scholarly journals Yeast Cell-Based Transport Assay for the Functional Characterization of Human 4F2hc-LAT1 and ‐LAT2, and LAT1 and LAT2 Substrates and Inhibitors

2021 ◽  
Vol 8 ◽  
Author(s):  
Satish Kantipudi ◽  
Dimitrios Fotiadis
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Functional Characterization ◽  
Methylotrophic Yeast ◽  
Cell Types ◽  
Amino Acid Transporters ◽  
Cell Assays ◽  
Transport Assay ◽  
Compound Screening

In mammalian cells, the L-type amino acid transporters (LATs) LAT1 (SLC7A5) and LAT2 (SLC7A8) form heterodimeric amino acid transporters (HATs) with the ancillary protein 4F2hc and are involved in the cellular uptake of specific amino acids. The HAT 4F2hc-LAT1 is found upregulated in various cancer cell types, while 4F2hc-LAT2 is a transporter for non-cancer cells. Preclinical studies have highlighted that 4F2hc-LAT1 plays an important role in tumor progression representing a valid anticancer target. Consequently, current research is focusing on the development of potent and specific human 4F2hc-LAT1 inhibitors. On the other hand, 4F2hc-LAT2 is emerging as target of other diseases, thus also gaining clinical interest. To determine affinity and specificity of substrates and inhibitors for 4F2hc-LAT1 or 4F2hc-LAT2, robust transport cell assays are indispensable. We have optimized and validated a transport assay using cells of the methylotrophic yeast Pichia pastoris stably overexpressing the human HATs 4F2hc-LAT1 or -LAT2, and the LATs LAT1 or LAT2 alone. The radioligand [3H]L-leucine was used as reporter and the substrates L-leucine, triiodothyronine (T3) and thyroxine (T4) as well as the inhibitors BCH and JPH203 (KYT-0353) for assay validation. Obtained half-maximal inhibitory concentrations also provided new insights, e.g., into the LAT specificity of the potent inhibitor JPH203 and on the potency of the thyroid hormones T3 and T4 to inhibit transport through human 4F2hc-LAT2. The LAT1 and LAT2 assays are of particular interest to determine possible implications and influences of 4F2hc in ligand binding and transport. In summary, the presented assays are valuable for characterization of ligands, e.g., towards 4F2hc-LAT1 specificity, and can also be applied for compound screening. Finally, our established approach and assay would also be applicable to other HATs and LATs of interest.

scholarly journals Functional characterization of the alanine-serine-cysteine exchanger of Carnobacterium sp AT7

2019 ◽  
Vol 151 (4) ◽  
pp. 505-517 ◽  
Author(s):  
Paola Bartoccioni ◽  
Joana Fort ◽  
Antonio Zorzano ◽  
Ekaitz Errasti-Murugarren ◽  
Manuel Palacín
Keyword(s):  
Amino Acid ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Functional Characterization ◽  
Physiological Role ◽  
Cell Uptake ◽  
Amino Acid Transporters ◽  
Inhibitor Selectivity ◽  
Substrate Binding Sites

Many key cell processes require prior cell uptake of amino acids from the environment, which is facilitated by cell membrane amino acid transporters such as those of the L-type amino acid transporter (LAT) subfamily. Alterations in LAT subfamily amino acid transport are associated with several human diseases, including cancer, aminoacidurias, and neurodegenerative conditions. Therefore, from the perspective of human health, there is considerable interest in obtaining structural information about these transporter proteins. We recently solved the crystal structure of the first LAT transporter, the bacterial alanine-serine-cysteine exchanger of Carnobacterium sp AT7 (BasC). Here, we provide a complete functional characterization of detergent-purified, liposome-reconstituted BasC transporter to allow the extension of the structural insights into mechanistic understanding. BasC is a sodium- and proton-independent small neutral amino acid exchanger whose substrate and inhibitor selectivity are almost identical to those previously described for the human LAT subfamily member Asc-1. Additionally, we show that, like its human counterparts, this transporter has apparent affinity asymmetry for the intra- and extracellular substrate binding sites—a key feature in the physiological role played by these proteins. BasC is an excellent paradigm of human LAT transporters and will contribute to our understanding of the molecular mechanisms underlying substrate recognition and translocation at both sides of the plasma membrane.

scholarly journals Molecular and Functional Characterization of a Family of Amino Acid Transporters from Arabidopsis

2004 ◽  
Vol 136 (2) ◽  
pp. 3104-3113 ◽  
Author(s):  
Yan-Hua Su ◽  
Wolf B. Frommer ◽  
Uwe Ludewig
Keyword(s):  
Amino Acid ◽  
Functional Characterization ◽  
Amino Acid Transporters

