2019 Fiscal Year Research-status Report
Molecular mechanism of the pathogenic protein interaction at the C-terminus of amino acid transporter b0,+AT/SLC7A9 in Japanese-type cystinuria
| Project/Area Number |
19K07373
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| Research Institution | Nara Medical University |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
コンプラシャ ポーンパン 奈良県立医科大学, 医学部, 助教 (70817767)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | Transporter / Cystinuria / Cystine / Amino acids / SLC7 / Kidney disease |
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| Outline of Annual Research Achievements |
P482L is a mutation on the amino acid transporter b0,+AT (SLC7A9). This pathogenic mutation causes Japanese-type cystinuria. b0,+AT forms heterodimeric complex with a regulatory protein called rBAT (SLC3A1). To establish and apply multiple analysis system to determine P482L pathogenic mechanism, I planned to purify both wild-type b0,+AT and the P482L mutant along with their heterodimeric partner rBAT. I applied serum-free cell suspension culture system for the large-scale protein production. The transfection method and cell culturing condition were optimized. cDNAs of rBAT-b0,+AT and P482L mutant were cloned and the sequences were confirmed. The cDNA clones were transfected into suspension cells. The results showed that all proteins were well expressed. Protein purification was performed by the method which we successfully established and applied to purify another member of heterodimeric amino acid transporter (HAT) family. With this method, high yield of membrane proteins can be obtained. Currently, we have succeeded to purified all proteins and have been continuedly producing them in a large scale for adequate protein materials. The purified proteins will be used in all analysis.
Determination of P482L pathogenic mechanism will be examined by using multiple analytical assays. Optimization of analytical methods are well progressed. The functional study of the purified the HAT protein provides us not only the purification method but also good tips on analytical assays.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In overall, the research has exceeding progress. The steps of protein production and optimization of analytical systems are proceeded faster than the original plan. This is due to the success of structural and functional study of another member of heterodimeric amino acid transporter (HAT) family. The HAT member shares conserved sequences with rBAT-b0,+AT, probably 3D structure and transport cycle as well. Our research teams and collaborators have established methods for protein expression, protein purification and functional analysis. Purified HAT has been produced and the function was well characterized along with the structure which was solved at the high resolution. Achievement of this study was recently published. Based on the study, we are able to apply several methods and optimize them to be suitable for the study of rBAT-b0,+AT and rBAT-P482L in this research plan. The current status is in the optimization processes of analytical methods.
Some of our analytical assays were originally planned to be conducted at the collaborator’s laboratories (in Osaka and USA). This part needs to be postponed due to the restriction on the traveling regarding the emergency control of COVID-19 pandemic. To solve the problem, we rearrange our schedule to focus on other assays which we are able to proceed at our laboratory, Nara Medical University. Moreover, we are developing a novel assay. Combination of multiple assay systems will convince the results and outcome.
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| Strategy for Future Research Activity |
The future work and strategies are planned as described in the original research plan without major change.
The research plan has two scientific purposes. To clarify these purposes, we aim to utilize several strategies as described in the original research plan. Most of them were planned to be conducted at Nara Medical University while other few were originally planned to be conducted in Osaka and USA. All strategies were formerly planned to be started in FY2019 and partially finished in FY2020. In this regard, some strategies are on-going as planned and will be taken as priorities. However, few experiments which were planned to be conducted in Osaka and USA will be postponed from FY2020 to FY2021 due to the current travel restriction regarding COVID-19 pandemic.
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| Causes of Carryover |
A part of the budget was originally planned to be used for traveling fee in order to conduct an experiment in the collaborator's laboratory. However, due to the traveling restriction regarding COVID-19 pandemic controlling, this traveling plan is postponed to FY2020 instead.
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[Journal Article] Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc2019
Author(s)
Lee Y, Wiriyasermkul P, Jin C, Quan L, Ohgaki R, Okuda S, Kusakizako T, Nishizawa T, Oda K, Ishitani R, Yokoyama T, Nakane T, Shirouzu M, Endou H, Nagamori S, Kanai Y, Nureki O
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Journal Title
Nature Structural & Molecular Biology
Volume: 26(6)
Pages: 510-517
DOI
Peer Reviewed / Int'l Joint Research
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