这也算化学？
饶议科学约 41 分钟2025年1月21日，学术刊物ACS Sensors（《美国化学学会 感受器》）在线发表：
基因编码开发一种用于检测精氨酸的探针
王纯1，张小雪1，毛浩雨1,2，咸逸2,3, *，饶毅1,2,3, *
1首都医科大学、首都医学科学创新中心
2北京大学 化学与分子工程学院、北大/清华生命科学联合中心、生命科学学院、医学部药学院、麦戈文脑科学研究所
3北京脑科学研究所
*通讯作者
摘要
L-精氨酸（Arg）在多种代谢和生理过程中发挥重要作用，其浓度的变化与病理过程密切相关。尽管直接且实时测量生物系统中的Arg水平非常重要，但现有的Arg探针对L-鸟氨酸或L-赖氨酸也有响应。本文报道了一种新的Arg探针ArgS1。该探针对Arg表现出浓度依赖性的Ex488/405比率增加，其表观亲和力约为64 μM，动态范围（ΔR/R0）为3。ArgS1在细胞质和亚细胞器中对Arg均有响应。ArgS1成功监测了MDA-MB-231细胞（一种缺乏Arg合成关键酶——精氨琥珀酸合成酶1（ASS1）的乳腺癌细胞系，且适用于Arg耗竭疗法）中的Arg水平。研究发现，当细胞外Arg被耗竭后，MDA-MB-231细胞中的Arg水平下降，同时细胞活力也随之降低。当细胞中过表达ASS1时，Arg水平上升，细胞活力也得到增强。因此，ArgS1是一种在生理和病理相关动态范围内实时监测人类细胞中Arg水平的有效工具。
L-精氨酸（Arg）是一种条件性必需氨基酸，在人体中具有重要的生理作用 [1,2]。Arg的主要来源包括蛋白质降解、膳食摄入和从头合成[3]。Arg的合成涉及两种关键酶：精氨琥珀酸合成酶（ASS），它将L-瓜氨酸（Cit）转化为精氨琥珀酸；以及精氨琥珀酸裂解酶（ASL），它将精氨琥珀酸裂解为Arg[4,5]。Arg在一氧化氮合酶（NOS）的催化下生成Cit和一氧化氮（NO），Arg是NO的唯一直接前体[6-9]。Arg还参与尿素循环，在精氨酸酶（ARGs）的作用下被水解为尿素和L-鸟氨酸（Orn）[10,11]，这对于氨的解毒至关重要[12]。精氨酸：甘氨酸脒基转移酶（AGAT）是一种线粒体酶，它催化Arg生成胍基乙酸，这是肌酸合成的直接前体[13-15] 。Arg代谢紊乱与多种疾病相关[5,16]。精氨酸酶1（ARG1）缺乏会导致高精氨酸血症，这是一种以进行性神经系统症状为特征的罕见遗传性代谢疾病[17] 。Arg还与多种疾病的治疗相关，例如勃起功能障碍[18,19]、高血压[20]和心力衰竭[21]。
癌细胞的生存和生长需要氨基酸[22,24]，其中Arg是肿瘤微环境中的重要组成部分[25-27] 。某些类型的癌症，如乳腺癌[28]、黑色素瘤[29]、肝细胞癌[30]、急性淋巴细胞白血病（ALL）[31] 和急性髓系白血病（AML）[32]，其ASS表达缺失。这些类型的癌细胞依赖摄取细胞外Arg以维持生存，因此限制Arg供应已被探索为癌症治疗中的一种潜在辅助治疗策略[27,33]。在培养基中去除Arg或添加ADI-PEG20（聚乙二醇化精氨酸脱亚氨酶）可导致ASS1缺陷型癌细胞在体外被清除[28,34]。系统性给予ADI或精氨酸酶以限制Arg可用性，已在肿瘤治疗的II期临床试验中进行了研究[35]。这些研究表明，ASS1缺陷是AML患者对ADI-PEG20单药治疗产生反应的必要条件，但并非充分条件。Arg的测量是确定患者是否适合接受ADI-PEG20治疗AML的重要指标。
传统的Arg测量方法包括样品的微透析[36,37] 和化学分析[38]，这些方法无法研究细胞内分布的变化，也无法以必要的空间和时间分辨率分析Arg。荧光探针提供了非侵入性实时测量浓度及其时空变化的可能性。现有的基因编码Arg荧光探针基于Förster共振能量转移（FRET），其动态范围相对较小[39-41]。这些探针在体外报道的最大动态范围仅为约0.6[40]。更大的问题在于其特异性：这些探针不仅对Arg有响应，还对其他氨基酸（如Orn、L-赖氨酸（Lys）、L-谷氨酰胺（Gln）和L-组氨酸（His））有响应[39-41] 。此外，这些探针对Arg的解离常数（Kd）（9.4或14 μM[39,40]）不适合检测生理水平的Arg，因为血浆[42]和细胞质[43]中的Arg浓度通常约为100 µM。
因此，需要开发具有大动态范围和合适Kd的基因编码Arg特异性荧光探针，以用于生理和病理条件下Arg的检测。
结果
Arg探针的设计、优化及体外表征
