2025年1月21日，學術刊物ACS Sensors（《美國化學學會 感受器》）線上發表：
基因編碼開發一種用於檢測精氨酸的探針
王純1，張小雪1，毛浩雨1,2，鹹逸2,3, *，饒毅1,2,3, *
1首都醫科大學、首都醫學科學創新中心
2北京大學 化學與分子工程學院、北大/清華生命科學聯合中心、生命科學學院、醫學部藥學院、麥戈文腦科學研究所
3北京腦科學研究所
*通訊作者
摘要
L-精氨酸（Arg）在多種代謝和生理過程中發揮重要作用，其濃度的變化與病理過程密切相關。儘管直接且即時測量生物系統中的Arg水平非常重要，但現有的Arg探針對L-鳥氨酸或L-賴氨酸也有響應。本文報道了一種新的Arg探針ArgS1。該探針對Arg表現出濃度依賴性的Ex488/405比率增加，其表觀親和力約為64 μM，動態範圍（ΔR/R0）為3。ArgS1在細胞質和亞細胞器中對Arg均有響應。ArgS1成功監測了MDA-MB-231細胞（一種缺乏Arg合成關鍵酶——精氨琥珀酸合成酶1（ASS1）的乳腺癌細胞系，且適用於Arg耗竭療法）中的Arg水平。研究發現，當細胞外Arg被耗竭後，MDA-MB-231細胞中的Arg水平下降，同時細胞活力也隨之降低。當細胞中過表達ASS1時，Arg水平上升，細胞活力也得到增強。因此，ArgS1是一種在生理和病理相關動態範圍內即時監測人類細胞中Arg水平的有效工具。
L-精氨酸（Arg）是一種條件性必需氨基酸，在人體中具有重要的生理作用 [1,2]。Arg的主要來源包括蛋白質降解、膳食攝入和從頭合成[3]。Arg的合成涉及兩種關鍵酶：精氨琥珀酸合成酶（ASS），它將L-瓜氨酸（Cit）轉化為精氨琥珀酸；以及精氨琥珀酸裂解酶（ASL），它將精氨琥珀酸裂解為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的唯一直接前體[6-9]。Arg還參與尿素迴圈，在精氨酸酶（ARGs）的作用下被水解為尿素和L-鳥氨酸（Orn）[10,11]，這對於氨的解毒至關重要[12]。精氨酸：甘氨酸脒基轉移酶（AGAT）是一種線粒體酶，它催化Arg生成胍基乙酸，這是肌酸合成的直接前體[13-15] 。Arg代謝紊亂與多種疾病相關[5,16]。精氨酸酶1（ARG1）缺乏會導致高精氨酸血癥，這是一種以進行性神經系統症狀為特徵的罕見遺傳性代謝疾病[17] 。Arg還與多種疾病的治療相關，例如勃起功能障礙[18,19]、高血壓[20]和心力衰竭[21]。
癌細胞的生存和生長需要氨基酸[22,24]，其中Arg是腫瘤微環境中的重要組成部分[25-27] 。某些型別的癌症，如乳腺癌[28]、黑色素瘤[29]、肝細胞癌[30]、急性淋巴細胞白血病（ALL）[31] 和急性髓系白血病（AML）[32]，其ASS表達缺失。這些型別的癌細胞依賴攝取細胞外Arg以維持生存，因此限制Arg供應已被探索為癌症治療中的一種潛在輔助治療策略[27,33]。在培養基中去除Arg或新增ADI-PEG20（聚乙二醇化精氨酸脫亞氨酶）可導致ASS1缺陷型癌細胞在體外被清除[28,34]。系統性給予ADI或精氨酸酶以限制Arg可用性，已在腫瘤治療的II期臨床試驗中進行了研究[35]。這些研究表明，ASS1缺陷是AML患者對ADI-PEG20單藥治療產生反應的必要條件，但並非充分條件。Arg的測量是確定患者是否適合接受ADI-PEG20治療AML的重要指標。
