Introduction Cells have developed complex interconnecting biosynthetic pathways to regulate intracellular NAD+ levels. Aberrant NAD+ metabolism has been linked to cancer, metabolic disorders and neurodegenerative diseases. The role played by NAD+ metabolism in protection from protein misfolding induced aging has been studied in wild type yeast cells (YWT) in comparison with in yeast cells expressing polyQ huntingtin (YPQ), designed to recapitulate the crucial events that are manifested during the course of human aging, including protein misfolding and aggregation. Methods In the yeast model the protein responsible of cellular toxicity consists of an amino-terminal FLAG-tagged construct containing the first 17 amino acids of Htt followed by the polyQ tract and green fluorescent protein (GFP). The lifespan of yeast cultures were followed both by monitoring the absorbance of liquid cultures at 600 nm, and by counting the number of colonies grown in agar plates. Quantification of NAD related metabolites has been performed by RP-HPLC in yeast cells extracts, after extraction with boiling buffered ethanol. Nicotinamide mononucleotide adenylyltransferase (NMNAT) enzymatic activity has been determined through a spectrophotometric assay. Results The growth curves of both yeast cells expressing polyQ huntingtin (YPQ) and wild type yeast cells (YWT) have shown that the mutant protein is not lethal in the proliferative life span, while can affect negatively the stationary phase. The evaluation of the NAD metabolites in yeast cells has shown a significant decrease of nicotinamide and NAD in YPQ cells in comparison with the control yeast cells, while NMNAT specific activity in YPQ cells was higher than in YWT cells. Conclusions The presence of polyQ huntingtin can affect the chronological life span of S. cerevisiae yeast cells. The observed response of NMNAT enzymatic activity in YPQ cells could be related to a possible role of the enzyme as molecular chaperone.
Role of NAD+ metabolism in protection from protein misfolding induced aging in a yeast model.
MICOZZI, DANIELA;CARPI, FRANCESCO MARTINO;LUCCI, CRISTIANO;VINCENZETTI, Silvia;POLZONETTI, Valeria;MORESCHINI, BENEDETTA;BALLARINI, Patrizia;PUCCIARELLI, Stefania
2015-01-01
Abstract
Introduction Cells have developed complex interconnecting biosynthetic pathways to regulate intracellular NAD+ levels. Aberrant NAD+ metabolism has been linked to cancer, metabolic disorders and neurodegenerative diseases. The role played by NAD+ metabolism in protection from protein misfolding induced aging has been studied in wild type yeast cells (YWT) in comparison with in yeast cells expressing polyQ huntingtin (YPQ), designed to recapitulate the crucial events that are manifested during the course of human aging, including protein misfolding and aggregation. Methods In the yeast model the protein responsible of cellular toxicity consists of an amino-terminal FLAG-tagged construct containing the first 17 amino acids of Htt followed by the polyQ tract and green fluorescent protein (GFP). The lifespan of yeast cultures were followed both by monitoring the absorbance of liquid cultures at 600 nm, and by counting the number of colonies grown in agar plates. Quantification of NAD related metabolites has been performed by RP-HPLC in yeast cells extracts, after extraction with boiling buffered ethanol. Nicotinamide mononucleotide adenylyltransferase (NMNAT) enzymatic activity has been determined through a spectrophotometric assay. Results The growth curves of both yeast cells expressing polyQ huntingtin (YPQ) and wild type yeast cells (YWT) have shown that the mutant protein is not lethal in the proliferative life span, while can affect negatively the stationary phase. The evaluation of the NAD metabolites in yeast cells has shown a significant decrease of nicotinamide and NAD in YPQ cells in comparison with the control yeast cells, while NMNAT specific activity in YPQ cells was higher than in YWT cells. Conclusions The presence of polyQ huntingtin can affect the chronological life span of S. cerevisiae yeast cells. The observed response of NMNAT enzymatic activity in YPQ cells could be related to a possible role of the enzyme as molecular chaperone.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
