Heterologous Expression of Human SLC1A5v2 as a Functional Glutamine Transporter in Escherichia coli
2
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Neutral and non-essential amino acid, glutamine (Gln), plays an essential role in supplying nitrogen to all the amino acids and nucleotides in the mammalian body. Gln is also the most important carbon source that provides intermediates for gluconeogenesis and fatty acid synthesis and supplements the tricarboxylic acid cycle in fast-growing cancer cells. Among the known 14 Gln transporter genes, soluted carrier family 1 member 5 (SLC1A5) has been reported to be closely associated with cancer cell growth. Three variants (v1, v2, and v3) have been derived from SLC1A5. Here, we established a heterologous gene expression system for the active form of human SLC1A5 variant-2 (hSLC1A5v2) in Escherichia coli. v2 is the smallest variant that has not yet been studied. Four expression systems were investigated: pBAD, pCold, pET, and pQE. We also addressed the problem of codon usage bias. Although pCold and pET overexpressed hSLC1A5v2 in E. coli, they were functionally inactive. hSLC1A5v2 using the pBAD system was able to catalyze the successful transport of Gln, even if it was not highly expressed. Initial activity of hSLC1A5v2 for [14C] Gln uptake in E. coli reached up to 6.73 μmole·min-1·gDW-1 when the cell was induced with 80 mM L-arabinose. In this study, we demonstrated a heterologous expression system for the human membrane protein, SLC1A5, in E. coli. Our results can be used for the functional comparison of SLC1A5 variants (v1, v2, and v3) in future studies, to facilitae the developement of SLC1A5 inhibitors as effective anticancer drugs.
Abstract
Neutral and non-essential amino acid, glutamine (Gln), plays an essential role in supplying nitrogen to all the amino acids and nucleotides in the mammalian body. Gln is also the most important carbon source that provides intermediates for gluconeogenesis and fatty acid synthesis and supplements the tricarboxylic acid cycle in fast-growing cancer cells. Among the known 14 Gln transporter genes, soluted carrier family 1 member 5 (SLC1A5) has been reported to be closely associated with cancer cell growth. Three variants (v1, v2, and v3) have been derived from SLC1A5. Here, we established a heterologous gene expression system for the active form of human SLC1A5 variant-2 (hSLC1A5v2) in Escherichia coli. v2 is the smallest variant that has not yet been studied. Four expression systems were investigated: pBAD, pCold, pET, and pQE. We also addressed the problem of codon usage bias. Although pCold and pET overexpressed hSLC1A5v2 in E. coli, they were functionally inactive. hSLC1A5v2 using the pBAD system was able to catalyze the successful transport of Gln, even if it was not highly expressed. Initial activity of hSLC1A5v2 for [14C] Gln uptake in E. coli reached up to 6.73 μmole·min-1·gDW-1 when the cell was induced with 80 mM L-arabinose. In this study, we demonstrated a heterologous expression system for the human membrane protein, SLC1A5, in E. coli. Our results can be used for the functional comparison of SLC1A5 variants (v1, v2, and v3) in future studies, to facilitae the developement of SLC1A5 inhibitors as effective anticancer drugs.
참고문헌 (24)
- Watford M, "Encyclopedia of Biological Chemistry III", pp.56~70 (2021), Elsevier
- Newsholme P, "Glutamine and glutamate—their central role in cell metabolism and function", Cell. Biochem. Funct, 21, pp.1~9 (2003)
- Nelson DL, "Lehninger Principles of Biochemistry", pp.859~901 (2017), W. H. Freeman
- Moffatt BA, "Purine and pyrimidine nucleotide synthesis and metabolism", pp.1~ (2002), Arabidopsis Book
- Anastasiou D, "Breathless cancer cells get fat on glutamine", Cell Res, 22, pp.443~446 (2012)
- Vander Heiden MG, "Understanding the Warburg effect: The metabolic requirements of cell proliferation", Science, 324, pp.1029~1033 (2009)
- Wang Y, "Coordinative metabolism of glutamine carbon and nitrogen in proliferating cancer cells under hypoxia", Nat. Commun, 10, pp.1~14 (2019)
- Hensley CT, "Glutamine and cancer:Cell biology, physiology, and clinical opportunities", J. Clin. Invest, 123, pp.3678~3684 (2013)
- Durán RV, "Glutaminolysis activates Rag-mTORC1signaling", Mol. Cell, 47, pp.349~358 (2012)
- Liu Y, "High expression of solute carrier family 1, member 5 (SLC1A5) is associated with poor prognosis in clear-cell renal cell carcinoma", Sci. Rep, 5, pp.1~10 (2015)
- Utsunomiya-Tate N, "Cloning and functional characterization of a system ASC-like Na+-dependent neutral amino acid transporter", J. Biol. Chem, 271, pp.14883~14890 (1996)
- Oppedisano F, "The glutamine", amino acid transporter (ASCT2) reconstituted in liposomes:Transport mechanism, regulation by ATP and characterization of the glutamine, Biochim. Biophys. Acta Biomembr, pp.2007, 1768[DOI]
- Reynolds MR, "Control of glutamine metabolism by the tumor suppressor Rb", Oncogene, 33, pp.556~566 (2014)
- Wise DR, "Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction", Proc. Natl. Acad. Sci. USA, 105, pp.18782~18787 (2008)
- Chen Z, "Cross-talk between ER and HER2 regulates c-MYC-mediated glutamine metabolism in aromatase inhibitor resistant breast cancer cells", J. Steroid Biochem. Mol. Biol, 149, pp.118~127 (2015)
- Yoo HC, "A variant of SLC1A5 is a mitochondrial glutamine transporter for metabolic reprogramming in cancer cells", Cell Metab, 31, pp.267~283 (2020)
- Kim OB, "The L-tartrate", succinate antiporter TtdT (YgjE) of L-tartrate fermentation in Escherichia coli, 2007, pp.189, 1597~1603
- Galluccio M, "Low temperature bacterial expression of the neutral amino acid transporters SLC1A5 (ASCT2) and SLC6A19 (B0AT1)", Mol. Biol. Rep, 47, pp.7283~7289 (2020)
- van Heeswijk WC, "Nitrogen assimilation in Escherichia coli: Putting molecular data into a systems perspective", Microbiol. Mol. Biol. Rev, 77, pp.628~695 (2013)
- Velappan N, "Plasmid incompatibility: More compatible than previously thought?", Protein Eng. Des. Sel, 20, pp.309~313 (2007)
- Spadiut O, "Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from Trametes multicolor in E. coli", Microb. Cell Fact, 9, pp.1~9 (2010)
- Yu X, "Cryo-EM structures of the human glutamine transporter SLC1A5 (ASCT2) in the outward-facing conformation", Elife, 8, pp.e48120~ (2019)
- Jiang Y, "Computational methods for protein localization prediction", Comput. Struct. Biotechnol. J, 19, pp.5834~5844 (2021)
- Vögtle FN, "Global analysis of the mitochondrial Nproteome identifies a processing peptidase critical for protein stability", Cell, 139, pp.428~439 (2009)