我们基于大肠杆菌（E. coli）的artJ（一种周质结合蛋白，PBP）设计了一种基因编码的荧光Arg探针。artJ在结合Arg时会发生构象变化[44-46]。与其他探针的设计类似 [47-50]，我们将来自GRABDA2m的环状置换绿色荧光蛋白（cpEGFP）[51] 整合到artJ中（图1A、B）。根据artJ和argT（一种Arg、Lys和Orn结合蛋白）的晶体结构，其叶II的环区和铰链区在底物结合时会发生显著构象变化[45,46,52-56]，这为cpEGFP的插入提供了潜在的位点。首先，我们在纯化蛋白上筛选了cpEGFP的插入位点（图1C）。artJ与cpEGFP之间的连接肽设计为柔性（Gly-Gly）或刚性（Pro-Pro）。基于初步筛选结果，我们选择了动态范围大于0.1的探针（图1D）。考虑到基于蛋白纯化的筛选方法耗时较长，且在体外响应的探针在细胞环境中可能表现不同，我们转而使用HEK293T细胞进行筛选。通过将探针与iSeroSnFR的膜靶向序列融合[57]，我们将探针靶向到人胚胎肾293T（HEK293T）细胞的细胞膜上，以优化连接肽的长度和氨基酸组成，从而最小化细胞内Arg对探针响应的影响。我们发现，在HEK293T细胞膜上表现出良好响应的探针的响应值分别为0.4和0.6，分别命名为ArgS0.1和ArgS0.2（图1C、D，图S1C）。随后，我们优化了连接肽邻近位点，发现响应最佳的探针的响应值为0.9，命名为ArgS1（图1D，图S1C）。总结而言，通过对1200多种不同变体的筛选，我们最终确定了三种探针，分别命名为ArgS0.1、ArgS0.2和ArgS1（图1D）。
图1 ArgS探针的体外测量
A, B ArgS探针的设计。通过不同的插入位点将cpEGFP整合到大肠杆菌artJ蛋白中。我们将cpEGFP与artJ之间的氨基酸连接肽称为N端连接肽和C端连接肽。该模型使用AlphaFold2生成。C ArgS探针的筛选流程。第1步是在纯化蛋白上进行插入位点筛选，第2至第4步是在HEK293T细胞膜上进行筛选，其中第2步和第3步分别优化连接肽的长度和组成，第4步优化连接肽邻近位点。D ArgS探针的筛选结果。ArgS0.1、ArgS0.2和ArgS1探针分别用洋红色、蓝色和绿色突出显示。E ArgS1探针的氨基酸特异性。对1 mM各种氨基酸的响应归一化为对1 mM L-Arg的响应。每组数据点n = 3；数据以均值±标准误（SEM）表示。F ArgS1探针在有无1 mM Arg条件下的激发和发射光谱。G ArgS1探针在不同浓度Arg暴露下的剂量响应曲线。每组数据点n = 6；数据以均值±标准误（SEM）表示。
我们将这三种探针分别在大肠杆菌中表达并纯化，随后测试了它们对各种氨基酸的特异性。结果显示，所有三种ArgS探针均特异性地响应L-Arg，而对其他氨基酸的L型或D型均无响应（图1E，图S2A、B）。这三种ArgS探针均表现出比率特性，具有两个激发峰（分别位于400纳米（nm）和500 nm附近）和一个发射峰（位于515 nm附近）。当与1 mM Arg结合时，三种探针的400 nm激发峰降低，而500 nm激发峰增加（图1F，图S2C、D）。我们还测量了ArgS0.1、ArgS0.2和ArgS1的Arg结合亲和力，其Kd值分别为73 μM、367 μM和1141 μM，荧光峰值变化（ΔR/R0）分别为1.9、4.6和19（图1G，图S2E、F）。同时，我们评估了ArgS探针对精氨酸相关代谢物的特异性。ArgS0.1对胍丁胺、Cit和精氨琥珀酸表现出可检测的响应，而ArgS0.2和ArgS1仅对Cit和精氨琥珀酸有响应（图S3A、D、G）。在瓜氨酸（Cit）结合实验中，ArgS0.1、ArgS0.2和ArgS1的Kd分别为312 μM、1162 μM和2912 μM，其荧光峰值变化约为Arg的一半（图S3B、E、H）。对于精氨琥珀酸的结合，所有探针的Kd值均超过300 μM（图S3C、F、G）。为了减少Arg相关代谢物的潜在干扰，我们通过LC-MS定量分析了HEK293T和MDA-MB-231细胞中这些代谢物的细胞内浓度。在两种细胞系中，Cit和精氨琥珀酸的浓度均低于5 μM（HEK293T细胞中Cit为1.290 μM，MDA-MB-231细胞中Cit为3.920 μM；HEK293T细胞中精氨琥珀酸为1.556 μM，MDA-MB-231细胞中精氨琥珀酸为4.156 μM），均低于ArgS探针的检测阈值（图S3J）。因此，在检测HEK293T或MDA-MB-231细胞中的Arg水平时，Cit或精氨琥珀酸的潜在干扰可忽略不计。此外，基于嗜热脂肪土芽孢杆菌（Geobacillus stearothermophilus）artJ的晶体结构[45]，我们开发了三种ArgS1探针的突变版本。其中，双点突变版本ArgS1-F51L E114L对Arg几乎无响应（图S4C）。因此，该版本探针ArgS1-F51L E114L被命名为ArgS1-C，并作为后续实验的对照。为了研究pH依赖性，我们在pH 4至9的范围内测试了ArgS1和ArgS1-C。ArgS1在pH 7至8之间对Arg的响应最佳（图S4A），而ArgS1和ArgS1-C的apo形式在整个测试范围内表现出相似的pH依赖性行为（图S4B）。
