Human ABC Proteins Research Articles

Targeting breast cancer resistance protein (BCRP/ABCG2): Functional inhibitors and expression modulators

The primary source of failure of cancer therapies is multidrug resistance (MDR), which can be caused by different mechanisms, including the overexpression of ABC transporters in cancer cells. Among the 48 human ABC proteins, the breast cancer resistance protein (BCRP/ABCG2) has been described as a pivotal player in cancer resistance. The use of functional inhibitors and expression modulators is a promising strategy to overcome the MDR caused by ABCG2. Despite the lack of clinical trials using ABCG2 inhibitors, many compounds have already been discovered. This review presents an overview about various ABCG2 inhibitors that have been identified, discussing some chemical aspects and the main experimental methods used to identify and characterize the mechanisms of new inhibitors. In addition, some biological requirements to pursue preclinical tests are described. Finally, we discuss the potential use of ABCG2 inhibitors in cancer stem cells (CSC) for improving the objective response rate and the mechanism of ABCG2 modulators at transcriptional and protein expression levels.

The human YAE1-ORAOV1 complex of the cytosolic iron-sulfur protein assembly machinery binds a [4Fe-4S] cluster

Iron-sulfur (Fe-S) clusters are among the most versatile cofactors in biology. Although Fe-S clusters formation can be achieved spontaneously in vitro with inorganic iron and sulfur sources, the in vivo behaviour is more complex and requires the so-called Fe-S biogenesis machineries. In the cytosol, the biogenesis of Fe-S proteins is assisted by the cytosolic Fe-S protein assembly machinery, which comprises at least thirteen known proteins, among which there are human ORAOV1 and YAE1. A hetero-complex formed by the two latter proteins facilitates Fe-S cluster insertion in the human ABC protein ABCE1 within a chain of binding events that are still not well understood. In the present work, ORAOV1 and the YAE1-ORAOV1 complex were produced and their structural and cluster binding properties spectroscopically investigated. It resulted that both ORAOV1 and the YAE1-ORAOV1 complex are characterized by well-structured, α -helical regions and by unstructured, flexible regions, and are both able to bind a [4Fe-4S]2+ cluster. Bioinformatics and site-directed mutagenesis studies indicated that the [4Fe-4S] cluster in ORAOV1 is bound by a conserved cluster binding motif, while YAE1, which does not have a metal-binding consensus motif, is not essential for the [4Fe-4S]2+ cluster binding in the YAE1-ORAOV1 hetero-complex. Overall, these results support a model that the YAE1-ORAOV1 complex might actively participate in the Fe-S cluster insertion into ABCE1 thanks to the [4Fe-4S]2+ cluster binding properties of ORAOV1.

In vitro transport of methotrexate by Drosophila Multidrug Resistance-associated Protein.

Methotrexate (MTX) is a widely used chemotherapeutic agent, immune suppressant and antimalarial drug. It is a substrate of several human ABC proteins that confer multidrug resistance to cancer cells and determine compartmentalization of a wide range of physiological metabolites and endo or xenobiotics, by their primary active transport across biological membranes. The substrate specificity and tissue distribution of these promiscuous human ABC transporters show a high degree of redundancy, providing robustness to these key physiological and pharmacological processes, such as the elimination of toxins, e.g. methotrexate from the body. A similar network of proteins capable of transporting methotrexate has been recently suggested to exist in Drosophila melanogaster. One of the key players of this putative network is Drosophila Multidrug-resistance Associated Protein (DMRP). DMRP has been shown to be a highly active and promiscuous ABC transporter, capable of transporting various organic anions. Here we provide the first direct evidence that DMRP, expressed alone in a heterologous system lacking other, potentially functionally overlapping D. melanogaster organic anion transporters, is indeed able to transport methotrexate. Our in vitro results support the hypothesized but debated role of DMRP in in vivo methotrexate excretion.

Leishmania tarentolae as a host for heterologous expression of functional human ABCB6 transporter

The need for large amounts of reproducibly produced and isolated protein arises not only in structural studies, but even more so in biochemical ones, and with regard to ABC transporters it is especially pressing when faced with the prospect of enzymatic/transport activity studies, substrate screening etc. Thus, reliable heterologous expression systems/model organisms for large and complex proteins are at a premium. We have verified the applicability of the recently established novel eukaryotic expression system, using Leishmania tarentolae as a host, for human ABC protein overexpression. We succeeded in overexpressing human ABCB6, a protein with controversial subcellular localization and multiple proposed cellular functions. We were able to demonstrate its efficient expression in the expected subcellular locations as well as biochemical activity of the overexpressed protein (ATPase activity and porphyrin-like substrate transport). This activity was absent in cells overexpressing the catalytically inactive variant of ABCB6 (K629M). We demonstrate the possibility of applying a cost-effective expression system to study the activity of membrane transporters from the ABC superfamily.

