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Molecular Determinants for Nitric Oxide Regulation of the Murine Cationic Amino Acid Transporter CAT-2A
Biochemistry ( IF 2.9 ) Pub Date : 2020-11-02 , DOI: 10.1021/acs.biochem.0c00729
Ruifang Zheng 1 , Gabriela da Rosa 2, 3, 4 , Pablo D. Dans 3, 4 , R. Daniel Peluffo 1, 4
Affiliation  

Cationic amino acid transporters (CATs) supply cells with essential and semiessential dibasic amino acids. Among them, l-arginine is the substrate for nitric oxide synthases (NOS) to produce nitric oxide (NO), a key signaling molecule and second messenger. In cardiac preparations, we showed that NO acutely and directly modulates transport activity by noncompetitively inhibiting these CATs. We hypothesize that this NO regulation occurs through modification of cysteine residues in CAT proteins. Homology modeling and a computational chemistry approach identified Cys347 as one of two putative targets for NO binding, of 15 Cys residues present in the low-affinity mouse CAT-2A (mCAT-2A). To test this prediction, mammalian cell lines overexpressing mCAT-2A were used for site-directed mutagenesis and uptake studies. When Cys347 was replaced with alanine (Cys347Ala), mCAT-2A became insensitive to inhibition by NO donors. In addition, the transport capacity of this variant decreased by >50% compared to that of the control, without affecting membrane expression levels or apparent affinities for the transported amino acids. Interestingly, replacing Cys347 with serine (Cys347Ser) restored uptake levels to those of the control while retaining NO insensitivity. Other Cys residues, when replaced with Ala, still produced a NO-sensitive CAT-2A. In cells co-expressing NOS and mCAT-2A, exposure to extracellular l-arginine inhibited the uptake activity of control mCAT-2A, via NO production, but not that of the Cys347Ser variant. Thus, the -SH moiety of Cys347 is largely responsible for mCAT-2A inhibition by NO. Because of the endogenous NO effect, this modulation is likely to be physiologically relevant and a potential intervention point for therapeutics.

中文翻译:

一氧化氮调节小鼠阳离子氨基酸转运蛋白CAT-2A的分子决定因素。

阳离子氨基酸转运蛋白(CAT)为细胞提供必需和半必需的二元氨基酸。其中,1-精氨酸是一氧化氮合酶(NOS)产生一氧化氮(NO),关键信号分子和第二信使的底物。在心脏制剂中,我们显示NO通过非竞争性抑制这些CAT来直接和直接调节转运活性。我们假设这种NO调节是通过修饰CAT蛋白中的半胱氨酸残基而发生的。同源建模和计算化学方法鉴定为Cys 347低亲和力小鼠CAT-2A(mCAT-2A)中存在的15个Cys残基是NO结合的两个推定靶标之一。为了检验这一预测,将过度表达mCAT-2A的哺乳动物细胞系用于定点诱变和摄取研究。当将Cys 347替换为丙氨酸(Cys 347 Ala)时,mCAT-2A对NO供体的抑制不敏感。另外,与对照相比,该变体的转运能力降低了> 50%,而没有影响膜表达水平或对转运氨基酸的表观亲和力。有趣的是,用丝氨酸取代Cys 347(Cys 347Ser)将吸收水平恢复至对照水平,同时保持NO不敏感。当其他Cys残基替换为Ala时,仍然会产生对NO敏感的CAT-2A。在共表达NOS和mCAT-2A的细胞中,暴露于细胞外1-精氨酸可通过NO产生抑制对照mCAT-2A的摄取活性,但不能抑制Cys 347 Ser变体的摄取活性。因此,Cys 347的-SH部分在很大程度上负责NO对mCAT-2A的抑制作用。由于内源性NO的作用,这种调节可能与生理相关,并且可能成为治疗的潜在干预点。
更新日期:2020-11-12
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