Dataset Information

Cellular Depletion of Major Cathepsin Proteases in HeLa and SH-SY5Y Cells

ABSTRACT: Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans there are 15 lysosomal cathepsins (CTS) that act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant lysosomal proteinases and are often linked to autophagy, lysosomal storage and neurodegenerative diseases. Their concerted functions in proteolysis in the lysosome, their individual substrates, cleavage specificity, functional redundancy, and possible sequential action on substrate proteins are not well understood. To address these open points, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of semitryptic peptides and of lysosome/autophagy-associated proteins but also of potentially endocytosed membrane proteins like the amyloid precursor protein (APP). Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that APP is regularly processed by multiple CTS-mediated cleavage events.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Stephan Mueller

LAB HEAD: Stefan F. Lichtenthaler

PROVIDER: PXD048603 | Pride | 2024-05-24

REPOSITORIES: Pride

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Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis.

Gallwitz Lisa L Bleibaum Florian F Voss Matthias M Schweizer Michaela M Spengler Katharina K Winter Dominic D Zöphel Frederic F Müller Stephan S Lichtenthaler Stefan S Damme Markus M Saftig Paul P

Cellular and molecular life sciences : CMLS 20240522 1

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have b ...[more]

PMID: 38775843

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Project description:Background Transforming growth factor β1 (TGF-β1) has a neuroprotective function in traumatic brain injury (TBI) through its anti-inflammatory and immunomodulatory properties. In our previous study, we found that TGF-β1 played a critical role in inhibiting apoptosis and increasing neuronal activity in murine cortical neurons following trauma. However, the precise mechanisms underlying the neuroprotective actions of TGF-β1 on the cortex require further investigation. Methods Thus, in this study, we were aimed to investigate the regulatory function of TGF-β1 on neuronal autophagy and apoptosis using an in vitro primary cortical neuron trauma-injury model. Results To establish the landscape of differentially expressed genes (DEGs) with or without TGF-β1 (10ng/ml) treatment for 24 hours, we performed RNA-sequencing. We observed significant enrichment of DEGs related to autophagy, apoptosis, and the lysosome pathway in trauma-injured cortical neurons. Additionally, transmission electron microscopy (TEM) confirmed the presence of autophagosomes as well as autophagolysosomes. Western blot analysis revealed upregulation of autophagy-related protein light chain 3 (LC3)-Ⅱ/LC3-Ⅰ, sequestosome 1 (SQSTM1)/p62, along with apoptosis-related protein Cleaved-caspase3 in trauma-injured primary cortical neurons. Furthermore, mechanically injured neurons showed an upregulation of lysosomal marker protein lysosomal marker protein (LAMP1) and lysosomal enzyme mature cathepsin D (mCTSD), but a decrease in the activity of CTSD enzyme. These results indicated that apoptosis was up-regulated in mechanically injured neurons at 24 hours, accompanied by lysosomal dysfunction and impaired autophagic flux. Notably, TGF-β1 significantly reversed these changes. Conclusions Therefore, our findings suggested that TGF-β1 exerted neuroprotective effects on mechanically injured neurons by reducing lysosomal dysfunction, decreasing the accumulation of autophagosomes and autophagolysosomes, and enhancing autophagic flux.

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