傳統的Arg測量方法包括樣品的微透析[36,37] 和化學分析[38]，這些方法無法研究細胞內分佈的變化，也無法以必要的空間和時間解析度分析Arg。熒光探針提供了非侵入性即時測量濃度及其時空變化的可能性。現有的基因編碼Arg熒光探針基於Förster共振能量轉移（FRET），其動態範圍相對較小[39-41]。這些探針在體外報道的最大動態範圍僅為約0.6[40]。更大的問題在於其特異性：這些探針不僅對Arg有響應，還對其他氨基酸（如Orn、L-賴氨酸（Lys）、L-谷氨醯胺（Gln）和L-組氨酸（His））有響應[39-41] 。此外，這些探針對Arg的解離常數（Kd）（9.4或14 μM[39,40]）不適合檢測生理水平的Arg，因為血漿[42]和細胞質[43]中的Arg濃度通常約為100 µM。
因此，需要開發具有大動態範圍和合適Kd的基因編碼Arg特異性熒光探針，以用於生理和病理條件下Arg的檢測。
結果
Arg探針的設計、最佳化及體外表徵
我們基於大腸桿菌（E. coli）的artJ（一種周質結合蛋白，PBP）設計了一種基因編碼的熒光Arg探針。artJ在結合Arg時會發生構象變化[44-46]。與其他探針的設計類似 [47-50]，我們將來自GRABDA2m的環狀置換綠色熒光蛋白（cpEGFP）[51] 整合到artJ中（圖1A、B）。根據artJ和argT（一種Arg、Lys和Orn結合蛋白）的晶體結構，其葉II的環區和鉸鏈區在底物結合時會發生顯著構象變化[45,46,52-56]，這為cpEGFP的插入提供了潛在的位點。首先，我們在純化蛋白上篩選了cpEGFP的插入位點（圖1C）。artJ與cpEGFP之間的連線肽設計為柔性（Gly-Gly）或剛性（Pro-Pro）。基於初步篩選結果，我們選擇了動態範圍大於0.1的探針（圖1D）。考慮到基於蛋白純化的篩選方法耗時較長，且在體外響應的探針在細胞環境中可能表現不同，我們轉而使用HEK293T細胞進行篩選。透過將探針與iSeroSnFR的膜靶向序列融合[57]，我們將探針靶向到人胚胎腎293T（HEK293T）細胞的細胞膜上，以最佳化連線肽的長度和氨基酸組成，從而最小化細胞內Arg對探針響應的影響。我們發現，在HEK293T細胞膜上表現出良好響應的探針的響應值分別為0.4和0.6，分別命名為ArgS0.1和ArgS0.2（圖1C、D，圖S1C）。隨後，我們優化了連線肽鄰近位點，發現響應最佳的探針的響應值為0.9，命名為ArgS1（圖1D，圖S1C）。總結而言，透過對1200多種不同變體的篩選，我們最終確定了三種探針，分別命名為ArgS0.1、ArgS0.2和ArgS1（圖1D）。
圖1 ArgS探針的體外測量
A, B ArgS探針的設計。透過不同的插入位點將cpEGFP整合到大腸桿菌artJ蛋白中。我們將cpEGFP與artJ之間的氨基酸連線肽稱為N端連線肽和C端連線肽。該模型使用AlphaFold2生成。C ArgS探針的篩選流程。第1步是在純化蛋白上進行插入位點篩選，第2至第4步是在HEK293T細胞膜上進行篩選，其中第2步和第3步分別最佳化連線肽的長度和組成，第4步最佳化連線肽鄰近位點。D ArgS探針的篩選結果。ArgS0.1、ArgS0.2和ArgS1探針分別用洋紅色、藍色和綠色突出顯示。E ArgS1探針的氨基酸特異性。對1 mM各種氨基酸的響應歸一化為對1 mM L-Arg的響應。每組資料點n = 3；資料以均值±標準誤（SEM）表示。F ArgS1探針在有無1 mM Arg條件下的激發和發射光譜。G ArgS1探針在不同濃度Arg暴露下的劑量響應曲線。每組資料點n = 6；資料以均值±標準誤（SEM）表示。
