P38/MAPK is required for protein aggregate formation and removal by autophagy upon proteasome dysfunction

Abstract

AbstractPurposeAge‐related macular degeneration (AMD) can be classified as an aggregation disease where cellular protein waste starts to accumulate in the retinal pigment epithelial (RPE) cells. Decline of autophagy and dysfunction of proteasomes have been identified as one the main reasons for the eventually detrimental protein aggregation. p38/mitogen‐activated protein kinases (MAPK) seem to play a regulatory role in autophagy. However, the function of p38/MAPK in proteasome dysfunction remains unveiled.MethodsARPE‐19 cells were treated with proteasome inhibitor MG‐132 (5 µM) to create a need for autophagosomal removal of protein waste. Simultaneously, commercial p38/MAPK inhibitor PD169316 (5 µM) were introduced to the cells to study it’s role in autophagy regulation. Autophagy flux was inhibited with Bafilomycin A1 (50 nM). Western blot for ubiquitin (marker of degradation‐destined proteins), p62 (protein aggregation marker) and LC3 (autophagosome marker) was performed. Similarly, immunocytochemistry of ubiquitin, p62 and LC3 to reveal their expression levels and cellular localization was conducted.ResultsProteasome inhibition resulted strong accumulation of ubiquitin‐tagged proteins, protein aggregates (p62) and autophagosomes (LC3‐II). p38/MAPK inhibition upon proteasome dysfunction blocked aggregate formation as p62 remained at basal level. Furthermore, despite the need for autophagosomal removal of protein waste due to proteasome inhibition, p38/MAPK inhibitor treated cells did not show increased autophagy. Immunocytochemistry revealed spread of ubiquitin‐ and p62‐tagged proteins throughout the cytosol upon p38/MAPK inhibition, autophagy‐related perinuclear accumulation of protein aggregates was not observed.Conclusionsp38/MAPK inhibition seemed to prevent protein aggregate formation upon proteasome inhibition and thereby autophagic degradation. Securing correct function of p38/MAPK may be a potential therapy target for aggregation diseases such as AMD.