Increased expression of an amino acid transporter LAT1 in healing of acetic acid-induced chronic gastric ulcer in rats

SLC6A19 (B0AT1) is a neutral amino acid transporter, the loss of function of which results in Hartnup disease. SLC6A19 is also believed to have an important role in amino acid homeostasis, diabetes, and weight control. A small-molecule inhibitor of human SLC6A19 (hSLC6A19) was identified using two functional cell-based assays: a fluorescence imaging plate reader (FLIPR) membrane potential (FMP) assay and a stable isotope-labeled neutral amino acid uptake assay. A diverse collection of 3440 pharmacologically active compounds from the Microsource Spectrum and Tocriscreen collections were tested at 10 µM in both assays using MDCK cells stably expressing hSLC6A19 and its obligatory subunit, TMEM27. Compounds that inhibited SLC6A19 activity in both assays were further confirmed for activity and selectivity and characterized for potency in functional assays against hSLC6A19 and related transporters. A single compound, cinromide, was found to robustly, selectively, and reproducibly inhibit SLC6A19 in all functional assays. Structurally related analogs of cinromide were tested to demonstrate structure–activity relationship (SAR). The assays described here are suitable for carrying out high-throughput screening campaigns to identify modulators of SLC6A19.

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Large amino acid transporter 1 mediated glutamate modified mesoporous silica nanoparticles for chemophotothermal therapy

Journal of Drug Delivery Science and Technology ◽

10.1016/j.jddst.2019.05.043 ◽

2019 ◽

Vol 52 ◽

pp. 784-793 ◽

Cited By ~ 1

Author(s):

Da Wang ◽

Qinfu Zhao ◽

Tongying Jiang ◽

Luping Sha ◽

Siling Wang ◽

...

Keyword(s):

Amino Acid ◽

Mesoporous Silica ◽

Silica Nanoparticles ◽

Mesoporous Silica Nanoparticles ◽

Amino Acid Transporter ◽

Acid Transporter

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Clinicopathological Significance of L-type Amino Acid Transporter 1 (LAT1) Expression in Patients with Adenoid Cystic Carcinoma

Pathology & Oncology Research ◽

10.1007/s12253-013-9624-2 ◽

2013 ◽

Vol 19 (4) ◽

pp. 649-656 ◽

Cited By ~ 8

Author(s):

Kyoichi Kaira ◽

Minoru Toyoda ◽

Masato Shino ◽

Koichi Sakakura ◽

Katsumasa Takahashi ◽

...

Keyword(s):

Amino Acid ◽

Adenoid Cystic Carcinoma ◽

Amino Acid Transporter ◽

Adenoid Cystic ◽

Clinicopathological Significance ◽

Acid Transporter

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B0AT1 amino acid transporter complexed with SARS-CoV-2 receptor ACE2 forms a heterodimer functional unit: in situ conformation using radiation inactivation analysis

Function ◽

10.1093/function/zqab027 ◽

2021 ◽

Author(s):

Bruce R Stevens ◽

J Clive Ellory ◽

Robert L Preston

Keyword(s):

Amino Acid ◽

Membrane Trafficking ◽

Functional Unit ◽

Membrane Vesicles ◽

High Energy ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

Transport Activity ◽

Acid Transporter

Abstract The SARS-CoV-2 receptor, Angiotensin Converting Enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared to all other human tissues. Enterocyte ACE2 is co-expressed as the apical membrane trafficking partner obligatory for expression and activity of the B0AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2: B0AT1]2 dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2: B0AT1] heterodimer pair constitutes a physiological “functional unit.” This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles that were exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on established target theory, the results indicated the presence of Na+-dependent neutral amino acid influx transport activity functional unit with target size mw = 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2: B0AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2: B0AT1]2 dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut-lung axis approaches to understanding COVID-19. These findings advance the understanding of the physiology of B0AT1 interaction with ACE2 in the gut, and thereby potentially contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul Post-Acute Sequelae of SARS-CoV-2 (PASC).

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Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

Methods and Protocols ◽

10.3390/mps4030051 ◽

2021 ◽

Vol 4 (3) ◽

pp. 51

Author(s):

Satish Kantipudi ◽

Daniel Harder ◽

Sara Bonetti ◽

Dimitrios Fotiadis ◽

Jean-Marc Jeckelmann

Keyword(s):

Amino Acid ◽

Pichia Pastoris ◽

Protein Complexes ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Expression Host ◽

Amino Acids And Derivatives ◽

Heterodimeric Complex ◽

Acid Transporter ◽

And Function

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

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