我們將這三種探針分別在大腸桿菌中表達並純化，隨後測試了它們對各種氨基酸的特異性。結果顯示，所有三種ArgS探針均特異性地響應L-Arg，而對其他氨基酸的L型或D型均無響應（圖1E，圖S2A、B）。這三種ArgS探針均表現出比率特性，具有兩個激發峰（分別位於400奈米（nm）和500 nm附近）和一個發射峰（位於515 nm附近）。當與1 mM Arg結合時，三種探針的400 nm激發峰降低，而500 nm激發峰增加（圖1F，圖S2C、D）。我們還測量了ArgS0.1、ArgS0.2和ArgS1的Arg結合親和力，其Kd值分別為73 μM、367 μM和1141 μM，熒光峰值變化（ΔR/R0）分別為1.9、4.6和19（圖1G，圖S2E、F）。同時，我們評估了ArgS探針對精氨酸相關代謝物的特異性。ArgS0.1對胍丁胺、Cit和精氨琥珀酸表現出可檢測的響應，而ArgS0.2和ArgS1僅對Cit和精氨琥珀酸有響應（圖S3A、D、G）。在瓜氨酸（Cit）結合實驗中，ArgS0.1、ArgS0.2和ArgS1的Kd分別為312 μM、1162 μM和2912 μM，其熒光峰值變化約為Arg的一半（圖S3B、E、H）。對於精氨琥珀酸的結合，所有探針的Kd值均超過300 μM（圖S3C、F、G）。為了減少Arg相關代謝物的潛在干擾，我們透過LC-MS定量分析了HEK293T和MDA-MB-231細胞中這些代謝物的細胞內濃度。在兩種細胞系中，Cit和精氨琥珀酸的濃度均低於5 μM（HEK293T細胞中Cit為1.290 μM，MDA-MB-231細胞中Cit為3.920 μM；HEK293T細胞中精氨琥珀酸為1.556 μM，MDA-MB-231細胞中精氨琥珀酸為4.156 μM），均低於ArgS探針的檢測閾值（圖S3J）。因此，在檢測HEK293T或MDA-MB-231細胞中的Arg水平時，Cit或精氨琥珀酸的潛在干擾可忽略不計。此外，基於嗜熱脂肪土芽孢桿菌（Geobacillus stearothermophilus）artJ的晶體結構
[45]
，我們開發了三種ArgS1探針的突變版本。其中，雙點突變版本ArgS1-F51L E114L對Arg幾乎無響應（圖S4C）。因此，該版本探針ArgS1-F51L E114L被命名為ArgS1-C，並作為後續實驗的對照。為了研究pH依賴性，我們在pH 4至9的範圍內測試了ArgS1和ArgS1-C。ArgS1在pH 7至8之間對Arg的響應最佳（圖S4A），而ArgS1和ArgS1-C的apo形式在整個測試範圍內表現出相似的pH依賴性行為（圖S4B）。
Arg探針在HEK293T細胞質及亞細胞器中檢測Arg變化
為了研究Arg探針是否能夠在哺乳動物細胞中檢測Arg，我們在HEK293T細胞的細胞質中表達了ArgS0.1、ArgS0.2和ArgS1（圖2A）。Arg轉運蛋白SLC7A1[58]和SLC7A2[59]在HEK293T細胞中高表達（圖S5A）。當細胞外Arg濃度升高至1 mM時，我們立即觀察到ArgS1、ArgS0.1和ArgS0.2在405 nm處的熒光強度下降，而在488 nm處的熒光強度增加（圖2B，圖S6A）。這表明所有三種探針（ArgS1、ArgS0.1和ArgS0.2）均對Arg的增加作出了響應，其最大動態範圍分別為3.3、0.3和1.1（圖2D，圖S6D、E）。當Arg探針達到其峰值動態範圍後，我們將灌注液切換回磷酸鹽緩衝液（PBS），觀察到三種探針在405 nm處的熒光強度立即增加，而在488 nm處的熒光強度下降，其響應恢復到基線值（圖2C，圖S6B、C）。
圖2 ArgS1探針在HEK293T細胞質中對Arg濃度變化的響應
