Nanosensor detection of an immunoregulatory tryptophan influx/kynurenine efflux cycle.
Nanosensor detection of an immunoregulatory tryptophan influx/kynurenine efflux cycle.
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DOI：
10.1371/journal.pbio.0050257
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发表时间：
2007-10
期刊：
影响因子：
9.8
通讯作者：
Frommer WB
中科院分区：
文献类型：
--
作者：
Kaper T;Looger LL;Takanaga H;Platten M;Steinman L;Frommer WB
哺乳动物细胞依赖于细胞对必需氨基酸色氨酸的摄取。通过上调色氨酸降解的关键酶吲哚胺2，3-双加氧酶（IDO），例如，在癌症和炎症中，被认为通过T细胞饥饿抑制免疫反应。此外，排泄的色氨酸catenine（犬尿氨酸）诱导淋巴细胞凋亡。虽然色氨酸转运系统已被确定，但犬尿氨酸输出的分子性质仍然未知。为了测量细胞溶质色氨酸稳态水平和流量在真实的时间，我们开发了基因编码的荧光共振能量转移纳米传感器（FLIPW）。通过FLIPW在人口腔癌细胞系KB细胞和COS-7细胞中检测到的转运特性涉及LAT 1，一种存在于增殖组织如癌症中的转运蛋白，参与色氨酸摄取。重要的是，我们发现这种转运系统介导色氨酸/犬尿氨酸交换。色氨酸内流/犬尿氨酸外流循环将色氨酸饥饿与犬尿氨酸血清水平的升高偶联，提供了邻近细胞中细胞凋亡的双管齐下的诱导。严格偶联保护过度产生IDO的细胞免于犬尿氨酸积累。因此，这种机制可能有助于参与自身免疫和肿瘤免疫逃逸的免疫抑制。尽管免疫系统的调节性抑制可以防止自身免疫，并且在怀孕期间保护胎儿或器官移植后阻止移植物排斥反应是重要的，但如果被肿瘤吸收以逃避检测，则可能是有害的。免疫系统的T细胞通常识别并破坏异常细胞，包括癌组织和移植组织。这个过程需要色氨酸。外来移植物和癌细胞可以通过使T细胞缺乏色氨酸来抑制免疫反应，这是通过一种机制实现的，该机制涉及使用一种称为吲哚胺-2，3-双加氧酶（IDO）的酶摄取和转化为犬尿氨酸。独立于色氨酸饥饿，犬尿氨酸通过未知机制排泄时诱导T细胞死亡。我们构建了荧光纳米传感器，报告两个永生化细胞系，COS-7细胞和人口腔癌KB细胞中的色氨酸水平。使用单细胞实时成像分析，我们证明L-氨基酸转运蛋白（LAT 1）将色氨酸交换为其犬尿氨酸降解产物。LAT 1与IDO一起形成代谢微生物群，其将色氨酸饥饿与犬尿氨酸诱导的细胞死亡偶联，提供相邻细胞的双管齐下的失活。严格偶联保护过度产生IDO的细胞免于积累犬尿氨酸。这种循环可能有助于抑制自身免疫性疾病和癌症的免疫反应。基因编码的FRET纳米传感器被开发用于测量胞质色氨酸稳态水平和真实的时间通量。
Mammalian cells rely on cellular uptake of the essential amino acid tryptophan. Tryptophan sequestration by up-regulation of the key enzyme for tryptophan degradation, indoleamine 2,3-dioxygenase (IDO), e.g., in cancer and inflammation, is thought to suppress the immune response via T cell starvation. Additionally, the excreted tryptophan catabolites (kynurenines) induce apoptosis of lymphocytes. Whereas tryptophan transport systems have been identified, the molecular nature of kynurenine export remains unknown. To measure cytosolic tryptophan steady-state levels and flux in real time, we developed genetically encoded fluorescence resonance energy transfer nanosensors (FLIPW). The transport properties detected by FLIPW in KB cells, a human oral cancer cell line, and COS-7 cells implicate LAT1, a transporter that is present in proliferative tissues like cancer, in tryptophan uptake. Importantly, we found that this transport system mediates tryptophan/kynurenine exchange. The tryptophan influx/kynurenine efflux cycle couples tryptophan starvation to elevation of kynurenine serum levels, providing a two-pronged induction of apoptosis in neighboring cells. The strict coupling protects cells that overproduce IDO from kynurenine accumulation. Consequently, this mechanism may contribute to immunosuppression involved in autoimmunity and tumor immune escape. Although regulated suppression of the immune system prevents autoimmunity and is important during pregnancy to protect the fetus or after organ transplant to block graft rejection, it can be harmful if co-opted by tumors to escape detection. T cells of the immune system normally recognize and destroy abnormal cells, including cancerous and grafted tissues. This process requires the amino acid tryptophan. Foreign grafts and cancer cells can dampen the immune response by starving T cells of tryptophan through a mechanism involving uptake and conversion to kynurenines using an enzyme called indoleamine-2,3-dioxygenase (IDO). Independent of tryptophan starvation, kynurenines induce T cell death when they are excreted through an unknown mechanism. We constructed fluorescent nanosensors that report tryptophan levels in two immortalized cell lines, COS-7 cells and human oral cancer KB cells. Using single-cell, real-time imaging analysis, we demonstrate that an L-amino acid transporter protein (LAT1) exchanges tryptophan for its kynurenine degradation products. LAT1 together with IDO forms a metabolic minicycle that couples tryptophan starvation with kynurenine-induced cell death, providing a two-pronged inactivation of neighboring cells. The strict coupling protects the cells that overproduce IDO from accumulating kynurenine. This cycle may contribute to suppression of the immune response in autoimmune diseases and cancer. Genetically encoded FRET nanosensors were developed to measure cytosolic tryptophan steady-state levels and flux in real time.
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影响因子：
2.7
作者：
Fehr, M;Lalonde, S;Frommer, WB
通讯作者：
Frommer, WB
影响因子：
4.4
作者：
Kim, DK;Kanai, Y;Endou, H
通讯作者：
Endou, H
影响因子：
3.9
作者：
Bröer, S;Cavanaugh, JA;Rasko, JEJ
通讯作者：
Rasko, JEJ
DOI：
10.1073/pnas.142089199
发表时间：
2002-07-23
影响因子：
11.1
作者：
Fehr, M;Frommer, WB;Lalonde, S
通讯作者：
Lalonde, S
影响因子：
3.5
作者：
LaMorte, VJ;Zoumi, A;Tromberg, BJ
通讯作者：
Tromberg, BJ