Publications by authors named "Boglárka Zámbó"

7 Publications

  • Page 1 of 1

Precision-engineered reporter cell lines reveal ABCG2 regulation in live lung cancer cells.

Biochem Pharmacol 2020 05 4;175:113865. Epub 2020 Mar 4.

Research Centre for Natural Sciences, Institute of Enzymology, Budapest, Hungary. Electronic address:

Expression of the ABCG2 multidrug transporter is a marker of cancer stem cells and a predictor of recurrent malignant disease. Understanding how human ABCG2 expression is modulated by pharmacotherapy is crucial in guiding therapeutic recommendations and may aid rational drug development. Genome edited reporter cells are useful in investigating gene regulation and visualizing protein activity in live cells but require precise targeting to preserve native regulatory regions. Here, we describe a fluorescent reporter assay that allows the noninvasive assessment of ABCG2 regulation in human lung adenocarcinoma cells. Using CRISPR-Cas9 gene editing coupled with homology-directed repair, we targeted an EGFP coding sequence to the translational start site of ABCG2, generating ABCG2 knock-out and in situ tagged ABCG2 reporter cells. Using the engineered cell lines, we show that ABCG2 is upregulated by a number of anti-cancer medications, HDAC inhibitors, hypoxia-mimicking agents and glucocorticoids, supporting a model in which ABCG2 is under the control of a general stress response. To our knowledge, this is the first description of a fluorescent reporter assay system designed to follow the endogenous regulation of a human ABC transporter in live cells. The information gained may guide therapy recommendations and aid rational drug design.
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http://dx.doi.org/10.1016/j.bcp.2020.113865DOI Listing
May 2020

Molecular Diversity of Plasma Membrane Ca Transporting ATPases: Their Function Under Normal and Pathological Conditions.

Adv Exp Med Biol 2020 ;1131:93-129

2nd Department of Pathology, Semmelweis University, Budapest, Hungary.

Plasma membrane Ca transport ATPases (PMCA1-4, ATP2B1-4) are responsible for removing excess Ca from the cell in order to keep the cytosolic Ca ion concentration at the low level essential for normal cell function. While these pumps take care of cellular Ca homeostasis they also change the duration and amplitude of the Ca signal and can create Ca gradients across the cell. This is accomplished by generating more than twenty PMCA variants each having the character - fast or slow response, long or short memory, distinct interaction partners and localization signals - that meets the specific needs of the particular cell-type in which they are expressed. It has become apparent that these pumps are essential to normal tissue development and their malfunctioning can be linked to different pathological conditions such as certain types of neurodegenerative and heart diseases, hearing loss and cancer. In this chapter we summarize the complexity of PMCA regulation and function under normal and pathological conditions with particular attention to recent developments of the field.
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http://dx.doi.org/10.1007/978-3-030-12457-1_5DOI Listing
October 2019

Cellular Processing of the ABCG2 Transporter-Potential Effects on Gout and Drug Metabolism.

Cells 2019 10 8;8(10). Epub 2019 Oct 8.

Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudosok krt. 2, 1117 Budapest, Hungary.

The human ABCG2 is an important plasma membrane multidrug transporter, involved in uric acid secretion, modulation of absorption of drugs, and in drug resistance of cancer cells. Variants of the ABCG2 transporter, affecting cellular processing and trafficking, have been shown to cause gout and increased drug toxicity. In this paper, we overview the key cellular pathways involved in the processing and trafficking of large membrane proteins, focusing on ABC transporters. We discuss the information available for disease-causing polymorphic variants and selected mutations of ABCG2, causing increased degradation and impaired travelling of the transporter to the plasma membrane. In addition, we provide a detailed in silico analysis of an as yet unrecognized loop region of the ABCG2 protein, in which a recently discovered mutation may actually promote ABCG2 membrane expression. We suggest that post-translational modifications in this unstructured loop at the cytoplasmic surface of the protein may have special influence on ABCG2 processing and trafficking.
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http://dx.doi.org/10.3390/cells8101215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830335PMC
October 2019

Cellular expression and function of naturally occurring variants of the human ABCG2 multidrug transporter.

