR proteins in S. cerevisiae cells, that can be used to model and study the activity of MDR human proteins using cheaper yeast cells. Also, the data obtained might be used to develop new directions of modulating the activity of these proteins in yeast, that is aimed at ethanol bio-production increase.
Base on the literature review this analysis has not been done before and represents a new method identification of intraspecific and interspecies functional analogs of proteins. In addition, this method is easy to use and no time-consuming, and allows to minimize the range of proteins for experimental validation of their predicted functions in future.
Section snippets
Materials and methods
The protein sequence of ABC-transporters of S. cerevisiae and MDR proteins of human identified from UniProt. Information about the amino acid sequence position of NBDs and TMDs of human MDR proteins (p-glycoprotein, MRP1, BCRP) and yeast ABC proteins are presented in the Table A1 (Appendices).
The full sequences, NBDs, TMDs, Walker A and Walker B motifs, Q-loop were used for local alignment withBLASTp.InformationabouttheaminoacidsequencepositionofABCsignature, Walker A and Walker B motifs,
Identification of potential MDR proteins of yeast ABCB subfamily
Four ABC proteins of S. cerevisiae MDL1, MDL2, ATM1 and STE6 belong to ABCB subfamily. MDL1exports peptides formed upon proteolysis of mitochondrial proteins, whereas the function of MDL2 remains unknown (Paumi et al., 2009). ATM1 performs biogenesis of iron‑sulfur (Fe/S) clusters, STE6takes part in mating of α factor secretion protein (Srinivasan et al., 2014), (Kölling and Hollenberg, 1994). Recently, functioning of members of ABCB subfamily as MDR proteins has not been covered in the
Discussion
The most convenient approach for the identification of potential MDR proteins has been found because there is the growth of data on the functions of ABC-transporters in human cells, namely, their participation in drug transfer. The disadvantages of using animal and human cell lines are the necessity of standardization of the environment, difficulties of medium preparation and a limited number of passages. An attempt of conduct a cluster analysis of three subfamilies of ABC-transporters of S.
Funding
Our publication was supported for this work by the Belarusian Republican Foundation for Fundamental Research (grant agreement № М19МС-033 (02.05.2019)). Registration number 20200121.
Author statement
All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript.
Declaration of Competing Interest
Authors declare that they have no conflict of interests.
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