A ArgS探針在HEK293T細胞質中成像的示意圖。在HEK293T細胞的細胞質中，C端融合IRES-mCherry序列的ArgS探針被轉染，隨後在不同溶劑灌注條件下使用共聚焦顯微鏡進行成像。B代表性影像顯示ArgS1探針在HEK293T細胞質中對PBS或1 mM Arg變化在不同時間點（0分鐘、10分鐘、30分鐘和50分鐘）的響應。ArgS1探針的熒光在兩個通道中可見：ArgS1 Ex488（激發波長488 nm）顯示為綠色，而ArgS1 Ex405（激發波長405 nm）顯示為藍色。此外，透過IRES序列融合到ArgS探針C端的mCherry在mCherry通道中以紅色顯示。ArgS1探針的Ex488/Ex405比率變化進一步在ΔR/R0通道中視覺化。比例尺：10 μm。C ArgS1在PBS或1 mM Arg中熒光響應的平均軌跡。我們在0分鐘時將1 mM Arg灌注到表達ArgS1探針的HEK293T細胞中，並在35分鐘時切換為PBS緩衝液。ArgS1探針在405 nm激發波長下的ΔF/F0以藍色表示，而在488 nm波長下的ΔF/F0以綠色表示。mCherry的ΔF/F0以紅色表示。紫色表示ΔR/R0（右側Y軸）。每組n = 20個細胞；資料以均值±標準誤（SEM）表示。D ArgS1探針對Arg灌注的最大響應。每組n = 20個細胞；資料以均值±標準誤（SEM）表示。E ArgS1探針在HEK293T細胞質中的劑量依賴曲線及相應的Kd值。每組n = 600個細胞（來自6個孔）；資料以均值±標準誤（SEM）表示。F HEK293T細胞中Arg濃度的藥理學改變。ArgS1探針的488/405比率在分別與500 μM抑制劑孵育12、24、36和48小時前後的變化。對照組以黑色等邊三角形表示，單獨L-NMMA組以灰色菱形表示，單獨AI1組以灰色圓形表示，L-NMMA和AI1聯合組以深灰色方形表示。每組n = 600個細胞（來自6個孔）；資料以均值±標準誤（SEM）表示。
為了在HEK293T細胞質中進行原位滴定以測量ArgS1的Arg結合親和力，我們用毛地黃皂苷（digitonin）透化細胞膜，並向細胞中新增不同濃度（1至∼60000 μM）的Arg（圖S5B）。透過繪製表達ArgS1的細胞平均響應與Arg濃度的關係，獲得了原位校準曲線。因此，我們測量了ArgS1在HEK293T細胞質中的Arg結合親和力，其Kd值為64 μM（圖2E）。此外，為了最小化非特異性熒光訊號變化，我們在HEK293T細胞中表達了非結合對照探針ArgS1-C。如圖S5D所示，ArgS1-C對1 mM Arg灌注無響應。
為了確定Arg探針是否能夠檢測由藥物干預引起的HEK293T細胞中Arg的變化，我們在這些細胞中表達了ArgS1。我們利用精氨酸酶I和II抑制劑（Arginase Inhibitor 1, AI1）[60] 和一氧化氮合酶（NOS）抑制劑L-NMMA [61-65]來調控Arg水平。如圖2F所示，使用ArgS1檢測到AI1或AI1與L-NMMA聯合應用引起的細胞內Arg水平升高；然而，單獨使用L-NMMA並未誘導類似的響應。這些結果表明，在HEK293T細胞中，精氨酸酶途徑在Arg降解中的作用比NOS途徑更為重要。這一結論與我們的轉錄組測序結果一致。在HEK293T細胞內，ARG2的表達水平最高，而ARG1和NOS2的表達水平非常低，NOS1則未表達（圖S5C）。因此，ArgS1探針有效地監測了HEK293T細胞中藥理干預引起的Arg濃度變化。
圖3 ArgS1探針在HEK293T細胞器中對1 mM Arg灌注的響應
