ENT3 utilizes a pH Sensing Mechanism for Transport.
ENT3 utilizes a pH Sensing Mechanism for Transport.
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ENT3 利用 pH 传感机制进行运输。
DOI：
10.1080/19336950.2017.1389581
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Govindarajan,Rajgopal
中科院分区：
文献类型：
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作者：
Singh,Anusha;Govindarajan,Rajgopal
平衡核苷转运蛋白 3 (ENT3) 由 SLC29A3 基因编码，是主要的酸性 pH 依赖性核苷转运蛋白，负责维持溶酶体和潜在线粒体区室中的核苷稳态。 1, 2 再加上这一特征，它也是最常见突变的核苷转运蛋白之一，并导致许多人类遗传性疾病，包括 H 综合征、胰岛素依赖型糖尿病 (PHID)、费萨拉巴德组织细胞增多症 (FHC)、伴有大量淋巴结病的窦组织细胞增多症 (SHML)、罗赛·多夫曼病 (RDD) 和骨骼营养不良。 3 尽管最近的研究认识到许多上述 ENT3 疾病为溶酶体储存或线粒体样疾病，但 ENT3 转运的分子基础以及因此 ENT3 疾病的分子发病机制尚不清楚。在我们最近发表的研究中，4我们在注射了 ENT 突变 RNA 的非洲爪蟾卵母细胞中采用了定点诱变、同源建模和 3H-腺苷通量测量，以确定 pH 依赖性 ENT3 内源核苷转运背后的分子机制。正如早期研究 2、5 中所见，我们通过删除 N 端 36 个氨基酸，使 ENT3 能够定位在细胞表面，研究了 ENT3 对 pH 的响应，并注意到在 pH 范围高于 6.5 时，ENT3 的活性急剧下降。为了分析 ENT 家族不同成员的 pH 敏感性，我们得出了 r 值，即 pH 7.4 时的运输通量/pH 5.5 时的运输通量，阐明 pH 依赖性最强的核苷转运蛋白是 ENT3。矛盾的是，虽然 ENT3 主要在酸性条件下发挥作用，但注射的 hENT1 和 2 卵母细胞的功能即使在酸性 pH 值下仍然强劲，这与它们在细胞膜中表达且通常遇到较高 pH 范围的事实相反。尽管发现动质体原生动物中的ENT3是产电的，但人类ENT3是否利用质子共转运进行渗透易位尚不清楚。我们的研究认识到 ENT3 作为非产电转运蛋白的作用，并支持质子转运与腺苷转运不同时进行。这使我们推测 ENT3 中质子和氨基酸之间的相互作用，而不是生电性，负责酸性 pH 激活的转运性。因此，我们测试了 ENT3 中组氨酸残基的 pH 传感能力，因为它们在 ENT3 发挥作用的精确 pH 范围内表现出 pKa。在 pH 范围 5.5 和 7.4 下，用不可电离的氨基酸 Ala 替代会导致 3H-腺苷转运至卵母细胞的能力丧失。然而，为了确保这种可转运性的减弱是由于 His 无法感知 pH 值造成的，而不是由于其他可能的属性（例如底物识别丢失和膜靶向不当）造成的，我们进行了后续替代研究，检查了 ENT3 在 pH 5.5 和 7.4 中这些位置上的带正电、带负电和中性氨基酸。出乎意料的是，我们的结果表明组氨酸在 pH 传感中发挥相对较小的作用。接下来，我们
Equilibrative nucleoside transporter 3 (ENT3), encoded by the SLC29A3 gene, is the major acidic pH dependent nucleoside transporter responsible for maintaining nucleoside homeostasis in lysosomal, and potentially, in mitochondrial compartments. 1, 2 Coupled with that characteristic, it is also amongst the most frequently mutated nucleoside transporters, and is responsible for numerous human genetic disorders including H syndrome, Insulin-dependent diabetes (PHID), Faisalabad histiocytosis (FHC), Sinus histiocytosis with massive lymphadenopathy (SHML), Rosai Dorfman Disease (RDD) and skeletal dystrophy. 3 Despite, recent studies recognizing many of the above mentioned ENT3 disorders as lysosomal-storage or mitochondria-like disorders, the molecular basis of ENT3 transport, and thus, the molecular pathogenesis of ENT3 disorders are unknown. In our recently published study, 4 we employed sitedirected mutagenesis, homology modeling and 3H-adenosine flux measurements in ENT mutant RNA-injected Xenopus oocytes to identify the molecular mechanisms behind pH-dependent ENT3 transport of endogenous nucleosides. As seen in earlier studies, 2, 5 we investigated the response of ENT3 to pH by deleting the N-terminal 36 amino acids, enabling ENT3 to localize on the cell surface, and noticed a sharp decline in the activity of ENT3 in pH ranges above 6.5. To analyze pH sensitivity of the different members of the ENT family we derived an r value, Transport Flux at pH 7.4/Transport flux at pH 5.5, elucidating that the most pH dependent nucleoside transporter is ENT3.Paradoxically, while ENT3 primarily functioned in acidic conditions, the functioning of hENT1 and 2 injected oocytes was still robust even at acidic pH values, contrary to the fact they are expressed in the cell membrane and typically encounter a higher pH range. Although it was discovered that ENT3 in kinetoplastoid protozoans is electrogenic, it was unknown whether human ENT3 utilized proton co-transport for permeant translocation. Our study discerned the role of ENT3 as a non-electrogenic transporter and supported that proton transport is not concurrent with adenosine transport. This led us to hypothesize that the interactions between protons and amino acids in ENT3, rather than electrogenicity, are responsible for acidic pH-activated transportability. Consequently, we tested the pH sensing capacity of histidine residues in ENT3 since they exhibit a pKa at the precise pH range that ENT3 functions. Substitution with nonionizable amino acid Ala caused a loss of transportability of 3H-adenosine into oocytes at pH ranges 5.5 and 7.4. However, in order to ensure that this attenuation of transportability resulted from the inability of His to sense pH, and not due to other possible attributes such as loss of substrate recognition and improper membrane targeting, we carried out subsequent substitution studies examining the positively charged, negatively charged and neutral amino acids at these positions in ENT3 in both pH 5.5 and 7.4. Unexpectedly, our results suggested that histidines play a relatively minor role in pH sensing. Next, we