Human ATG13 blocking peptide

Autophagy is an important protein quality control mechanism for cells under stress conditions to promote cell survival. Modulation of autophagy on biomaterial substrates is rarely reported. In this study, the autophagy of adipose-derived stem cells (ADSCs) cultured on chitosan (CS) substrates was examined. Compared to the traditional monolayer culture, ADSCs cultured on CS substrates showed spheroid formation as well as a prolonged upregulation of autophagosomal marker-microtubule-associated protein 1 light chain 3 (LC3) II protein expression. In addition, the green fluorescent protein tagged-LC3 (GFP-LC3) expressing ADSCs also revealed more GFP-LC3 puncta on CS substrates. The enhanced autophagy on CS substrates was associated with Ca2+, while ethylene glycol tetraacetic acid (EGTA), a Ca2+ chelator, repressed the autophagy in a dose-dependent manner. Moreover, ADSC spheroids on CS substrates demonstrated a higher survival rate and autophagy response upon H2O2 treatment. The upstream components of autophagy signal pathway-UNC51-like kinase 1 (Ulk1), autophagy-related protein 13 (Atg13), and autophagy/beclin-1 regulator 1 (Ambra1) genes were more highly expressed in ADSC spheroids before and after adding H2O2 than those in the conventional culture. EGTA also decreased the cell viability and autophagy-associated gene expression for ADSC spheroids on CS substrates after H2O2 treatment. Therefore, we suggest that three-dimensional (3D) cell culture on CS may confer ADSCs the ability to increase the autophagic flux in response to stimulations in a Ca2+-dependent manner.

Misfolded and aggregated proteins are eliminated to maintain protein homeostasis. Autophagy contributes to the removal of protein aggregates. However, if and how proteotoxic stress induces autophagy is poorly understood. Here we show that proteotoxic stress after treatment with azetidine-2-carboxylic acid (AZC), a toxic proline analog, induces autophagy in budding yeast. AZC treatment attenuated target of rapamycin complex 1 (TORC1) activity, resulting in the dephosphorylation of Atg13, a key factor of autophagy. By contrast, AZC treatment did not affect target of rapamycin complex 2 (TORC2). Proteotoxic stress also induced TORC1 inactivation and autophagy in fission yeast and human cells. This study suggested that TORC1 is a conserved key factor to cope with proteotoxic stress in eukaryotic cells. (C) 2019 Elsevier Inc. All rights reserved.