A ArgS1探針在不同細胞器中表達的示意圖：線粒體、內質網（ER）、溶酶體和細胞核。B ArgS1探針定位在不同細胞區室的影像：線粒體外膜的胞質側（Mito-ArgS1）、ER膜的胞質側（ER-ArgS1）、溶酶體膜的胞質側（Lyso-ArgS1）以及細胞核內（Nuc-ArgS1）。ArgS1探針的熒光在ArgS1通道中以綠色表示。細胞器特異性標記物，包括線粒體的Mito-Tracker、ER的ER-Tracker、溶酶體的Lyso-Tracker和細胞核的NucRed，在細胞器標記通道中以紅色表示。ArgS1探針熒光與細胞器標記熒光的合併影像顯示在Merge通道中。ArgS1探針對Arg灌注的響應在ΔR/R0通道中表示。比例尺：5 μm。C-F定位在不同細胞區室的ArgS1在PBS或1 mM Arg中熒光響應的平均軌跡。我們在0分鐘時將1 mM Arg灌注到表達Mito-ArgS1、ER-ArgS1、Lyso-ArgS1和Nuc-ArgS1探針的HEK293T細胞中，並在達到最大響應後分別切換為PBS緩衝液。ArgS1探針在405 nm激發波長下的ΔF/F0以藍色表示，而在488 nm波長下的ΔF/F0以綠色表示。ΔR/R0以紫色表示。每組n = 20個細胞；資料以均值±標準誤（SEM）表示。G-J Mito-ArgS1、ER-ArgS1、Lyso-ArgS1和Nuc-ArgS1對1 mM Arg灌注的最大響應。每組n = 20個細胞；資料以均值±標準誤（SEM）表示。
為了進一步研究Arg探針是否能夠響應亞細胞器中的Arg，我們將ArgS1（圖3）和ArgS0.1（圖S7）靶向到不同的細胞器。我們將ArgS1和ArgS0.1分別與AKAP1的N端30個氨基酸引導序列
[66]
、p450的內質網（ER）靶向基序
[67]
、LAMP1衍生的序列
[68]
或核定位訊號
[69]
融合，以定位到線粒體胞質側（Mito-ArgS1和Mito-ArgS0.1）、ER胞質側（ER-ArgS1和ER-ArgS0.1）、溶酶體（Lyso-ArgS1和Lyso-ArgS0.1）或細胞核（Nuc-ArgS1和Nuc-ArgS0.1）（圖3A）。為了評估ArgS1線上粒體、ER、溶酶體和細胞核中的定位，我們分別使用Mito-Tracker、ER-Tracker、Lyso-Tracker和NucRed作為對照。結果顯示，ArgS探針與相應的細胞器標記物高度共定位（圖3B，圖S7A）。當向培養HEK293T細胞的培養基中灌注1 mM Arg時，定位在細胞器中的ArgS探針也對Arg水平的變化作出了響應（圖3C-F，圖S7B-E）。Mito-ArgS1、ER-ArgS1、Lyso-ArgS1和Nuc-ArgS1探針的最大動態範圍分別為3.7、3.8、3.2和3.8（圖3G-J），而Mito-ArgS0.1、ER-ArgS0.1、Lyso-ArgS0.1和Nuc-ArgS0.1探針的最大動態範圍分別為0.5、0.5、0.4和0.6（圖S7F-I）。這些結果表明，ArgS探針線上粒體、ER和溶酶體附近區域以及細胞核內的響應相似。因此，我們成功開發了能夠監測哺乳動物細胞質和亞細胞器中Arg的ArgS探針。
細胞外Arg剝奪導致癌細胞細胞內Arg水平下降