A sensitive tool to measure CFTR channel activity

cystic fibrosis transmembrane conductance regulator(CFTR, ABCC7) is a member of the human ABC protein fam-ily. The characteristic feature of ABC proteins is the similararchitecture of the ATP binding sites and the common mecha-nism of ATP binding and hydrolysis. In contrast to most ofthe ABC proteins, CFTR is not an active transporter, rather itis a phosphorylation and ATP hydrolysis gated chloride ionchannel and channel regulator (1,2). Mutations in both copiesof the CFTR gene may lead to a heritable genetic disease, i.e.cystic fibrosis (CF) with variable severity depending on thesite of the mutation. Certain mutations alter biogenesis or cel-lular processing of the CFTR protein, while others affect itschannel activity. Chemical chaperons, also called correctors(e.g., VX-809) can partially rescue the misprocessing, mostlikely by improving the folding of the protein at the endoplas-mic reticulum (2). Activators of CFTR may function throughelevating cytosolic cAMP (promoting CFTR phosphoryla-tion), inhibiting phosphatase activity (thus blocking CFTR de-phosphorylation), and/or interacting directly with the channelprotein (1). Since CFTR is expressed in the epithelial cells ofseveral organs, CF is a multi-organ disease that affects thesinopulmonary and male urogenital systems, alters pancreaticand biliary secretion, therefore the current average lifespan ofCF patients is approximately 40 years of age (1,2). In mostcases, the primary cause of early onset of death is the progres-sion of lung disease (2,3). The mechanisms by which CFTRmutations cause lung disease in CF patients are not fullyunderstood. It may include altered ion and water transportacross the airway epithelium and aberrant inflammatory andimmune responses to pathogens within the airways (3,4).Several techniques have been developed to study CFTRfunction or to search for CFTR activators or modulatorsincluding halide sensitive fluorescent dyes, electrophysiologi-cal approaches such as patch clamp, short-circuit measure-ments in Ussing chambers and influx or efflux measurementsapplying radioactive ions. In addition to the above techniquesa halide sensitive mutant form of yellow fluorescent protein(YFP) has been also utilized to probe CFTR function meas-uring the iodide-mediated quenching rate of YFP, since theopen state of CFTR is also permeable to iodide ions (5). YFPfluorescence intensity is both dependent on the intracellulariodide concentration and YFP expression level. Therefore, flu-orescence intensities measured in the presence of iodideshould be normalized to YFP fluorescence intensities meas-ured in the absence of iodide to decrease experimental vari-ability related to the differences in sensor expression levelamong individual cells.However, normalization to the un-quenched YFP fluores-cence intensity is problematic especially in flow cytometricassays. The co-expression of a halide insensitive fluorescentprotein can overcome the above problem as was demonstratedby Vijftigschild et al. published in the current issue of Cytome-try Part A (page 576). When the two fluorescent proteins wereexpressed as a fusion protein formation of aggregates wasobserved. To avoid this problem the two proteins were con-nected by an auto-cleavable polypeptide chain (6) resulting inphysically not connected proteins. Theoretically, the expres-sion level of the two proteins may change upon time due to

Cholesterol fill-in model: mechanism for substrate recognition by ABC proteins

Many of the 48 or 49 human ABC proteins are involved in lipid homeostasis and in defence against hydrophobic substances in food and the environment. Defects in their functions cause various diseases, suggesting that they play very important roles in human health; however, the mechanism of how they handle enormous numbers of hydrophobic compounds with various structures and molecular weights, or phospholipids and cholesterol, major components of cellular membranes, is not known. We compared the functions of drug-transporting and lipid-transporting ABC proteins, and found that (1) ABC proteins, either lipid or drug transporters, have a similar substrate binding site which recognizes PL and cholesterol, or drugs and cholesterol; (2) Cholesterol in membranes binds to various ABC proteins together with PL or drugs, and plays an important role in substrate recognition, especially by ABCB1/MDR1, where cholesterol fills the empty space in the substrate binding site when small drugs bind to it. ABC proteins exert very flexible substrate recognition, i.e., one-to-many interaction rather than the conventional rigid one-to-one interaction. We propose calling the mechanism the "cholesterol fill-in model".

Membrane topology of human ABC proteins

In this review, we summarize the currently available information on the membrane topology of some key members of the human ABC protein subfamilies, and present the predicted domain arrangements. In the lack of high-resolution structures for eukaryotic ABC transporters this topology is based only on prediction algorithms and biochemical data for the location of various segments of the polypeptide chain, relative to the membrane. We suggest that topology models generated by the available prediction methods should only be used as guidelines to provide a basis of experimental strategies for the elucidation of the membrane topology.

Correspondence of function and phylogeny of ABC proteins based on an automated analysis of 20 model protein data sets

Using our BLAST-based procedure RiPE (Retrieval-induced Phylogeny Environment), which automates the evolutionary analysis of a protein family, we assembled a set of 1138 ABC protein components [adenosine triphosphate (ATP)-binding cassette and transmembrane domain] from the protein data sets of 20 model organisms and subjected them to phylogenetic and functional analysis. For maximum speed, we based the alignment directly on a homology search with a profile of all known human ABC proteins and used neighbor-joining tree estimation. All but 11 sequences from Homo sapiens, Arabidopsis thaliana, Drosophila melanogaster, and Saccharomyces cerevisiae were placed into the correct subtree/subfamily, reproducing published classifications of the individual organisms. By following a simple "function transfer rule", our comparative phylogenetic analysis successfully predicted the known function of human ABC proteins in 19 of 22 cases. Three functional predictions did not correspond, and 10 were novel. Predictions based on BLAST alone were inferior in five cases and superior in two. Bacterial sequences were placed close to the root of most subtrees. This placement coincides with domain architecture, suggesting an early diversification of the ABC family before the kingdoms split apart. Our approach can, in principle, be used to annotate any protein family of any organism included in the study.