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Merck

Inhibition of class I PI3K enhances chaperone-mediated autophagy.

The Journal of cell biology (2020-10-14)
S Joseph Endicott, Zachary J Ziemba, Logan J Beckmann, Dennis N Boynton, Richard A Miller
ABSTRACT

Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, where individual peptides, recognized by a consensus motif, are translocated directly across the lysosomal membrane. CMA regulates the abundance of many disease-related proteins, with causative roles in neoplasia, neurodegeneration, hepatosteatosis, and other pathologies relevant to human health and aging. At the lysosomal membrane, CMA is inhibited by Akt-dependent phosphorylation of the CMA regulator GFAP. The INS-PI3K-PDPK1 pathway regulates Akt, but its role in CMA is unclear. Here, we report that inhibition of class I PI3K or PDPK1 activates CMA. In contrast, selective inhibition of class III PI3Ks does not activate CMA. Isolated liver lysosomes from mice treated with either of two orally bioavailable class I PI3K inhibitors, pictilisib or buparlisib, display elevated CMA activity, and decreased phosphorylation of lysosomal GFAP, with no change in macroautophagy. The findings of this study represent an important first step in repurposing class I PI3K inhibitors to modulate CMA in vivo.

MATERIALS
Product Number
Brand
Product Description

Roche
cOmplete, Mini Protease Inhibitor Cocktail, Tablets provided in a glass vial
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OptiPrep Density Gradient Medium, used for cell and subcellular organelle isolation
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Hoechst 33258 solution, 1 mg/mL in H2O, ≥98.0% (HPLC)
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Adenosine 5′-triphosphate disodium salt hydrate, 99%
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Dexamethasone, powder, BioReagent, suitable for cell culture, ≥97%
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Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
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Trifluoroacetic acid, ReagentPlus®, 99%
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Bafilomycin A1 Ready Made Solution, 0.16 mM in DMSO, from Streptomyces griseus
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Leupeptin, Hemisulfate, Synthetic, Synthetic version of the microbial-derived inhibitor Leupeptin against trypsin-like and cysteine protease activities.