Arg探针在HEK293T细胞质及亚细胞器中检测Arg变化
为了研究Arg探针是否能够在哺乳动物细胞中检测Arg，我们在HEK293T细胞的细胞质中表达了ArgS0.1、ArgS0.2和ArgS1（图2A）。Arg转运蛋白SLC7A1[58]和SLC7A2[59]在HEK293T细胞中高表达（图S5A）。当细胞外Arg浓度升高至1 mM时，我们立即观察到ArgS1、ArgS0.1和ArgS0.2在405 nm处的荧光强度下降，而在488 nm处的荧光强度增加（图2B，图S6A）。这表明所有三种探针（ArgS1、ArgS0.1和ArgS0.2）均对Arg的增加作出了响应，其最大动态范围分别为3.3、0.3和1.1（图2D，图S6D、E）。当Arg探针达到其峰值动态范围后，我们将灌注液切换回磷酸盐缓冲液（PBS），观察到三种探针在405 nm处的荧光强度立即增加，而在488 nm处的荧光强度下降，其响应恢复到基线值（图2C，图S6B、C）。
图2 ArgS1探针在HEK293T细胞质中对Arg浓度变化的响应
A ArgS探针在HEK293T细胞质中成像的示意图。在HEK293T细胞的细胞质中，C端融合IRES-mCherry序列的ArgS探针被转染，随后在不同溶剂灌注条件下使用共聚焦显微镜进行成像。B代表性图像显示ArgS1探针在HEK293T细胞质中对PBS或1 mM Arg变化在不同时间点（0分钟、10分钟、30分钟和50分钟）的响应。ArgS1探针的荧光在两个通道中可见：ArgS1 Ex488（激发波长488 nm）显示为绿色，而ArgS1 Ex405（激发波长405 nm）显示为蓝色。此外，通过IRES序列融合到ArgS探针C端的mCherry在mCherry通道中以红色显示。ArgS1探针的Ex488/Ex405比率变化进一步在ΔR/R0通道中可视化。比例尺：10 μm。C ArgS1在PBS或1 mM Arg中荧光响应的平均轨迹。我们在0分钟时将1 mM Arg灌注到表达ArgS1探针的HEK293T细胞中，并在35分钟时切换为PBS缓冲液。ArgS1探针在405 nm激发波长下的ΔF/F0以蓝色表示，而在488 nm波长下的ΔF/F0以绿色表示。mCherry的ΔF/F0以红色表示。紫色表示ΔR/R0（右侧Y轴）。每组n = 20个细胞；数据以均值±标准误（SEM）表示。D ArgS1探针对Arg灌注的最大响应。每组n = 20个细胞；数据以均值±标准误（SEM）表示。E ArgS1探针在HEK293T细胞质中的剂量依赖曲线及相应的Kd值。每组n = 600个细胞（来自6个孔）；数据以均值±标准误（SEM）表示。F HEK293T细胞中Arg浓度的药理学改变。ArgS1探针的488/405比率在分别与500 μM抑制剂孵育12、24、36和48小时前后的变化。对照组以黑色等边三角形表示，单独L-NMMA组以灰色菱形表示，单独AI1组以灰色圆形表示，L-NMMA和AI1联合组以深灰色方形表示。每组n = 600个细胞（来自6个孔）；数据以均值±标准误（SEM）表示。
为了在HEK293T细胞质中进行原位滴定以测量ArgS1的Arg结合亲和力，我们用毛地黄皂苷（digitonin）透化细胞膜，并向细胞中添加不同浓度（1至∼60000 μM）的Arg（图S5B）。通过绘制表达ArgS1的细胞平均响应与Arg浓度的关系，获得了原位校准曲线。因此，我们测量了ArgS1在HEK293T细胞质中的Arg结合亲和力，其Kd值为64 μM（图2E）。此外，为了最小化非特异性荧光信号变化，我们在HEK293T细胞中表达了非结合对照探针ArgS1-C。如图S5D所示，ArgS1-C对1 mM Arg灌注无响应。
为了确定Arg探针是否能够检测由药物干预引起的HEK293T细胞中Arg的变化，我们在这些细胞中表达了ArgS1。我们利用精氨酸酶I和II抑制剂（Arginase Inhibitor 1, AI1）[60] 和一氧化氮合酶（NOS）抑制剂L-NMMA [61-65]来调控Arg水平。如图2F所示，使用ArgS1检测到AI1或AI1与L-NMMA联合应用引起的细胞内Arg水平升高；然而，单独使用L-NMMA并未诱导类似的响应。这些结果表明，在HEK293T细胞中，精氨酸酶途径在Arg降解中的作用比NOS途径更为重要。这一结论与我们的转录组测序结果一致。在HEK293T细胞内，ARG2的表达水平最高，而ARG1和NOS2的表达水平非常低，NOS1则未表达（图S5C）。因此，ArgS1探针有效地监测了HEK293T细胞中药理干预引起的Arg浓度变化。
图3 ArgS1探针在HEK293T细胞器中对1 mM Arg灌注的响应
