A novel mathematical model describing adaptive cellular drug metabolism and toxicity in the chemoimmune system

Cells cope with the threat of xenobiotic stress by activating a complex molecular network that recognizes and eliminates chemically diverse toxic compounds. This "chemoimmune system" consists of cellular Phase I and Phase II metabolic enzymes, Phase 0 and Phase III ATP Binding Cassette (ABC) membrane transporters, and nuclear receptors regulating these components. In order to provide a systems biology characterization of the chemoimmune network, we designed a reaction kinetic model based on differential equations describing Phase 0-III participants and regulatory elements, and characterized cellular fitness to evaluate toxicity. In spite of the simplifications, the model recapitulates changes associated with acquired drug resistance and allows toxicity predictions under variable protein expression and xenobiotic exposure conditions. Our simulations suggest that multidrug ABC transporters at Phase 0 significantly facilitate the defense function of successive network members by lowering intracellular drug concentrations. The model was extended with a novel toxicity framework which opened the possibility of performing in silico cytotoxicity assays. The alterations of the in silico cytotoxicity curves show good agreement with in vitro cell killing experiments. The behavior of the simplified kinetic model suggests that it can serve as a basis for more complex models to efficiently predict xenobiotic and drug metabolism for human medical applications

Jump into a new fold-A homology based model for the ABCG2/BCRP multidrug transporter

ABCG2/BCRP is a membrane protein, involved in xenobiotic and endobiotic transport in key pharmacological barriers and drug metabolizing organs, in the protection of stem cells, and in multidrug resistance of cancer. Pharmacogenetic studies implicated the role of ABCG2 in response to widely used medicines and anticancer agents, as well as in gout. Its Q141K variant exhibits decreased functional expression thus increased drug accumulation and decreased urate secretion. Still, there has been no reliable molecular model available for this protein, as the published structures of other ABC transporters could not be properly fitted to the ABCG2 topology and experimental data. The recently published high resolution structure of a close homologue, the ABCG5-ABCG8 heterodimer, revealed a new ABC transporter fold, unique for ABCG proteins. Here we present a structural model of the ABCG2 homodimer based on this fold and detail the experimental results supporting this model. In order to describe the effect of mutations on structure and dynamics, and characterize substrate recognition and cholesterol regulation we performed molecular dynamics simulations using full length ABCG2 protein embedded in a membrane bilayer and in silico docking simulations. Our results show that in the Q141K variant the introduced positive charge diminishes the interaction between the nucleotide binding and transmembrane domains and the R482G variation alters the orientation of transmembrane helices. Moreover, the R482 position, which plays a role the substrate specificity of the transporter, is located in one of the substrate binding pockets identified by the in silico docking calculations. In summary, the ABCG2 model and in silico simulations presented here may have significant impact on understanding drug distribution and toxicity, as well as drug development against cancer chemotherapy resistance or gout. © 2016 László et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

This chapter deals with the interactions of two medically important multidrug ABC transporters (MDR-ABC), ABCB1 and ABCG2, with lipid molecules. Both ABCB1 and ABCG2 are capable of transporting a wide range of hydrophobic drugs and xenobiotics and are involved in cancer chemotherapy resistance. Therefore, the exploration of their mechanism of action has major therapeutic consequences. As discussed here in detail, both ABCB1 and ABCG2 are significantly affected by various lipid compounds especially those residing in their close proximity in the plasma membrane. ABCB1 is capable of transporting lipids and lipid derivatives, and thus may alter the general membrane composition by "flopping" membrane lipid constituents, while there is no such information regarding ABCG2. Still, both ABCB1 and ABCG2 show complex interactions with a variety of lipid molecules, and the transporters are significantly modulated by cholesterol and cholesterol derivatives at the posttranslational level. In this chapter, we explore the molecular details of the direct transporter-lipid interactions, the potential role of lipid-sensor domains within the proteins, as well as the application of experimental site-directed mutagenesis, detailed structural studies, and in silico modeling for examining these interactions. We also discuss the regulation of ABCB1 and ABCG2 expression at the transcriptional level, occurring through nuclear receptors involved in lipid sensing. The better understanding of lipid interactions with these medically important MDR-ABC transporters may significantly improve further drug development and clinical treatment options

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter

Inconsistencies in the red blood cell membrane proteome analysis: generation of a database for research and diagnostic applications.

Based on recent results, the determination of the easily accessible red blood cell (RBC) membrane proteins may provide new diagnostic possibilities for assessing mutations, polymorphisms or regulatory alterations in diseases. However, the analysis of the current mass spectrometry-based proteomics datasets and other major databases indicates inconsistencies-the results show large scattering and only a limited overlap for the identified RBC membrane proteins. Here, we applied membrane-specific proteomics studies in human RBC, compared these results with the data in the literature, and generated a comprehensive and expandable database using all available data sources. The integrated web database now refers to proteomic, genetic and medical databases as well, and contains an unexpected large number of validated membrane proteins previously thought to be specific for other tissues and/or related to major human diseases. Since the determination of protein expression in RBC provides a method to indicate pathological alterations, our database should facilitate the development of RBC membrane biomarker platforms and provide a unique resource to aid related further research and diagnostics

Alterations of membrane protein expression in red blood cells of Alzheimer's disease patients

Preventive measures, prognosis, or selected therapy in multifactorial maladies, including Alzheimer's disease (AD), require the application of a wide range of diagnostic assays. There is a large unmet need for relatively simple, blood-based biomarkers in this regard. We have recently developed a rapid and reliable flow cytometry and antibody-based method for the quantitative measurement of various red blood cell (RBC) membrane proteins from a drop of blood. Here, we document that the RBC expression of certain membrane proteins, especially that of the GLUT1 transporter and the insulin receptor (INSR), is significantly higher in AD patients than in age-matched healthy subjects. The observed differences may reflect long-term metabolic alterations relevant in the development of AD. These findings may pave the way for a diagnostic application of RBC membrane proteins as relatively stable and easily accessible personalized biomarkers in AD. © 2015 The Authors