Arg飢餓正被探索為一種針對ASS1缺陷型癌症的潛在治療策略[28,70]（圖4A）。據報道，乳腺癌細胞系MDA-MB-231不表達ASS1[28] （圖4B）。我們首先驗證了Arg剝奪對細胞活力的影響。Arg剝奪24小時後，MDA-MB-231細胞的活力下降（圖4C），這與之前的報道一致 [28]。此外，過表達ASS1能夠回補MDA-MB-231細胞對Arg飢餓的敏感性（圖4B、C）。為了觀察細胞內Arg水平，我們開發了在MDA-MB-231細胞中穩定表達ArgS1的細胞系（圖4A）。我們發現，Arg剝奪後，ArgS1探針的488/405比率及其衰減速率均顯著下降，表明MDA-MB-231細胞內的Arg水平顯著低於對照組（圖4D、E，圖S8B）。此外，Arg剝奪後，ASS1過表達組的488/405比率高於ASS1缺陷組，表明透過ASS1過表達回補了Arg水平（圖4D、E）。此外，為了進一步減少非特異性熒光訊號變化，我們在MDA-MB-231細胞中表達了ArgS1-C。如圖S8A所示，ArgS1-C對1 mM Arg灌注也無響應。因此，ArgS1探針可用於監測癌細胞中的Arg水平。
圖4 MDA-MB-231細胞中Arg的成像
A ArgS1探針在MDA-MB-231細胞中表達的示意圖，包括有和無Arg飢餓條件下的情況。B MDA-MB-231細胞和ASS1過表達的MDA-MB-231細胞中ASS1的表達水平。C MDA-MB-231細胞（圓形）和ASS1過表達細胞（方形）在Arg飢餓（深紅色）或完全DMEM對照組（黑色）處理24小時後的細胞活力。Arg飢餓組的細胞活力均歸一化為完全DMEM對照組。每組n = 600個細胞（來自6個孔）；資料以均值±標準誤（SEM）表示。D ArgS1探針在MDA-MB-231細胞和ASS1過表達的MDA-MB-231細胞中的熒光強度和響應變化在Arg飢餓30分鐘前後成像。比例尺：10 μm。E ArgS1探針在MDA-MB-231細胞和ASS1過表達的MDA-MB-231細胞中的488/405比率變化在Arg飢餓2小時前和24小時後每15分鐘成像一次。每組n = 600個細胞（來自6個孔）；資料以均值±標準誤（SEM）表示。
討論
為了比較ArgS探針與其他Arg探針，我們整理了各種Arg探針的特性，如表S1所示。例如，基於glnH的QBP/Citrine/ECFP探針對Arg的Kd為2.1 mM，動態範圍為0.3[39]。此外，基於argT的FLIP-cpargT194探針對Arg的Kd為48 μM，動態範圍為0.5[40]。然而，這些探針並非對Arg完全特異：QBP/Citrine/ECFP還對Orn有響應，而FLIP-cpargT194對Orn和Lys均有響應[39,40]。相比之下，FLIP-cpartJ185和FLIPR探針對Arg具有特異性，其Kd值分別為9.4 μM和14 μM，動態範圍分別為0.5和0.3[40,41]。由於這些探針對Arg的響應和特異性有限，它們並不適合檢測哺乳動物細胞內Arg濃度的變化。相比之下，ArgS探針——ArgS0.1、ArgS0.2和ArgS1的Kd值分別為73 μM、367 μM和1141 μM，熒光峰值變化（ΔR/R0）分別為1.9、4.6和19。這些響應優於以往的Arg探針，且ArgS探針特異性地響應Arg，而不會與其他氨基酸發生交叉反應。最近，研究人員報道了一種名為STAR的基因編碼探針，它特異性地響應Arg，並能夠在體外和體內監測Arg動態[71]。與STAR相比，ArgS1的熒光響應略大（ΔR/R0：19 vs. ~16，體外）。然而，與ArgS1不同，STAR不具備比率特性，因此需要額外的對照來校正探針表達水平的變化。
為了增強ArgS探針的最大響應，我們篩選了不同長度和組成的連線肽。結果表明，N端連線肽為2或3個氨基酸、C端連線肽為2個氨基酸時，響應更大，且N端連線肽的首個氨基酸優選為Gly。此外，在篩選過程的第4步中，測試了N端連線肽鄰近的氨基酸殘基。例如，將該殘基替換為Ile顯著提高了響應，從而開發出了ArgS1。儘管我們篩選了1200多個候選變體，但透過進一步擴充套件篩選範圍以包括更多鄰近連線肽位點，仍有可能實現進一步改進。