Real-time functional characterization of cationic amino acid transporters using a new FRET sensor

2015 ◽  
Vol 468 (4) ◽  
pp. 563-572 ◽  
Author(s):  
Liviu Vanoaica ◽  
Alok Behera ◽  
Simone M. R. Camargo ◽  
Ian C. Forster ◽  
François Verrey
Keyword(s):  
Amino Acid ◽  
Real Time ◽  
Functional Characterization ◽  
Amino Acid Transporters ◽  
Cationic Amino Acid

scholarly journals The Heavy Chain 4F2hc Modulates the Substrate Affinity and Specificity of the Light Chains LAT1 and LAT2

2020 ◽  
Vol 21 (20) ◽  
pp. 7573
Author(s):  
Satish Kantipudi ◽  
Jean-Marc Jeckelmann ◽  
Zöhre Ucurum ◽  
Patrick D. Bosshart ◽  
Dimitrios Fotiadis
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Heavy Chain ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Expression System ◽  
Functional Characterization ◽  
Light Chains ◽  
Substrate Affinity ◽  
Amino Acid Transporters

The human L-type amino acid transporters LAT1 and LAT2 mediate the transport of amino acids and amino acid derivatives across plasma membranes in a sodium-independent, obligatory antiport mode. In mammalian cells, LAT1 and LAT2 associate with the type-II membrane N-glycoprotein 4F2hc to form heteromeric amino acid transporters (HATs). The glycosylated ancillary protein 4F2hc is known to be important for successful trafficking of the unglycosylated transporters to the plasma membrane. The heavy (i.e., 4F2hc) and light (i.e., LAT1 and LAT2) chains belong to the solute carrier (SLC) families SLC3 and SLC7, and are covalently linked by a conserved disulfide bridge. Overexpression, absence, or malfunction of certain HATs is associated with human diseases and HATs are therefore considered therapeutic targets. Here, we present a comparative, functional characterization of the HATs 4F2hc-LAT1 and 4F2hc-LAT2, and their light chains LAT1 and LAT2. For this purpose, the HATs and the light chains were expressed in the methylotrophic yeast Pichia pastoris and a radiolabel transport assay was established. Importantly and in contrast to mammalian cells, P. pastoris has proven useful as eukaryotic expression system to successfully express human LAT1 and LAT2 in the plasma membrane without the requirement of co-expressed trafficking chaperone 4F2hc. Our results show a novel function of the heavy chain 4F2hc that impacts transport by modulating the substrate affinity and specificity of corresponding LATs. In addition, the presented data confirm that the light chains LAT1 and LAT2 constitute the substrate-transporting subunits of the HATs, and that light chains are also functional in the absence of the ancillary protein 4F2hc.

scholarly journals Structural and Functional Characterization of SARS-CoV-2 RBD Domains Produced in Mammalian Cells

2021 ◽  
Author(s):  
Christoph Gstöttner ◽  
Tao Zhang ◽  
Anja Resemann ◽  
Sophia Ruben ◽  
Stuart Pengelley ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Functional Characterization

scholarly journals Cloning and functional characterization of the human fractalkine receptor promoter regions

2002 ◽  
Vol 368 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Alexandre GARIN ◽  
Philippe PELLET ◽  
Philippe DETERRE ◽  
Patrice DEBRÉ ◽  
Christophe COMBADIÈRE
Keyword(s):  
Functional Characterization ◽  
Cell Types ◽  
Promoter Regions ◽  
Exon 2 ◽  
Reading Frame ◽  
Fractalkine Receptor ◽  
A Cell ◽  
Exon 4 ◽  
Coronary Events

We have previously shown that reduced expression of the fractalkine receptor, CX3CR1, is correlated with rapid HIV disease progression and with reduced susceptibility to acute coronary events. In order to elucidate the mechanisms underlying transcriptional regulation of CX3CR1 expression, we structurally and functionally characterized the CX3CR1 gene. It consists of four exons and three introns spanning over 18kb. Three transcripts are produced by splicing the three untranslated exons with exon 4, which contains the complete open reading frame. The transcript predominantly found in leucocytes corresponds to the splicing of exon 2 with exon 4. Transcripts corresponding to splicing of exons 1 and 4 are less abundant in leucocytes and splicing of exons 3 and 4 are rare longer transcripts. A constitutive promoter activity was found in the regions extending upstream from untranslated exons 1 and 2. Interestingly, exons 1 and 2 enhanced the activity of their respective promoters in a cell-specific manner. These data show that the CX3CR1 gene is controlled by three distinct promoter regions, which are regulated by their respective untranslated exons and that lead to the transcription of three mature messengers. This highly complex regulation may allow versatile and precise expression of CX3CR1 in various cell types.

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