Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory for Physical Chemistry of Solid Surfaces, The Key Laboratory for Chemical Biology of Fujian Province, and The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Xiamen University, Xiamen 361005, China.
College of Life Science and State Key Laboratory for Cell Stress, Xiamen University, Xiamen 361005, China.
Anal Chem. 2022 Jul 12;94(27):9903-9910. doi: 10.1021/acs.analchem.2c01892. Epub 2022 Jun 27.
Lysosomal rupture engaged in diverse diseases remains poorly discerned from lysosomal membrane permeabilization (LMP). We herein reported biocapture-directed chemical labeling (BCCL) for the discern of lysosomal rupture by tracking the release of optically labeled cathepsins from damaged lysosomes into the cytosol. BCCL entails covalent anchoring of an azide-tagged suicide substrate (Epo-LeuTyr) to the enzyme active site and bioorthogonal ligation of the introduced azide with RC, a ratiometric sensor featuring an acidity-reporting red emissive X-rhodamine-lactam (ROX), blue emissive coumarin (CM) inert to pH, and DBCO reactive to azide. Aided with fluorescein isocyanate-labeled sialic acid (FITC-Sia), a probe remained in pH-elevated lysosomes but dissipated from LMP lysosomes, BCCL enables optical discern of four states of lysosomes: ruptured lysosomes (blue in cytosol), LMP lysosomes (blue in lysosomes), pH-elevated lysosomes (blue and green in lysosomes), and physiological lysosomes (blue, green and red in lysosomes). This approach could find applicability to study lysosome rupture over LMP in diseases and to evaluate lysosome rupture-inducing drugs.
溶酶体破裂在多种疾病中的作用仍未被很好地区分，与溶酶体膜通透性（LMP）不同。本文报道了一种通过跟踪受损溶酶体中的光标记组织蛋白酶释放到细胞质中来区分溶酶体破裂的生物捕获导向化学标记（BCCL）方法。BCCL 需要将带有叠氮化物标记的自杀底物（Epo-LeuTyr）共价锚定到酶的活性位点，并通过生物正交连接将引入的叠氮化物与 RC 连接，RC 是一种比率型传感器，具有报告酸度的红色发射 X-罗丹明内酰胺（ROX）、对 pH 不敏感的蓝色发射香豆素（CM）和对叠氮化物反应的 DBCO。借助荧光素异氰酸酯标记的唾液酸（FITC-Sia），该探针仍留在 pH 升高的溶酶体中，但从 LMP 溶酶体中消失，BCCL 能够光学区分溶酶体的四种状态：破裂的溶酶体（细胞质中呈蓝色）、LMP 溶酶体（溶酶体中呈蓝色）、pH 升高的溶酶体（溶酶体中呈蓝色和绿色）和生理溶酶体（溶酶体中呈蓝色、绿色和红色）。这种方法可以应用于研究疾病中溶酶体破裂与 LMP 的关系，并评估诱导溶酶体破裂的药物。