Cell Mol Life Sci 2020 Jan 28;77(2):365-378. Epub 2019 Jun 28.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudosok krt. 2, Budapest, 1117, Hungary.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics; thus the relatively frequent polymorphic and mutant ABCG2 variants in the population may significantly alter disease conditions and pharmacological effects. Low-level or non-functional ABCG2 expression may increase individual drug toxicity, reduce cancer drug resistance, and result in hyperuricemia and gout. In the present work we have studied the cellular expression, trafficking, and function of nine naturally occurring polymorphic and mutant variants of ABCG2. A comprehensive analysis of the membrane localization, transport, and ATPase activity, as well as retention and degradation in intracellular compartments was performed. Among the examined variants, R147W and R383C showed expression and/or protein folding defects, indicating that they could indeed contribute to ABCG2 functional deficiency. These studies and the applied methods should significantly promote the exploration of the medical effects of these personal variants, promote potential therapies, and help to elucidate the specific role of the affected regions in the folding and function of the ABCG2 protein.
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http://dx.doi.org/10.1007/s00018-019-03186-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971004PMC
January 2020

Clinically relevant mutations in the ABCG2 transporter uncovered by genetic analysis linked to erythrocyte membrane protein expression.

Sci Rep 2018 05 10;8(1):7487. Epub 2018 May 10.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudosok krt. 2, Budapest, 1117, Hungary.

The ABCG2 membrane protein is a key xeno- and endobiotic transporter, modulating the absorption and metabolism of pharmacological agents and causing multidrug resistance in cancer. ABCG2 is also involved in uric acid elimination and its impaired function is causative in gout. Analysis of ABCG2 expression in the erythrocyte membranes of healthy volunteers and gout patients showed an enrichment of lower expression levels in the patients. By genetic screening based on protein expression, we found a relatively frequent, novel ABCG2 mutation (ABCG2-M71V), which, according to cellular expression studies, causes reduced protein expression, although with preserved transporter capability. Molecular dynamics simulations indicated a stumbled dynamics of the mutant protein, while ABCG2-M71V expression in vitro could be corrected by therapeutically relevant small molecules. These results suggest that personalized medicine should consider this newly discovered ABCG2 mutation, and genetic analysis linked to protein expression provides a new tool to uncover clinically important mutations in membrane proteins.
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http://dx.doi.org/10.1038/s41598-018-25695-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945641PMC
May 2018

Decreased calcium pump expression in human erythrocytes is connected to a minor haplotype in the ATP2B4 gene.

Cell Calcium 2017 07 3;65:73-79. Epub 2017 Feb 3.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudosok krt. 2, Budapest, 1117, Hungary; MTA-SE Molecular Biophysics Research Group, Department of Biophysics and Radiation Biology, Semmelweis University, Tuzolto u. 37-43., Budapest, 1094, Hungary. Electronic address:

Plasma membrane Ca-ATPases are key calcium exporter proteins in most tissues, and PMCA4b is the main calcium transporter in the human red blood cells (RBCs). In order to assess the expression level of PMCA4b, we have developed a flow cytometry and specific antibody binding method to quantitatively detect this protein in the erythrocyte membrane. Interestingly, we found several healthy volunteers showing significantly reduced expression of RBC-PMCA4b. Western blot analysis of isolated RBC membranes confirmed this observation, and indicated that there are no compensatory alterations in other PMCA isoforms. In addition, reduced PMCA4b levels correlated with a lower calcium extrusion capacity in these erythrocytes. When exploring the potential genetic background of the reduced PMCA4b levels, we found no missense mutations in the ATP2B4 coding regions, while a formerly unrecognized minor haplotype in the predicted second promoter region closely correlated with lower erythrocyte PMCA4b protein levels. In recent GWA studies, SNPs in this ATP2B4 haplotype have been linked to reduced mean corpuscular hemoglobin concentrations (MCHC), and to protection against malaria infection. Our data suggest that an altered regulation of gene expression is responsible for the reduced RBC-PMCA4b levels that is probably linked to the development of human disease-related phenotypes.
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http://dx.doi.org/10.1016/j.ceca.2017.02.001DOI Listing
July 2017

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

Alzheimers Dement (Amst) 2015 Sep 21;1(3):334-8. Epub 2015 Jul 21.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary; MTA-SE Molecular Biophysics Research Group, Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary.

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.
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http://dx.doi.org/10.1016/j.dadm.2015.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878320PMC
September 2015