JACS最新：精准锁定4类细胞器！湖南大学袁林团队等首创“双锁”荧光探针，揭秘LAP酶在亚细胞层面的“生死两重天”#

Figure 1. Schematic overview of organelle-gated reporters for imaging subcellular enzyme activity. (a) Enzymes often display distinct functions in different cellular compartments, yet the link between subcellular localization and activity remains poorly understood. (b) Conventional approaches for detecting enzyme activity in specific organelles. (c) Activatable organelle-gated reporters enable imaging of enzyme activity within defined organelles.#

Figure 2. Development and in vitro characterization of bioorthogonal-based subcellular enzyme biosensing system. (a) Schematic of the bioorthogonal-based subcellular enzyme reporters. Organelle-gated reporters consist of a rhodol fluorophore linked to an enzyme-specific substrate and a trans-cyclooctene (TCO) group. Fluorescence is restored only after sequential uncaging by organelle-localized tetrazine (Tz) and enzymatic activation. (b) Fluorescence enhancement of TCO-LAP, TCO-GGT, and TCO-MAO (5 μM) at 550 nm in the presence or absence of target enzymes and tetrazines. Incubation conditions: TCO-LAP with LAP (200 U/L) and Tz (10 μM) at 37 °C for 3 h; TCO-GGT with GGT (200 U/L) and Tz (10 μM) at 35 °C for 3 h; TCO-MAO with MAO-A (500 U/L) and Tz (10 μM) at 37 °C for 3 h. (c) Fluorescence emission spectra of TCO-LAP (5 μM) with LAP (100 U/L) in the presence of Mito-Tz, ER-Tz, Lyso-Tz, or Golgi-Tz (10 μM each). (d) Fluorescence emission spectra of TCO-LAP (5 μM) with LAP (100 U/L) and varying equivalents of ER-Tz (0, 0.3, 0.5, 0.8, 1, 1.5, 2, 5). (e) Time-dependent fluorescence response (550 nm) of TCO-LAP (5 μM) with ER-Tz and LAP (300 U/mL). R-LAP was used as a control. (f) Preparation of organelle enzyme reporters for confocal imaging and flow cytometry. (g–j) Flow cytometric analysis of live HeLa cells treated with TCO-LAP (5 μM; g), 4-TCO-LAP (5 μM; h), TCO-MAO (5 μM; i), or TCO-GGT (5 μM; j) after incubation with or without various organelle-targeted tetrazines (10 μM).#

为了验证该策略的普适性，团队针对LAP、γ-谷氨酰转肽酶（GGT）及单胺氧化酶A（MAO-A） 等多个酶家族开发了对应的探针。通过共聚焦成像实验证明，这些探针能够精准地分别在线粒体、溶酶体、内质网及高尔基体中产生强烈的荧光信号。这种极高的空间分辨率不仅避免了细胞质中的背景干扰，更允许研究者观察酶在特定环境下的动态响应。

Figure 3. Optimization of organelle-gated reporters for imaging subcellular enzyme activity. (a) Representative fluorescence images of HeLa cells incubated with TCO-LAP and organelle-targeted tetrazines (Tz), merged with commercial organelle trackers: Mito-Tracker Deep Red, Lyso-Tracker Red, ER-Tracker Blue, and Golgi-Tracker Red. (b) Schematic of a fluorogenic reporter that passively diffuses after enzyme activation, causing off-target localization. (c) Schematic of superior organelle enzyme reporters enabling enzymatic activation and localization via labeling of proximal proteins within the targeting organelle. (d) Chemical structure of TCO-LAP-F. (e) Fluorescent SDS-PAGE analysis. Proteins were incubated with or without TCO-LAP-F in PBS at 37 °C for 3 h. M: protein marker; 1: BSA (5 μg); 2: LAP (1 μg); 3: BSA + LAP; 4: LAP + TCO-LAP-F; 5: BSA + TCO-LAP-F; 6: LAP + BSA + Tz; 7: LAP + BSA + TCO-LAP + Tz; 8: BSA + TCO-LAP + Tz; 9: LAP + BSA + TCO-LAP-F + Tz. (f–i) Confocal images of live HeLa cells treated with TCO-LAP-F (2.5 μM) and organelle-targeted Tz (5 μM), costained with commercial organelle trackers: ER-Tz with ER-Tracker Blue (f), Lyso-Tz with Lyso-Tracker Red (g), Mito-Tz with Mito-Tracker Deep Red (h), Golgi-Tz with Golgi-Tracker Red (i). Scale bar: 20 μm.#

在生物学应用研究中，团队重点考察了LAP在应激状态下的亚细胞分工。实验发现，在化疗药物顺铂（CDDP）或喜树碱（CPT）的作用下，细胞内的LAP活性会显著升高。然而，利用OGRs技术进一步分析显示，这种升高具有明显的空间效应。如图3所示，线粒体中的LAP（Mito-LAP）通过调节细胞内的还原型谷胱甘肽水平，帮助肿瘤细胞抵抗氧化应激，从而在压力下“苟延残喘”。与之相反，内质网中的LAP（ER-LAP） 活性的提升则显著增强了肿瘤表面的抗原递呈效率，这相当于在肿瘤细胞上贴上了“向免疫系统举报”的标签，诱导T细胞对其进行精准杀伤。

