Download SSG1-1

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