Analysis of Chaperone-Mediated Autophagy - PubMed (original) (raw)

Analysis of Chaperone-Mediated Autophagy

Y R Juste et al. Methods Mol Biol. 2019.

Abstract

Chaperone-mediated autophagy (CMA) is a selective type of autophagy whereby a specific subset of intracellular proteins is targeted to the lysosome for degradation. These proteins are identified by a chaperone that targets them to lysosomes. There, they are translocated into the organelle lumen through a lysosomal membrane receptor/translocation complex. CMA plays an important role in maintaining cellular proteostasis by eliminating damaged and altered proteins. CMA also participates in the control of the cellular energetic balance through recycling of amino acids resulting from lysosomal proteolysis of the substrate proteins. Lastly, due to the intrinsic protein selectivity of CMA, this type of autophagy exerts regulatory functions by mediating timely degradation of key cellular proteins that participate in processes such as lipid and glucose metabolism, cell cycle, DNA repair, and cellular reprogramming, among others. Dysfunctional CMA occurs with age and has now been described in a growing list of human pathologies such as metabolic disorders, neurodegeneration, cancer, immunodeficiency, and diabetes. In this chapter, we describe current methodologies to quantitatively analyze CMA activity in different experimental models.

Keywords: Chaperones; Lysosomes; Proteolysis; Subcellular fractionation.

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Figures

Fig. 1

Fig. 1

Chaperone-mediated autophagy: schematic model of the steps in chaperone-mediated autophagy.1. Substrate binding by HSC70 and cochaperones and targeting to lysosomes. 2. Binding of the substrate to LAMP2A at the lysosomal membrane. 3. HSP90 binds to LAMP2A to stabilize it while it organizes into higher molecular weight complexes. 4. Substrate crosses the lysosomal membrane through a LAMP2A-enriched translocation complex, and translocation is complete by the action of luminal HSC70. 5. The substrate is rapidly degraded by luminal proteases (cathepsins). 6. Once substrate translocation is complete, LAMP2A dissociates into monomers in a process dependent on cytosolic HSC70. Red box: negative regulators of CMA at the lysosomal membrane. Green box: positive regulators of CMA at the lysosomal membrane

Fig. 2

Fig. 2

Measurement of CMA in vitro. Top: lysosomes active for CMA can be isolated by flotation in a discontinuous gradient of metrizamide. Bottom: incubation of CMA substrates with intact lysosomes pre-treated or not with inhibitors of lysosomal proteases allows to quantify substrate binding and translocation (uptake) inside the lysosomal lumen

Fig. 3

Fig. 3

Measurement of CMA in cultured cells and in vivo. Top: functional assays, expression of photoswitchable reporters (left) allows quantification of CMA in cultured cells as the number of fluorescent puncta (lysosomes) per cell. Injection of leupeptin in vivo (to block lysosomal proteolysis) and immunoblot for CMA substrate of lysosomes from injected and not injected mice allows quantification of substrate flux by CMA (right). Bottom: steady-state assays, quantification of number of lysosomes positive for LAMP2A and HSC70 allows for detecting changes in lysosomes capable of performing CMA (left). Immunoblot of isolated lysosomes for well-characterized CMA activator and inhibitor proteins. Profile of differences in specific protein levels in lysosomes with upregulated and downregulated CMA activity are shown

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