A ArgS1探针在不同细胞器中表达的示意图：线粒体、内质网（ER）、溶酶体和细胞核。B ArgS1探针定位在不同细胞区室的图像：线粒体外膜的胞质侧（Mito-ArgS1）、ER膜的胞质侧（ER-ArgS1）、溶酶体膜的胞质侧（Lyso-ArgS1）以及细胞核内（Nuc-ArgS1）。ArgS1探针的荧光在ArgS1通道中以绿色表示。细胞器特异性标记物，包括线粒体的Mito-Tracker、ER的ER-Tracker、溶酶体的Lyso-Tracker和细胞核的NucRed，在细胞器标记通道中以红色表示。ArgS1探针荧光与细胞器标记荧光的合并图像显示在Merge通道中。ArgS1探针对Arg灌注的响应在ΔR/R0通道中表示。比例尺：5 μm。C-F定位在不同细胞区室的ArgS1在PBS或1 mM Arg中荧光响应的平均轨迹。我们在0分钟时将1 mM Arg灌注到表达Mito-ArgS1、ER-ArgS1、Lyso-ArgS1和Nuc-ArgS1探针的HEK293T细胞中，并在达到最大响应后分别切换为PBS缓冲液。ArgS1探针在405 nm激发波长下的ΔF/F0以蓝色表示，而在488 nm波长下的ΔF/F0以绿色表示。ΔR/R0以紫色表示。每组n = 20个细胞；数据以均值±标准误（SEM）表示。G-J Mito-ArgS1、ER-ArgS1、Lyso-ArgS1和Nuc-ArgS1对1 mM Arg灌注的最大响应。每组n = 20个细胞；数据以均值±标准误（SEM）表示。
为了进一步研究Arg探针是否能够响应亚细胞器中的Arg，我们将ArgS1（图3）和ArgS0.1（图S7）靶向到不同的细胞器。我们将ArgS1和ArgS0.1分别与AKAP1的N端30个氨基酸引导序列[66]、p450的内质网（ER）靶向基序[67] 、LAMP1衍生的序列 [68]或核定位信号[69]融合，以定位到线粒体胞质侧（Mito-ArgS1和Mito-ArgS0.1）、ER胞质侧（ER-ArgS1和ER-ArgS0.1）、溶酶体（Lyso-ArgS1和Lyso-ArgS0.1）或细胞核（Nuc-ArgS1和Nuc-ArgS0.1）（图3A）。为了评估ArgS1在线粒体、ER、溶酶体和细胞核中的定位，我们分别使用Mito-Tracker、ER-Tracker、Lyso-Tracker和NucRed作为对照。结果显示，ArgS探针与相应的细胞器标记物高度共定位（图3B，图S7A）。当向培养HEK293T细胞的培养基中灌注1 mM Arg时，定位在细胞器中的ArgS探针也对Arg水平的变化作出了响应（图3C-F，图S7B-E）。Mito-ArgS1、ER-ArgS1、Lyso-ArgS1和Nuc-ArgS1探针的最大动态范围分别为3.7、3.8、3.2和3.8（图3G-J），而Mito-ArgS0.1、ER-ArgS0.1、Lyso-ArgS0.1和Nuc-ArgS0.1探针的最大动态范围分别为0.5、0.5、0.4和0.6（图S7F-I）。这些结果表明，ArgS探针在线粒体、ER和溶酶体附近区域以及细胞核内的响应相似。因此，我们成功开发了能够监测哺乳动物细胞质和亚细胞器中Arg的ArgS探针。
细胞外Arg剥夺导致癌细胞细胞内Arg水平下降
Arg饥饿正被探索为一种针对ASS1缺陷型癌症的潜在治疗策略[28,70]（图4A）。据报道，乳腺癌细胞系MDA-MB-231不表达ASS1[28] （图4B）。我们首先验证了Arg剥夺对细胞活力的影响。Arg剥夺24小时后，MDA-MB-231细胞的活力下降（图4C），这与之前的报道一致 [28]。此外，过表达ASS1能够回补MDA-MB-231细胞对Arg饥饿的敏感性（图4B、C）。为了观察细胞内Arg水平，我们开发了在MDA-MB-231细胞中稳定表达ArgS1的细胞系（图4A）。我们发现，Arg剥夺后，ArgS1探针的488/405比率及其衰减速率均显著下降，表明MDA-MB-231细胞内的Arg水平显著低于对照组（图4D、E，图S8B）。此外，Arg剥夺后，ASS1过表达组的488/405比率高于ASS1缺陷组，表明通过ASS1过表达回补了Arg水平（图4D、E）。此外，为了进一步减少非特异性荧光信号变化，我们在MDA-MB-231细胞中表达了ArgS1-C。如图S8A所示，ArgS1-C对1 mM Arg灌注也无响应。因此，ArgS1探针可用于监测癌细胞中的Arg水平。
图4 MDA-MB-231细胞中Arg的成像
A ArgS1探针在MDA-MB-231细胞中表达的示意图，包括有和无Arg饥饿条件下的情况。B MDA-MB-231细胞和ASS1过表达的MDA-MB-231细胞中ASS1的表达水平。C MDA-MB-231细胞（圆形）和ASS1过表达细胞（方形）在Arg饥饿（深红色）或完全DMEM对照组（黑色）处理24小时后的细胞活力。Arg饥饿组的细胞活力均归一化为完全DMEM对照组。每组n = 600个细胞（来自6个孔）；数据以均值±标准误（SEM）表示。D ArgS1探针在MDA-MB-231细胞和ASS1过表达的MDA-MB-231细胞中的荧光强度和响应变化在Arg饥饿30分钟前后成像。比例尺：10 μm。E ArgS1探针在MDA-MB-231细胞和ASS1过表达的MDA-MB-231细胞中的488/405比率变化在Arg饥饿2小时前和24小时后每15分钟成像一次。每组n = 600个细胞（来自6个孔）；数据以均值±标准误（SEM）表示。