具體而言，HEK293T細胞質中的ArgS1的Kd值為64 μM，高於體外測量的值。類似地，iGluSnFR探針在HEK293T細胞或培養的海馬神經元中的親和力也高於體外觀察到的值[47,49,72]。這種差異可能是由於蛋白質在不同環境和檢測系統中的暴露差異，也可能是細胞環境中的輔助因子增強了Arg結合的結果。然而，即使在體外實驗中加入細胞裂解液後，測得的Kd仍處於毫摩爾範圍，這表明需要進一步研究。因此，為了準確量化不同環境中的Arg水平，應在多種實驗條件下測量Kd以考慮這些因素。此外，ArgS1對Cit和精氨琥珀酸表現出可檢測的響應。在測量不同生物環境中的Arg水平時，必須確保Cit和精氨琥珀酸的濃度低於ArgS1探針的檢測閾值。
關於ArgS探針在不同亞細胞位置的動態特性，Nuc-ArgS1和ER-ArgS1的Arg灌注時間比其他位置短，這可能反映了亞細胞間Arg代謝的差異（圖3C-F）。此外，我們嘗試確定每個亞細胞位置的基線Arg濃度；然而，我們不確定灌注前的初始比率是否準確反映了基線濃度，因為探針遊離形式的熒光比率可能在不同亞細胞區室中有所不同。此外，在每個亞細胞位置除去Arg以獲得ArgS探針的遊離形式具有挑戰性。因此，我們選擇在獲得更可靠的方法來獲取這些區室中ArgS探針的遊離形式之前，不對亞細胞位置的Arg濃度進行量化。
Arg在生理和病理過程中發揮著關鍵作用；然而，其許多功能仍未得到充分理解。例如，星形膠質細胞和神經元之間Arg轉運的機制尚不清楚，其作為訊號分子的作用也未明確定義。此外，Arg濃度的變化可以與其他分子（如Ca²⁺、cAMP和Glu）一起使用互補的熒光探針進行監測。我們希望ArgS探針能夠有助於闡明Arg代謝的機制，併為其生理和病理作用提供更深入的見解。
結論
總之，ArgS1能夠即時檢測多種哺乳動物細胞中Arg水平的變化，為基礎研究和臨床應用提供了寶貴的工具。這是首個能夠動態監測哺乳動物細胞內Arg濃度變化的基因編碼熒光探針。此外，ArgS1在亞細胞器中對Arg表現出動態響應，並已成功用於監測MDA-MB-231乳腺癌細胞中的Arg水平。
致謝
我們感謝李毓龍教授提供GRABDA2m質粒。我們感謝國家蛋白質科學基礎設施——北京基地北京大學分設施在Operetta高內涵成像實驗提供的幫助。我們感謝北京腦科學研究所生物質譜中心於曉倩老師在LC-MS實驗提供的幫助。
附圖：
附圖 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.
附圖 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.
附圖 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.
附圖 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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材料與方法
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-AMPKAR68and 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,Kdis 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
