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

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 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.

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 Regiodivergent Biocatalytic Hydroxylation of L-Glutamine Facilitated by Characterization of Non-Heme Dioxygenases from Non-Ribosomal Peptide Biosyntheses

2021 ◽  
Author(s):  
Hans Renata ◽  
Emily Shimizu ◽  
Christian Zwick
Keyword(s):  
Amino Acid ◽  
Building Block ◽  
Solid Phase ◽  
Functional Characterization ◽  
Free Standing ◽  
Substrate Specificities ◽  
Total Turnover

We report the functional characterization of two iron- and a-ketoglutarate-dependent dioxygenases that are capable of hydroxylating free-standing glutamine at its C3 and C4 position respectively. In particular, the C4 hydroxylase, Q4Ox, catalyzes the reaction with approximately 4,300 total turnover numbers, facilitating synthesis of a solid-phase compatible building block and stereochemical elucidation at the C4 position of the hydroxylated product. This work will enable the development of novel synthetic strategies to prepare useful glutamine derivatives and stimulate further discoveries of new amino acid hydroxylases with distinct substrate specificities.<br>

scholarly journals Regiodivergent Biocatalytic Hydroxylation of L-Glutamine Facilitated by Characterization of Non-Heme Dioxygenases from Non-Ribosomal Peptide Biosyntheses

2021 ◽  
Author(s):  
Hans Renata ◽  
Emily Shimizu ◽  
Christian Zwick
Keyword(s):  
Amino Acid ◽  
Building Block ◽  
Solid Phase ◽  
Functional Characterization ◽  
Free Standing ◽  
Substrate Specificities ◽  
Total Turnover

We report the functional characterization of two iron- and a-ketoglutarate-dependent dioxygenases that are capable of hydroxylating free-standing glutamine at its C3 and C4 position respectively. In particular, the C4 hydroxylase, Q4Ox, catalyzes the reaction with approximately 4,300 total turnover numbers, facilitating synthesis of a solid-phase compatible building block and stereochemical elucidation at the C4 position of the hydroxylated product. This work will enable the development of novel synthetic strategies to prepare useful glutamine derivatives and stimulate further discoveries of new amino acid hydroxylases with distinct substrate specificities.<br>

Characterization of cationic amino acid transporters (hCATs) 1 and 2 in human skin

2008 ◽  
Vol 129 (3) ◽  
pp. 321-329 ◽  
Author(s):  
Kristin Jaeger ◽  
Friedrich Paulsen ◽  
Johannes Wohlrab
Keyword(s):  
Amino Acid ◽  
Human Skin ◽  
Amino Acid Transporters ◽  
Cationic Amino Acid
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