讨论
为了比较ArgS探针与其他Arg探针，我们整理了各种Arg探针的特性，如表S1所示。例如，基于glnH的QBP/Citrine/ECFP探针对Arg的Kd为2.1 mM，动态范围为0.3[39]。此外，基于argT的FLIP-cpargT194探针对Arg的Kd为48 μM，动态范围为0.5[40]。然而，这些探针并非对Arg完全特异：QBP/Citrine/ECFP还对Orn有响应，而FLIP-cpargT194对Orn和Lys均有响应[39,40]。相比之下，FLIP-cpartJ185和FLIPR探针对Arg具有特异性，其Kd值分别为9.4 μM和14 μM，动态范围分别为0.5和0.3[40,41]。由于这些探针对Arg的响应和特异性有限，它们并不适合检测哺乳动物细胞内Arg浓度的变化。相比之下，ArgS探针——ArgS0.1、ArgS0.2和ArgS1的Kd值分别为73 μM、367 μM和1141 μM，荧光峰值变化（ΔR/R0）分别为1.9、4.6和19。这些响应优于以往的Arg探针，且ArgS探针特异性地响应Arg，而不会与其他氨基酸发生交叉反应。最近，研究人员报道了一种名为STAR的基因编码探针，它特异性地响应Arg，并能够在体外和体内监测Arg动态[71]。与STAR相比，ArgS1的荧光响应略大（ΔR/R0：19 vs. ~16，体外）。然而，与ArgS1不同，STAR不具备比率特性，因此需要额外的对照来校正探针表达水平的变化。
为了增强ArgS探针的最大响应，我们筛选了不同长度和组成的连接肽。结果表明，N端连接肽为2或3个氨基酸、C端连接肽为2个氨基酸时，响应更大，且N端连接肽的首个氨基酸优选为Gly。此外，在筛选过程的第4步中，测试了N端连接肽邻近的氨基酸残基。例如，将该残基替换为Ile显著提高了响应，从而开发出了ArgS1。尽管我们筛选了1200多个候选变体，但通过进一步扩展筛选范围以包括更多邻近连接肽位点，仍有可能实现进一步改进。
具体而言，HEK293T细胞质中的ArgS1的Kd值为64 μM，高于体外测量的值。类似地，iGluSnFR探针在HEK293T细胞或培养的海马神经元中的亲和力也高于体外观察到的值[47,49,72]。这种差异可能是由于蛋白质在不同环境和检测系统中的暴露差异，也可能是细胞环境中的辅助因子增强了Arg结合的结果。然而，即使在体外实验中加入细胞裂解液后，测得的Kd仍处于毫摩尔范围，这表明需要进一步研究。因此，为了准确量化不同环境中的Arg水平，应在多种实验条件下测量Kd以考虑这些因素。此外，ArgS1对Cit和精氨琥珀酸表现出可检测的响应。在测量不同生物环境中的Arg水平时，必须确保Cit和精氨琥珀酸的浓度低于ArgS1探针的检测阈值。
关于ArgS探针在不同亚细胞位置的动态特性，Nuc-ArgS1和ER-ArgS1的Arg灌注时间比其他位置短，这可能反映了亚细胞间Arg代谢的差异（图3C-F）。此外，我们尝试确定每个亚细胞位置的基线Arg浓度；然而，我们不确定灌注前的初始比率是否准确反映了基线浓度，因为探针游离形式的荧光比率可能在不同亚细胞区室中有所不同。此外，在每个亚细胞位置除去Arg以获得ArgS探针的游离形式具有挑战性。因此，我们选择在获得更可靠的方法来获取这些区室中ArgS探针的游离形式之前，不对亚细胞位置的Arg浓度进行量化。
Arg在生理和病理过程中发挥着关键作用；然而，其许多功能仍未得到充分理解。例如，星形胶质细胞和神经元之间Arg转运的机制尚不清楚，其作为信号分子的作用也未明确定义。此外，Arg浓度的变化可以与其他分子（如Ca²⁺、cAMP和Glu）一起使用互补的荧光探针进行监测。我们希望ArgS探针能够有助于阐明Arg代谢的机制，并为其生理和病理作用提供更深入的见解。
结论
总之，ArgS1能够实时检测多种哺乳动物细胞中Arg水平的变化，为基础研究和临床应用提供了宝贵的工具。这是首个能够动态监测哺乳动物细胞内Arg浓度变化的基因编码荧光探针。此外，ArgS1在亚细胞器中对Arg表现出动态响应，并已成功用于监测MDA-MB-231乳腺癌细胞中的Arg水平。
致谢
我们感谢李毓龙教授提供GRABDA2m质粒。我们感谢国家蛋白质科学基础设施——北京基地北京大学分设施在Operetta高内涵成像实验提供的帮助。我们感谢北京脑科学研究所生物质谱中心于晓倩老师在LC-MS实验提供的帮助。
附图：
附图 S1 in vitro measurements of ArgS0.1 and ArgS0.2 sensors.
