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Stimulating S-adenosyl-L-methionine synthesis extends lifespan via activation of AMPK Speaker: TZU-CHUN, FANG Advisor: KUNG-YAO, CHANG Saccharomyces cerevisiae(S. cerevisiae) Replicative Life Span(RLS) Chronological Life Span(CLS) Dietary restriction is one of molecular strategies to extend lifespan • RAS-AC-PKA pathway: Ras-adenylate cyclase (AC)-Protein Kinase A (PKA) • TOR-Sch9 pathway: Target of Rapamycin (TOR)-the serine threonine kinase Sch9 The stress resistance transcription factors: Msn2, Msn4, Gis1 Rim15 protein kinase Gpr1:The G protein-coupled receptor Science(2010); 328(5976): 321–326 Lifespan extension Treating methionine restriction extended replicative life span of yeast PNAS (2004) vol. 101 no. 21 7999–8004 Motivation How does Met restriction influence the lifespan of yeast? Methionine metabolic cycle Methionine Synthase SAH SAM SSG1-1, a dominant mutation suppressed slow-growth of the sah1-1 strain 25℃, Incubated YMM222(sah1-1 mutant) in liquid YPD to Midlog phase Plated onto solid YPD 36℃ Suppressor mutants WT: MATa;sah1-1: MATa sah1-1;SSG1-1: MATa SSG1-1 15 Intragentic suppressors in sah1-1 101 dominant suppressor mutants→ SSG1 One colony(SSG1-1) Used chromosomal genomic library Sequencing The features of sah1-1 mutant: • Slow-growth • Accumulations of SAM and SAH • A shortened lifespan YHR032W • Ethionine resistance • The AdoMet accumulation WT: MATa sah1-1: MATa sah1-1 sah1-1 SSG1-1: MATa sah1-1 SSG1-1 SSG1-1: MATa SSG1-1 SSG1-1 suppressor mutant had relative high AdoMet levels SAM/SAH levels 25℃,Incubate cells in liquid YPD to log phase Wash, and extract by perchloric acid Run capillary electrophoresis(CE) Agilent Capillary Ion Analyzer nmol/mg-DCW: nanomole per milligram dry weight of cells SSG1-1 suppressor mutant can extand the chronological life span of yeasts CLS 28℃, Incubate cells in SDC overnight Plate onto YPD plates, Monitoring CFUs starting from day 3 SDC(synthetic dextrose complete) medium: YNB(without amino acid), 2% glucose, some amino acids CFU: colony-forming unit SSG1-1 cells showed increased stress resistances WT: MATa sah1-1: MATa sah1-1 sah1-1 SSG1-1: MATa sah1-1 SSG1-1 SDC, 25℃ YPD, 55℃ SSG1-1: MATa SSG1-1 SSG1-1 mutants contained higher levels of SAM and SAH than WT, and had a longer CLS. Methionine metabolic cycle Methionine Synthase Deleted AdoMet production by AdoMet synthetases decreased CLS WT: MATa sam1Δ: MATa sam1Δ::kanMX4 sam2Δ: MATa sam2Δ::kanMX4 SSG1-1 sam1Δ: MATa SSG1-1 sam1Δ::kanMX4 SSG1-1 sam2Δ: MATa SSG1-1 sam2Δ::kanMX4 The CLS extension by SSG1-1 was dependent on SAM synthesis. Increased AdoMet production by AdoMet synthetases extended CLS SGC medium: YNB(without amino acid), 2% galactose, some amino acids GAL1-SAM1/2: MATa GAL-SAM1 GAL-SAM2 Supplementing the medium with AdoHcy was able to extend the CLS of the WT cells 1mM AdoMet: the medium contains 1mM AdoMet. 1mM AdoHcy: the medium contains 1mM AdoHcy. The WT cells showed a higher accumulation of AdoMet than AdoHcy Stimulating SAM synthesis could promote longevity. Methionine metabolic cycle Methionine Synthase SSG1-1 cells required methionine or glucose for longevity Methionine + ATP Met 20 mg/l: the medium contains 20mg/l Methionine. Met 0 mg/l: the medium without Methionine. AdoMet Glu 2%: the medium contains 2% glucose. Glu 0.05%: the medium contains 0.05% glucose. Met levels in the SSG1-1 cells were lower than the WT cells but ATP levels Cell lysate Extracted by Methanol, centrifuged Boiled cell to extract intracellular ATP Centrifuged The lysed sample with ATP assay buffer The supernatant Ion-exchange chromatography amino acid analyzer Measured fluorescence LUMITESTER SSG1-1 might act to stimulate SAM synthesis by amplifying ATP levels. The CLS of the SSG1-1 strain was equivalent to the WT strain when growing under 0.5% glucose calorie restriction Glu 2%: the medium contains 2% glucose. Glu 0.5%: the medium contains 0.5% glucose. The expressions of Gene in the SSG1-1 cells were increased upper twofold more than in the WT cells Systematic name Standard name Ratio (SSG1-1 vs. WT) Description YLL062C MHT1 2.72 S-methylmethioninehomocysteine methyltransferase YHR092C HXT4 4.07 High-affinity glucose transporter SSG1-1 cell WT Extracted by hot phenol, DNase Cell lysate mRNA mRNA purification kit Labeled with cDNA Hybridized on the Gene Chip Yeast Genome The AMP-activated protein kinase (AMPK) ↑AMP:ATP (0.5% Glu,CR) • AMPK promotes catabolic pathways to generate more ATP. • A yeast sucrose non-fermenting 1 (Snf1) protein kinase is one of AMPK families. AMPK ATP SSG1-1 or the stimulated AdoMet synthesis associated with the activation of AMPK snf1Δ: MATa snf1Δ::kanMX6 GAL1-SAM1/2: MATa GAL-SAM1 GAL-SAM2 Primary antibody: Anti-Phospho-AMPKα (Thr172) Secondary antibody: Anti-His The lower ATP levels in SSG1-1 snf1Δ cells lead to synthesis lower levels of AdoMet SSG1-1 snf1Δ: MATa SSG1-1 snf1Δ::kanMX6 SNF1 might have been required for extending the CLS of the SSG1-1 cells snf1Δ: MATa snf1Δ::kanMX6 SSG1-1 snf1Δ: MATa SSG1-1 snf1Δ::kanMX6 Severe CR(Glu 0.05%) promoted SAM production dependent on SAM synthetases, leading to extended maximum CLS The AdoHcy level was also increased upon severe CR which contributed to the accumulation of AdoMet in WT Summary • Stimulating AdoMet synthesis, which consumes both ATP and Met could produce physiological conditions that mimicked CR. • Stimulating AdoMet synthesis led to AMPK activation and increased lifespan. Thanks for your attention! Homocysteine SMMHT