Quantifying autophagy using novel LC3B and p62 TR-FRET assays

被引：40

作者：

Bresciani, Alberto ^{[1
]}

Spiezia, Maria Carolina ^{[1
]}

Boggio, Roberto ^{[2
]}

Cariulo, Cristina ^{[1
]}

Nordheim, Anja ^{[3
]}

Altobelli, Roberta ^{[1
]}

Kuhlbrodt, Kirsten ^{[3
]}

Dominguez, Celia ^{[4
]}

Munoz-Sanjuan, Ignacio ^{[4
]}

Wityak, John ^{[4
]}

Fodale, Valentina ^{[1
]}

Marchionini, Deanna M. ^{[4
]}

Weiss, Andreas ^{[2
,3
]}

机构：

[1] IRBM Sci Pk, Rome, Italy

[2] IRBM Promidis, Rome, Italy

[3] Evotec AG, Manfred Eigen Campus, Hamburg, Germany

[4] CHDI Management CHDI Fdn, New York, NY 10001 USA

来源：

PLOS ONE | 2018年 / 13卷 / 03期

关键词：

MOUSE MODEL; SELECTIVE AUTOPHAGY; MUTANT HUNTINGTIN; DEGRADATION; P62/SQSTM1; DISEASE; MACROAUTOPHAGY; MATURATION; INHIBITOR; TOXICITY;

D O I：

10.1371/journal.pone.0194423

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.

引用

页数：18

共 32 条

[1] p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death [J].