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核心技术专利:CN118964589B侵权必究
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一种基于基因编码荧光的报告分子,用于时空研究甘露糖-6-磷酸途径。

A genetically-encoded fluorescence-based reporter to spatiotemporally investigate mannose-6-phosphate pathway.

机构信息

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.

出版信息

Mol Biol Cell. 2024 Aug 1;35(8):mr6. doi: 10.1091/mbc.E23-09-0344. Epub 2024 Jun 18.


DOI:10.1091/mbc.E23-09-0344
PMID:38888935
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11321044/
Abstract

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.

摘要

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。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce00/11321044/259337ef0fd8/mbc-35-mr6-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce00/11321044/a151bdb8b317/mbc-35-mr6-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce00/11321044/590f1a633f9e/mbc-35-mr6-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce00/11321044/76c676680a3f/mbc-35-mr6-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce00/11321044/afbd5cf296f9/mbc-35-mr6-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce00/11321044/259337ef0fd8/mbc-35-mr6-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce00/11321044/a151bdb8b317/mbc-35-mr6-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce00/11321044/590f1a633f9e/mbc-35-mr6-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce00/11321044/76c676680a3f/mbc-35-mr6-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce00/11321044/afbd5cf296f9/mbc-35-mr6-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce00/11321044/259337ef0fd8/mbc-35-mr6-g005.jpg

相似文献

[1]
A genetically-encoded fluorescence-based reporter to spatiotemporally investigate mannose-6-phosphate pathway.

Mol Biol Cell. 2024-8-1

[2]
LYSET/TMEM251/GCAF is critical for autophagy and lysosomal function by regulating the mannose-6-phosphate (M6P) pathway.

Autophagy. 2023-5

[3]
A shortcut to the lysosome: the mannose-6-phosphate-independent pathway.

Mol Genet Metab. 2012-7-20

[4]
LYSET/TMEM251- a novel key component of the mannose 6-phosphate pathway.

Autophagy. 2023-7

[5]
Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification.

Mol Cell Proteomics. 2018-9-20

[6]
Subcellular trafficking and activity of Hyal-1 and its processed forms in murine macrophages.

Traffic. 2014-3-11

[7]
Replication of Coxiella burnetii in a Lysosome-Like Vacuole Does Not Require Lysosomal Hydrolases.

Infect Immun. 2019-10-18

[8]
Transgenic expression of a ratiometric autophagy probe specifically in neurons enables the interrogation of brain autophagy in vivo.

Autophagy. 2018-10-26

[9]
The novel Drosophila lysosomal enzyme receptor protein mediates lysosomal sorting in mammalian cells and binds mammalian and Drosophila GGA adaptors.

J Biol Chem. 2005-4-1

[10]
Receptor-mediated transport of acid hydrolases to lysosomes.

Curr Top Cell Regul. 1985

本文引用的文献

[1]
Restoration of β-GC trafficking improves the lysosome function in Gaucher disease.

Traffic. 2023-10

[2]
Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation.

Nat Commun. 2023-7-3

[3]
Measuring Phagosomal pH by Fluorescence Microscopy.

Methods Mol Biol. 2023

[4]
Eat, prey, love: Pathogen-mediated subversion of lysosomal biology.

Curr Opin Immunol. 2023-8

[5]
The role of lysosomes in metabolic and autoimmune diseases.

Nat Rev Nephrol. 2023-6

[6]
GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway.

Nat Commun. 2022-9-12

[7]
Lysosomal enzyme trafficking factor LYSET enables nutritional usage of extracellular proteins.

Science. 2022-10-7

[8]
The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

Science. 2022-10-7

[9]
A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag.

Elife. 2022-8-8

[10]
KCTD7 mutations impair the trafficking of lysosomal enzymes through CLN5 accumulation to cause neuronal ceroid lipofuscinoses.

Sci Adv. 2022-8-5

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