Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, India.
Indian Institute of Science Education and Research, Tirupati 517619, India.
Mol Biol Cell. 2024 Aug 1;35(8):mr6. doi: 10.1091/mbc.E23-09-0344. Epub 2024 Jun 18.
Maintenance of a pool of active lysosomes with acidic pH and degradative hydrolases is crucial for cell health. Abnormalities in lysosomal function are closely linked to diseases, such as lysosomal storage disorders, neurodegeneration, intracellular infections, and cancer among others. Emerging body of research suggests the malfunction of lysosomal hydrolase trafficking pathway to be a common denominator of several disease pathologies. However, available conventional tools to assess lysosomal hydrolase trafficking are insufficient and fail to provide a comprehensive picture about the trafficking flux and location of lysosomal hydrolases. To address some of the shortcomings, we designed a genetically-encoded fluorescent reporter containing a lysosomal hydrolase tandemly tagged with pH sensitive and insensitive fluorescent proteins, which can spatiotemporally trace the trafficking of lysosomal hydrolases. As a proof of principle, we demonstrate that the reporter can detect perturbations in hydrolase trafficking, that are induced by pharmacological manipulations and pathophysiological conditions like intracellular protein aggregates. This reporter can effectively serve as a probe for mapping the mechanistic intricacies of hydrolase trafficking pathway in health and disease and is a utilitarian tool to identify genetic and pharmacological modulators of this pathway, with potential therapeutic implications.
维持具有酸性 pH 值和降解水解酶的活跃溶酶体池对于细胞健康至关重要。溶酶体功能异常与疾病密切相关,例如溶酶体贮积症、神经退行性疾病、细胞内感染和癌症等。越来越多的研究表明,溶酶体水解酶运输途径的功能障碍是几种疾病病理学的共同特征。然而,现有的常规工具来评估溶酶体水解酶的运输还不够充分,无法全面了解溶酶体水解酶的运输通量和位置。为了解决其中的一些缺点,我们设计了一种遗传编码的荧光报告子,其中包含一个溶酶体水解酶,该水解酶与 pH 敏感和不敏感的荧光蛋白串联标记,可以时空追踪溶酶体水解酶的运输。作为原理验证,我们证明该报告子可以检测到由药理学操作和细胞内蛋白质聚集等病理生理条件诱导的水解酶运输的扰动。该报告子可以有效地作为探针,用于绘制健康和疾病中水解酶运输途径的机制复杂性图谱,并且是识别该途径的遗传和药理学调节剂的有用工具,具有潜在的治疗意义。
Mol Genet Metab. 2012-7-20
Mol Cell Proteomics. 2018-9-20
Curr Top Cell Regul. 1985
Methods Mol Biol. 2023
Curr Opin Immunol. 2023-8
Nat Rev Nephrol. 2023-6