Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.
Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.
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DOI：
10.1371/journal.pone.0156312
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Naleway JJ
中科院分区：
文献类型：
--
作者：
Harlan FK;Lusk JS;Mohr BM;Guzikowski AP;Batchelor RH;Jiang Y;Naleway JJ
溶酶体是存在于所有有核哺乳动物细胞中的酸性细胞器，参与多种细胞过程，包括质膜修复、对病原体的防御、胆固醇稳态、骨重建、新陈代谢、细胞凋亡和细胞信号转导。溶酶体酶活性缺陷与多种神经系统疾病有关，包括帕金森氏病、溶酶体贮积症、阿尔茨海默病和亨廷顿病。合成了荧光溶酶体染色探针，用于以活细胞形式标记溶酶体和其他酸性细胞器，并被证明能够监测溶酶体的代谢活性。新的靶向底物是由具有低pKa值的荧光染料制备的，以便在溶酶体中发现的较低的生理pH下获得最佳的荧光。它们被改造成含有靶向基团，以指导它们在溶酶体中的积累，以及使用活细胞染色形式监测特定酶活性的酶切割功能。与已知的溶酶体染色LysoTracker®Red DND-99以及抗LAMP1抗体染色相比，应用于异染性脑白质营养不良、Krabbe和Gaucher病患者的血液和皮肤样本细胞以及健康人成纤维细胞和白细胞控制细胞的染色显示出对溶酶体的定位。当氯喹抑制细胞代谢时，酯酶底物的染色减少，表明该底物可用于测量细胞代谢。当应用于病变细胞时，染色强度反映了在病变细胞中发现的溶酶体酶水平。与未患病的细胞相比，高谢病或克雷伯病患者细胞系中针对酶缺陷的底物染色减少。新的溶酶体靶向荧光底物在溶酶体储存障碍和相关疾病的药物开发中将有助于研究、诊断和监测二级治疗药物对溶酶体酶活性的影响。
Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson’s Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and monitoring the effect of secondary therapeutic agents on lysosomal enzyme activity in drug development for the lysosomal storage disorders and allied diseases.