A and B, Excitation and emission spectra of ArgS0.1 and ArgS0.2 sensors with and without 1 mM Arg.
C, Dose-response curves of ArgS0.1 and ArgS0.2 sensors when exposed to varying concentrations of Arg. n = 6 per data point.
D and E, Amino acid specificities of ArgS0.1 and ArgS0.2 sensors. Responses to 1 mM of various amino acids was normalized to their responses to 1 mM Arg. n = 3 per data point.
附图 S2 Transcriptome sequencing results of HEK293T and MDA-MB-231.
A, Sequencing results of Arg transport in HEK293T cell, including SLC7A1-7. n = 3 per data point.
B, Sequencing results of ARG1, ARG2, NOS1, NOS2, NOS3 and AGAT in HEK293T cell. n = 3 per data point.
附图 S3 Responses of ArgS0.1 and ArgS0.2 sensors in HEK293T cytoplasm to Arg concentration changes.
A, Representative images showing responses of the ArgS0.1 and ArgS0.2 sensors in the cytoplasm of HEK293T cells to changes in PBS or 1mM Arg. The fluorescence of the ArgS0.1 and ArgS0.2 sensor were visible in two channels: Ex488 (excitation at 488 nm) appeared green, while Ex405 (excitation at 405 nm) appeared blue. Additionally, mCherry, fused to the C-terminus of ArgS sensors via an IRES sequence, was represented in the mCherry channels in red. The change in the Ex488/ Ex405 ratio of the ArgS1 sensor was further visualized in the ΔR/R0channels. Scale bar, 10 μm.
B and C, Average traces of fluorescence responses of ArgS0.1 and ArgS0.2 sensors measured in PBS or 1 mM Arg. ΔF/F0of the ArgS0.1 and ArgS0.2 sensors under a 405 nm excitation wavelength was represented in blue, while ΔF/F0 under a 488 nm wavelength was in green. The ΔF/F0 of mCherry was represented in red. Purple represented ΔR/R0 (the right Y-axis). n = 20 cells.
D and E, Maximum responses of ArgS0.1 and ArgS0.2 sensors to Arg perfusion. n = 20 cells.
附图 S4 Response of ArgS0.1 sensor in HEK293T organelles to 1 mM Arg perfusion.
A, Images of ArgS0.1 sensor localized in various cellular compartments: on the cytoplasmic side of the mitochondrial outer membrane (Mito-ArgS0.1), on the cytoplasmic side of the ER membrane (ER-ArgS0.1), on the cytoplasmic side of the lysosomal membrane (Lyso-ArgS0.1) and in the nucleus (Nuc-ArgS0.1). The fluorescence of the ArgS0.1 sensor was represented in the ArgS0.1 channels in green. Organelle-specific markers, Mito-Tracker for mitochondria, ER-Tracker for ER, Lyso-Tracker for lysosomes and NucRed for the nucleus, were represented in the organelle marker channel in red. The combined image of ArgS0.1 sensor fluorescence and organelle marker fluorescence were displayed in the Merge channels. The response of ArgS0.1 sensor to Arg perfusion were represented inΔR/R0 channels. Scale bar, 5 μm.
B, C, Localization of ArgSh sensor in mitochondria intermembrane space (mito-inter-ArgSh) , in mitochondria matrix (mito-matrix-ArgSh) and in ER matrix (er-matrix-ArgSh), respectively. The fluorescence of the ArgSh sensor was represented in the ArgSh channels in green. MitoTracker and ER Tracker were represented in the maker channel in red. The combined image of ArgSh sensor fluorescence and MitoTracker or ER Tracker fluorescence was displayed in the Merge channels. Scale bar, 5 μm.
D, E, Maximum responses of Mito-ArgS0.1, ER-ArgS0.1, LysoArgS0.1, and Nuc-ArgS0.1 to 1 mM Arg perfusion. n = 20 cells.
