Puromycin-sensitive aminopeptidase protects against aggregation-prone proteins via autophagy

Abstract

A major function of proteasomes and macroautophagy is to eliminate misfolded potentially toxic proteins. Mammalian proteasomes, however, cannot cleave polyglutamine (polyQ) sequences and seem to release polyQ-rich peptides. Puromycin-sensitive aminopeptidase (PSA) is the only cytosolic enzyme able to digest polyQ sequences. We tested whether PSA can protect against accumulation of polyQ fragments. In cultured cells, Drosophila and mouse muscles, PSA inhibition or knockdown increased aggregate content and toxicity of polyQ-expanded huntingtin exon 1. Conversely, PSA overexpression decreased aggregate content and toxicity. PSA inhibition also increased the levels of polyQ-expanded ataxin-3 as well as mutant α-synuclein and superoxide dismutase 1. These protective effects result from an unexpected ability of PSA to enhance macroautophagy. PSA overexpression increased, and PSA knockdown or inhibition reduced microtubule-associated protein 1 light chain 3-II (LC3-II) levels and the amount of protein degradation sensitive to inhibitors of lysosomal function and autophagy. Thus, by promoting autophagic protein clearance, PSA helps protect against accumulation of aggregation-prone proteins and proteotoxicity.

Cathepsins L and Z Are Critical in Degrading Polyglutamine-containing Proteins within Lysosomes

“…13) or CFP-LC3 (a generous gift from Prof. Lippincott-Schwartz). N1E-115 cells were transfected with ExGen500 (Fermentas) for 48 h with the plasmids for Htt74Q-mCherry and RNAi, either control or against cathepsin L or Z.…”

“…No gross evidence of necrosis or inflammation as a result of the transfection procedure was noted. Inclusion size and number were analyzed with Metamorph using fixed size and intensity thresholds as described (13).…”

“…In addition, there is increasing evidence that autophagy and thus lysosomal enzymes are important in the degradation of expanded polyQ-containing proteins (13,17). To determine if lysosomes have a greater capacity to hydrolyze such sequences than cytosolic enzymes, lysosomes from mouse liver were partially purified by differential centrifugation (20) and sonicated, and their capacity to hydrolyze several polyQ peptides was studied.…”

“…mutated SOD1). Surprisingly, puromycin-sensitive aminopeptidase was found not to simply catalyze the digestion of proteasome-derived fragments but to have the capacity to activate autophagy and lysosomal degradation through still unknown mechanisms (13). Moreover, a variety of studies suggest that the large aggregates of polyQ proteins are not degraded by proteasomes but instead tend to be destroyed by autophagy (13).…”

Glu121‐Lys319 salt bridge between catalytic and N‐terminal domains is pivotal for the activity and stability of Escherichia coli aminopeptidase N

“…For example, hAPN, LTA 4 H, and PSA are targeted by various inhibitor molecules to find cures for cancer, inflammation, and Alzheimers disease. 2,[16][17][18][19] Structure of ePepN can serve as a prototype example for understanding these important proteins. Crystal structures of ePepN have been reported for some product and inhibitor complexes that explain the basis for substrate recognition.…”