Comprehensive Analyses to Identify Specific Markers for Autophagic Degradation

Abstract

Objective: There is no convenient parameter for autophagic protein degradation. To ascertain specific marker(s) for autophagy, autophagic degradation products of liver autolysosomes as well as cultured cells were analyzed comprehensively.

Materials: Autolysosomes isolated from rat livers using Percoll density-gradient centrifugation and mouse embryonic fibroblasts (MEFs) in culture.

Methods: i) Autolysosomes were isolated from rat and mouse livers using Percoll-density gradient centrifugation. Lysosome-enriched fraction was separated by differential centrifugation from total lysate of MEFs that were cultured in starvation medium (Krebs-Ringer bicarbonate buffer) for 4 hr. ii) Free amino acids and degradation peptides in autolysosomes were determined using an amino acid analyzer (Hitachi L-8900FF) and a mass spectrometer (AB SCIEX, Triple TOF 5600).

Results: i) Peptides produced via autophagic degradation of some key metabolic enzymes were identified, using proteomic analyses of autolysosomes. These include argininosuccinate synthase and eukaryotic elongation factor 1A. Importantly, they were found excreted to culture media of MEFs. ii) In addition to 20 amino acids that were derived from autophagic degradation, β-alanine was found abundantly present in liver autolysosomes. It was also confirmed that β-alanine content in crude lysosomal fraction accounts for ～30% of total cellular β-alanine in cultured MEFs.

Conclusions: i) Antibodies raised against peptides derived from argininosuccinate synthase and eukaryotic elongation factor 1A can be used to trace autophagic degradation process or degradation markers. ii) Enrichment of β-alanine in autolysosomes suggests a mechanism for β-alanine transport into lysosomes. It is important to clarify the relationship between β-alanine accumulation in autolysosomes and autophagy.