Figure 4. Organelle-gated reporter for imaging subcellular LAP activity. (a) Organelle-gated reporter for probing LAP activity in distinct organelles under drug-induced oxidative stress. (b) Live-cell imaging workflow for spatial mapping of LAP activity under oxidative stress. (c) Schematic showing oxidative stress induced by β-lapachone (β-Lap)-mediated ROS generation via hNQO1 in cancer cells. (d) Fluorescence images of HepG2 cells treated with 10 μM organelle-targeted tetrazines (Mito-Tz, ER-Tz, and Golgi-Tz) and TCO-LAP-F (5 μM) after incubation with β-Lap (15 μM, 1 h). (e) Quantification of fluorescence intensity from panel d. (f) Western blot analysis of LAP levels in different organelles after β-Lap treatment as in panel d. (g) Schematic showing reductant depletion-induced oxidative stress. (h) Fluorescence images of HepG2 cells treated with organelle-targeted tetrazines (10 μM) and TCO-LAP-F (5 μM) after incubation with NEM (50 μM, 0.5 h). (i) Quantification of fluorescence intensity from panel h. (j) Western blot analysis of LAP levels in different organelles after NEM (100 μM) treatment as in panel h. (k) Schematic showing unknown LAP activity in oxidatively stressed organelles. (l) Fluorescence images of HepG2 cells treated with Mito-Tz and TCO-LAP-F after rotenone (10 μM) or DNP (1 mM), with ER-Tz and TCO-LAP-F after tunicamycin (Tm, 5 μM) or thapsigargin (Tg, 10 μM), and with Golgi-Tz and TCO-LAP-F after brefeldin A (BFA, 10 μM) or monensin (10 μM). (m) Quantification of relative fluorescence intensity from panel l. Scale bars, 20 μm. Data are mean ± SD (n = 5). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant (t-test).#

研究团队进一步通过药物联用实验验证了这一发现。当使用新开发的线粒体靶向LAP抑制剂时，肿瘤细胞的氧化应激防御被成功瓦解，化疗药物的杀伤效率显著提升。这一现象有力地证明了：针对同一蛋白质在不同亚细胞定位的异质性功能进行差异化干预，是提升癌症治疗效果的关键方向。

Figure 5. Relationship between LAP activity across organelles and drug stimulation. (a) Schematic illustrating the unknown changes in LAP activity within mitochondria, ER, and lysosomes during drug treatment of cancer cells. (b) Quantification of LAP activity in different organelles after stimulation of cancer cells with various drugs: β-Lap (15 μM, 1 h), NEM (50 μM, 0.5 h), rotenone (10 μM, 12 h) or DNP (1 mM, 12 h), Tm (5 μM, 12 h), Tg (1 μM, 12 h), BFA (10 μM, 6 h) or monensin (10 μM, 12 h), CPT (3 μM, 12 h) or CDDP (4 μg/mL, 12 h). (c) Fluorescence images of HepG2 cells treated with CPT (3 μM) or CDDP (4 μg/mL) for 12 h, or pretreated with NAC (100 μM) prior to CPT or CDDP incubation, followed by Mito-LAP activity imaging using TCO-LAP-F and Mito-Tz. (d–e) Quantification of fluorescence intensity of HepG2 cells from panel c. (f) Schematic diagram showing that chemotherapy drugs upregulate ER-LAP to enhance antigen presentation. (g) Fluorescence images of HCT116 and HCT116 p53–/– cells treated with CPT (3 μM) or CDDP (4 μg/mL) for 12 h followed by ER-LAP imaging using TCO-LAP-F and ER-Tz. (h**–**i) Quantification of fluorescence intensity of HCT116 cells (h) and HCT116 p53–/– cells (i) from panel g. (j) Immunofluorescence images of MHC-I expression in HCT116 and HCT116 p53–/– cells after CPT or CDDP treatment. (k–l) Quantification of MHC-I expression from panel j in HCT116 cells (k) and HCT116 p53–/– cells (l). (m) Immunofluorescence images of MHC-I in HepG2 cells treated with CPT or CDDP, Mito-stress inducer (DNP, 1 mM for 12 h), ER-stress inducer (Tg, 1 μM for 12 h), or Golgi-stress inducer (BFA, 10 μM for 6 h). (n) Schematic summarizing the dual role of antitumor agents: promoting pro-survival antioxidant responses in mitochondria while simultaneously enhancing ER-mediated antigen presentation, thereby facilitating immunological killing. Scale bar: 20 μm. Data are presented as mean ± SD (n = 5). Statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant (t-test).#

Figure 6. Development of an organelle-specific LAP inhibitor to enhance antitumor therapy. (a) Schematic of a mitochondria-targeted LAP inhibitor (mito-bestatin) designed to block mitochondrial antioxidant activity during drug treatment while preserving ER-LAP-mediated antigen presentation. Inset: chemical structure of mito-bestatin. (b) Molecular docking model of mito-bestatin bound to LAP. (c) Fluorescence images of live cells treated with bestatin (200 μM, 2 h) or mito-bestatin (200 μM, 2 h), followed by Mito-Tz and TCO-LAP-F. (d) Quantification of fluorescence intensity from panel c. (e) Immunofluorescence images of MHC-I in cells treated with bestatin or mito-bestatin (200 μM, 2 h) together with antitumor drugs CPT (3 μM, 12 h) or CDDP (4 μg/mL, 12 h). Scale bar: 20 μm. (f) Quantification of fluorescence intensity from panel e. (g) Immunofluorescence images of MHC-I in tumor sections from xenograft mouse models treated with LAP inhibitors and antitumor drugs. (h) Schematic workflow for evaluating the combination of mitochondria-specific LAP inhibitors (bestatin or mito-bestatin) (200 μM, 48 h) and antitumor drugs (CDDP) (4 μg/mL, 48 h) on cell viability. (i) Cell viability of cells treated with CDDP in combination with bestatin or mito-bestatin (200 μM, 48 h). (j) Percentage increase in CDDP treatment efficacy from the conditions in panel i. Data are presented as mean ± SD (n = 5). Statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant (t-test).#