Quantitative proteomic analysis of temporal lysosomal proteome and the impact of the KFERQ-like motif and LAMP2A in lysosomal targeting

Abstract

Autophagic pathways are regulated mechanisms that play important roles in lysosome-mediated cellular degradation. Yet, the contribution of different autophagic pathways in lysosomal targeting, and characterization of the extent and specificity in their degradome remains largely uncharacterized. By undertaking a multiplex quantitative mass spectrometry approach, we have previously analyzed the lysosomal proteome during chaperone-mediated autophagy (CMA)-stimulated conditions in cancer cells. Here, we have extended our multiplex quantitative mass spectrometry and bioinformatics analysis on the proteome from isolated lysosomes to gain a comprehensive view of the temporal enriched lysosomal content upon non-macroautophagy-activated conditions. In parallel, we describe the functional dependency of LAMP2A on, and to what degree the presence of KFERQ-like motifs in proteins influences, their lysosomal targeting. These findings establish a framework for a better understanding of the degradome mediated by autophagic pathways beyond macroautophagy, and present characterization of the impact of LAMP2A in lysosomal targeting in cancer cells.

Chaperone-Mediated Autophagy Controls Proteomic and Transcriptomic Pathways to Maintain Glioma Stem Cell Activity

“…Among them, ECM effectors were decreased both at the transcriptional and protein level, whereas immune system and mitochondrial metabolism related pathways were deregulated at proteome level, and PI3K-AKT and p53 pathways altered in RNAseq study. In the same way, previous analysis of CMA targetome in ovarian, breast, fibrosarcoma and lung cancer cells demonstrated alterations in processes such as translation and RNA regulation or intercellular transport (50,51), which are only partly observed in our model. These differences could suggest a cell-type dependent regulation of CMA, with some common processes in normal tumor bulk and cancer stem cells, but for the most part, very distinctive mechanism in these last ones.…”

Mitochondrial A‐kinase anchoring proteins in cardiac ventricular myocytes

“…Upon further analysis, D‐AKAP1, OPA1, and WAVE‐1 were found in both cytosolic and mitochondrial fractions, while D‐AKAP2, ACBD3, and Rab32 were primarily found in mitochondria enriched fractions. D‐AKAP1, ACBD3, and Rab32 appeared as single bands at the expected molecular weight (Das Banerjee et al, 2017 ; Gabrovsek et al, 2020 ; Hu et al, 2019 ; Kacal et al, 2021 ; Kalogeropulou et al, 2020 ; Tan et al, 2021 ; Teoule et al, 2013 ). The identification of multiple immunoreactive bands for D‐AKAP2 may be explained by the existence of splice variants as observed in mouse brain and lung extracts (Wang et al, 2001 ).…”

The roles of molecular chaperones in regulating cell metabolism

“…Bioinformatics studies identified that almost one-third of the human proteome contains this motif and might be substrates for CMA [192]; however, only a few proteins have been experimentally validated [193]. Recent proteomic studies have identified many metabolic enzymes that are degraded through CMA as a result of decreased glucose uptake using AC220, an inhibitor of the tyrosine kinase FLT3 [194,195]. Furthermore, the degradation of these metabolic enzymes is not constant but variable after periods of glucose starvation [194].…”