附表 S1
Table S1. Characteristics of Arg sensors. N/A, Not Available; Ex/Em, peak excitation/emission wavelengths; EYFP, yellow fluorescent protein; ECFP, enhanced cyan fluorescent protein; VFP, Venus fluorescent protein.
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材料与方法
Construction of expression plasmids
The artJ sequence was synthesized according to E. coli artJ from NCBI (NCBI Reference Sequence: NC_000913.3), while the cpEGFP sequence was synthesized according to a dopamine sensor GRABDA2m51. For bacterial expression, we cloned the ArgS sensors into the NcoI/XhoI sites of pET28b vectors (Novagen) using the ClonExpress Ultra One Step Cloning Kit (Vazyme). For expression in mammalian cell plasma, the ArgS sensors and the C-terminal fused IRES2-mCherry sequences were cloned into the HindIII/XhoI sites of pcDNA3.1 vectors (Thermo Fisher). To target plasma membrane expression, the ArgS sensors were fused with membrane-targeting sequences from the serotonin sensor iSeroSnFR-EnhancedExport57, and fused IRES2-mCherry-CAAX at C-terminus. For mitochondria, ER, lysosome and nucleus localization, ArgS sensors were fused with targeting sequences from mitoExRai-AKAR266, ER-GCaMP6f67, lysoExRai-AMPKAR68 and Nuc-ExRai-AktAR2 sensors69 respectively. All primers and gene synthesis, Sanger sequencing, and plasmid purification were carried out by Genewiz, Azenta Life Sciences.
Protein purification
We transformed the pET28b vectors containing the ArgS sensors into Transetta (DE3) competent cells (Transgene) using chemical transformation as per the manufacturer's instructions. After inducing with 0.25 mM IPTG (Yeasen) for 20 h at 16℃, the cells were collected, centrifuged, and lysed with B-PER Reagent (Thermo Fisher) for 15 min at room temperature. The soluble protein was purified using nickel affinity chromatography with HisSep Ni-NTA Agarose Resin 6FF (Yeasen), then concentrated using Amicon Ultra 15 mL centrifugal filters (30 kDa, Merck Millipore) in PBS buffer (Thermo Fisher), following established protocols73-74. To obtain the apo-form of ArgS sensors, we concentrated the purified protein into 20 mM NaOAc-HOAc buffer (pH = 5, Sigma-Aldrich) to reduce affinity for Arg53, then neutralized it by concentrating into PBS buffer.
In vitro measurements
We incubated 2 μL of concentrated protein with various concentrations of Arg (Sigma-Aldrich) in PBS buffer at pH 7.4, totaling 200 μL, for 5 min at room temperature in a Costar 3915 assay plate (Corning). Measurements were taken on an EnSpire plate reader (Perkin Elmer) at excitation wavelengths of 405 nm and 475 nm, and an emission wavelength of 515 nm. For dissociation constant determination, we calculated the excitation ratio (R) as
Given that the binding ratio of artJ and Arg is 1:145-46, 52-56, we fitted the excitation ratios at different Arg concentrations to a Hill equation (Equation 1) to calculate the dissociation constant, assuming a Hill slope of 1:
Here, R represents the excitation ratio, Bottom is the basal excitation ratio, Top is the maximum excitation ratio,Kd is the dissociation constant, and [L-arg] is the Arg concentration.
For sensor selectivity assays, we incubated the concentrated protein with 1 mMdifferent compounds in PBS buffer at pH 7.4. The excitation ratio (R) was recorded, while the ratio of the PBS buffer group was noted as R0. The response ΔR/R0was calculated as:
Then the responseΔR/R0 was normalized to the Arg group.
For excitation spectrum measurements, we incubated the protein with or without 1 mMArg, measuring it at excitation wavelengths from 400 nm to 500 nm (5 nm steps) and an emission wavelength of 515 nm. For emission spectrum measurements, we used an excitation wavelength of 475 nm and emission wavelengths from 500 to 600 nm (5 nm steps).
Cell culture and transfection
HEK293T and MDA-MB-231 cells were cultured in DMEM (high glucose, with pyruvate, Thermo Fisher) supplemented with 10% v/v FBS (Thermo Fisher) and 1% Pen Strep (Thermo Fisher) at 37℃ in a 5% CO2 environment. cells were plated on PDL (Thermo Fisher)-coated glass cover slips or Phenoplate 96-well plates (Perkin Elmer) 24 h before transfection. After transfection with Lipofectamine 3000 (Thermo Fisher) as per the manufacturer's instructions, cells were imaged with a Nikon confocal microscope or Operetta high-content imaging.
RNA sequencing
Total RNA was extracted from HEK293T and MDAMB231 cells (n=1*10^7) with TRIzol Reagent (Thermo Fisher Scientific Inc.) following the manufacturer’s protocol. Total RNA isolation, library preparation, sequencing and data analysis were conducted by GENEWIZ, Inc. (Suzhou, China) on an Illumina HiSeq/Novaseq platform.
