ABSTRACT
The lysosomal endoprotease cathepsin D (CatD) is an essential player in general protein turnover and specific peptide processing. CatD-deficiency is associated with neurodegenerative diseases, whereas elevated CatD levels correlate with tumor malignancy and cancer cell survival. Here, we show that the CatD ortholog of the budding yeast Saccharomyces cerevisiae (Pep4p) harbors a dual cytoprotective function, composed of an anti-apoptotic part, conferred by its proteolytic capacity, and an anti-necrotic part, which resides in the protein's proteolytically inactive propeptide. Thus, deletion of PEP4 resulted in both apoptotic and necrotic cell death during chronological aging. Conversely, prolonged overexpression of Pep4p extended chronological lifespan specifically through the protein's anti-necrotic function. This function, which triggered histone hypoacetylation, was dependent on polyamine biosynthesis and was exerted via enhanced intracellular levels of putrescine, spermidine and its precursor S-adenosyl-methionine. Altogether, these data discriminate two pro-survival functions of yeast CatD and provide first insight into the physiological regulation of programmed necrosis in yeast.
Subject(s)
Apoptosis/genetics , Aspartic Acid Endopeptidases , Cathepsin D/metabolism , Lysosomes/metabolism , Necrosis/metabolism , Protein Precursors/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Acetylation , Aspartic Acid Endopeptidases/biosynthesis , Aspartic Acid Endopeptidases/deficiency , Aspartic Acid Endopeptidases/genetics , Biogenic Polyamines/metabolism , Cathepsin D/genetics , Cell Survival , Cellular Senescence , Gene Deletion , Gene Expression , Histones/genetics , Histones/metabolism , Lysosomes/genetics , Necrosis/genetics , Plasmids , Protein Engineering/methods , Protein Precursors/genetics , Protein Structure, Tertiary/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid , TransfectionABSTRACT
This review focuses on the advances in electron capture mass spectrometry. Electron-capture (EC) is a sensitive ionisation technique for mass spectrometry providing selectivity towards electrophoric compounds. Recent advances in instrumentation have led to a more widespread application of this method in biomedical and pharmaceutical analysis. After a brief introduction to EC-mass spectrometry (MS), potential targets for EC-MS analysis are defined and enhancement of sensitivity by electrophoric derivatisation is discussed. A wide range of applications is selected, including prostanoid analysis in biomedical systems (with the oxidative stress indicators isoprostanes) and the trace level analysis of endogenous low-molecular weight compounds. Application to the trace level gas chromatography-negative ion chemical ionization MS (GC-NICI-MS) analysis of complex glucuronides is also demonstrated, as well as a wide range of drugs analysed in human blood. The review should point out the versatility and unique sensitivity of the technique, making it useful for basic research in medicinal chemistry, as well as clinical diagnosis, pharmaceutical and toxicological applications.
