{"title":"Cathepsin D inhibition during neuronal differentiation selectively affects individual proteins instead of overall protein turnover","authors":"","doi":"10.1016/j.biochi.2024.03.013","DOIUrl":null,"url":null,"abstract":"<div><div>Cathepsin D (CTSD) is a lysosomal aspartic protease and its inherited deficiency causes a severe pediatric neurodegenerative disease called neuronal ceroid lipofuscinosis (NCL) type 10. The lysosomal dysfunction in the affected patients leads to accumulation of undigested lysosomal cargo especially in none-dividing cells, such as neurons, resulting in death shortly after birth. To explore which proteins are mainly affected by the lysosomal dysfunction due to CTSD deficiency, Lund human mesencephalic (LUHMES) cells, capable of inducible dopaminergic neuronal differentiation, were treated with Pepstatin A. This inhibitor of “acidic” aspartic proteases caused accumulation of acidic intracellular vesicles in differentiating LUHMES cells. Pulse-chase experiments involving stable isotope labelling with amino acids in cell culture (SILAC) with subsequent mass-spectrometric protein identification and quantification were performed. By this approach, we studied the degradation and synthesis rates of 695 and 680 proteins during early and late neuronal LUHMES differentiation, respectively. Interestingly, lysosomal bulk proteolysis was not altered upon Pepstatin A treatment. Instead, the protease inhibitor selectively changed the turnover of individual proteins. Especially proteins belonging to the mitochondrial energy supply system were differentially degraded during early and late neuronal differentiation indicating a high energy demand as well as stress level in LUHMES cells treated with Pepstatin A.</div></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":"226 ","pages":"Pages 35-48"},"PeriodicalIF":3.3000,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochimie","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0300908424000713","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Cathepsin D (CTSD) is a lysosomal aspartic protease and its inherited deficiency causes a severe pediatric neurodegenerative disease called neuronal ceroid lipofuscinosis (NCL) type 10. The lysosomal dysfunction in the affected patients leads to accumulation of undigested lysosomal cargo especially in none-dividing cells, such as neurons, resulting in death shortly after birth. To explore which proteins are mainly affected by the lysosomal dysfunction due to CTSD deficiency, Lund human mesencephalic (LUHMES) cells, capable of inducible dopaminergic neuronal differentiation, were treated with Pepstatin A. This inhibitor of “acidic” aspartic proteases caused accumulation of acidic intracellular vesicles in differentiating LUHMES cells. Pulse-chase experiments involving stable isotope labelling with amino acids in cell culture (SILAC) with subsequent mass-spectrometric protein identification and quantification were performed. By this approach, we studied the degradation and synthesis rates of 695 and 680 proteins during early and late neuronal LUHMES differentiation, respectively. Interestingly, lysosomal bulk proteolysis was not altered upon Pepstatin A treatment. Instead, the protease inhibitor selectively changed the turnover of individual proteins. Especially proteins belonging to the mitochondrial energy supply system were differentially degraded during early and late neuronal differentiation indicating a high energy demand as well as stress level in LUHMES cells treated with Pepstatin A.
期刊介绍:
Biochimie publishes original research articles, short communications, review articles, graphical reviews, mini-reviews, and hypotheses in the broad areas of biology, including biochemistry, enzymology, molecular and cell biology, metabolic regulation, genetics, immunology, microbiology, structural biology, genomics, proteomics, and molecular mechanisms of disease. Biochimie publishes exclusively in English.
Articles are subject to peer review, and must satisfy the requirements of originality, high scientific integrity and general interest to a broad range of readers. Submissions that are judged to be of sound scientific and technical quality but do not fully satisfy the requirements for publication in Biochimie may benefit from a transfer service to a more suitable journal within the same subject area.