Confocal microscopy
cells were plated on PDL (Thermo Fisher)-coated12-mm glass coverslips 24 h before transfection and imaged using a Ti-E A1R confocal microscope (Nikon) equipped with a 10x/0.45 NA objective, a 20x/0.75 NA objective, a 40x/1.25 NA oil-immersion objective, a 100x/1.35 NA oil-immersion objective, a 405-nm laser, a 488-nm laser, and a 561-nm laser; green fluorescence and red fluorescence were recorded using a 525/50-nm, 525/50-nm and 595/50-nm emission filter, respectively. A custom-made perfusion system was used for imaging cells cultured on 12-mm coverslips. Arg and PBS were added at the indicated times. Image analysis for time-lapse imaging was done using custom ImageJ. Regions of interest (ROI) were randomly selected in cells throughout the field of view. For localized biosensors, ROIs were selected by mitochondria, lysosomes, endoplasmic reticulum and nucleus dye. Ratios were normalized to values before Arg stimulation. Maximum ratio changes (ΔR/R0) were calculated as (Rmax − R0)/R0, where R is cpEGFP excitation ratios (Ex480/405)，which were calculated for each time point. Image traces were generated by GraphPad Prism 10.1.
IntracellularKd measurements
Firstly, the cells were exposed to 0.0005% Digitonin (MCE) to permeabilize the cell membranes. After permeabilization for 10 mins, the cells were transferred to PBS and the PBS was replaced every 5 mins during the subsequent period. During this time, the excitation ratio of ArgS1 was monitored until the excitation ratio value stabilized and no longer decreased. Subsequently, different concentrations of Arg were added to the cells, and the excitation ratio of the ArgS1 was measured accordingly. The measurement of theKd mentioned above refers to In vitro measurements.
Operetta high-content imaging
cells were seeded in PDL-coated black-wall, clear-bottom Phenoplate 96-well plates (Perkin Elmer) 24 h before transfection. Cells grown in 96-well plates were imaged using an Opera Phenix high-content screening system (PerkinElmer) equipped with a 20x/0.4 NA objective, a 40x/1.15 NA water-immersion objective, a 405-nm laser, and a 488-nm laser and a 561-nm laser; green fluorescence were all recorded using a 525/50-nm emission filter; red fluorescence was recorded using a 595/50-nm emission filter.
Arginase and NOS inhibition assays
ArgS1 sensor-expressing HEK293T cells were incubated with 500 μM Arginase inhibitor 1 (MCE) and/or L-NMMA (MCE) 0, 12, 24, 36 and 48 h before imaging.
Stable cell line construction
Recombinant lentivirus expressing ArgS1 sensor were produced by Vigene Biosciences and applied to MDA-MB-231 cells (ATCC) following the manufacturer’s instructions. After24 h of lentiviral infection, puromycin (5 μg/mL for MDA-MB-231 cells, Thermo Fisher) was applied 7 days to remove uninfected cells and obtained cells that fully expressed GFP. Recombinant lentivirus expressing ASS1 (red fluorescent protein, RFP) was applied to MDA-MB-231 cells expressing ArgS1 sensor. After 24 h of lentiviral infection, the cells were subjected to flow cytometry analysis (BD Aria Fusion), resulting in the isolation of cells that co-express GFP and RFP.
Western analysis
cells were collected and lysed with 1 ml lysis buffer (1xprotease inhibitor cocktail (Roche), 1x phosphatase inhibitor II, 1x phosphatase inhibitor III (Sigma)), before centrifugation of cell lysates at 20000 rpm for 3 min at RT. Equivalent amounts of protein were electrophoresed on 4%–12% Novex precast gels (Invitrogen) and transferred to nitrocellulose membrane. After blocking for 2 h in 1x Tris-buffered saline/5% non-fat dried milk, membranes were incubated overnight at 4℃ in anti-ASS1 (PA5-82738, Invitrogen). Membranes were incubated with anti-immunoglobulin G-horseradish peroxidase and visualized by chemiluminescent detection (Touch Imager, e-BLOT). Immunoblotting for β-actin was performed as a loading control.
Arg starvation assay
MDA-MB-231 cells expressing ArgS1 sensor cells and MDA-MB-231 cells co-expressing ArgS1 sensor and ASS1 were seeded in PDL-coated Phenoplate 96-well plates. After 12 h, the culture medium was replaced with either normal DMEM or DMEM medium viabilityfor SILAC (Thermo Fisher) containing 10% v/v FBS, 1% v/v Pen Strep, and 0.8 mM Lys (Sigma-Aldrich), as previously reported34. Cells were prepared for transferred for Operetta high-content imaging or CCK8 assay.
Cell viability assays
According to the manufacturer's instructions, the CCK-8 reagent was mixed with DMEM medium in a ratio of 1:10 and then added to the cells that had been deprived of Arg for 20 hours. 100 µl of the mixture was added to each well. The reagent was also added to blank wells without cells as a background control. After 4 hours of incubation, the absorbance values were measured using EnSpire plate reader (Perkin Elmer). All values were subtracted from the background value.
Statistical analysis
All data were expressed in SEM. Statistical analysis was performed with a two-tailed t test or one way anova test using GraphPad Prism 10.1. The dose-dependent curves were fit to Hill equation by GraphPad Prism 10.1.
https://doi.org/10.1021/acssensors.4c03174