Speaker: TZU-CHUN, FANG Advisor: KUNG-YAO, CHANG Stimulating S-adenosyl-L-methionine synthesis extends lifespan via activation of AMPK Speaker: TZU-CHUN, FANG Advisor: KUNG-YAO, CHANG

Saccharomyces cerevisiae(S. cerevisiae) S.cerevisiae has relatively simple genetics, is well characterized, has a relatively rapid growth rate. 與其他系統相比，在酵母中可以量化壽命的相對容易性和快速性使得快速的進展是確定該生物體中老化的分子機制以及確定改變其壽命的幾十個因素。 酵母菌一生的分裂次數(壽命)稱為 Replicative Life Span ( RLS )，指的是母細胞在停止分裂前所產生的子代數目 酵母菌進行無性生殖的次數並非無所限制，隨著分裂次數越多，母細胞體內的老化因子也會隨之增加，最終導致死亡，雖然母細胞逐漸老化，但酵母菌會透過不對稱分裂( Asymmetric Segregation )的方式，將老化因子蓄積於母細胞體內，大幅減少老化對子代的影響。 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 衰老是分子、细胞与器官损伤不断积累的生理现象，它能够导致机体相关功能的丧失并使其更容 易遭受疾病的侵袭，以至于最终走向死亡 促生長和促衰老途徑以Tor和Sch9為中心，並與Ras-AC-PKA通路 TOR和Sch9都在營養物質可利用的下游途徑中起作用以調節CLS 0.5％葡萄糖DR對RLS的影響被認為涉及降低Ras-AC-PKA和Tor-Sch9通路的信號轉導，MSN2和Msn4的增加轉錄活性和PNC1，其調節NAD依賴性脫乙酰酶的Sir2（活性的煙酰胺脫氨酶的表達隨之6，16）。改變翻譯和自噬被認為也有助於復制長壽RLS Ras-AC-PKA和Tor-Sch9通路的抑制也參與CLS的DR依賴性延伸。 事實上，缺乏Rim15蛋白激酶或轉錄因子MSN2，MSN4和GIS1的三重缺失可逆轉飢餓或飲食限制對壽命的影響 Ras, adenylate cyclase (AC), and Protein Kinase A (PKA) target of Rapamycin (TOR) , the serine threonine kinase Sch9 The stress resistance transcription factors(阻抗轉錄因子): Msn2, Msn4, Gis1 Rim15 protein kinase 卡路里限制能顯著提高酵母、蠕虫、果蝇和哺乳动物的寿命 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

How does Met restriction influence the lifespan of yeast? Motivation How does Met restriction influence the lifespan of yeast?

Methionine metabolic cycle Methionine Synthase SAH SAM 過去20年來，膳食蛋氨酸限制（MR）已經成為有希望的DR模擬物，因為它在生命週期中產生了可比的延伸 Knockout SAH1 block破壞了內生性 Met的regeneration = mimick Met restriction

SSG1-1, a dominant mutation suppressed slow-growth of the sah1-1 strain One colony(SSG1-1) Used chromosomal genomic library 25℃, Incubated YMM222(sah1-1 mutant) in liquid YPD to Midlog phase Plated onto solid YPD 36℃ Suppressor mutants 15 Intragentic suppressors in sah1-1 101 dominant suppressor mutants→ SSG1 Sequencing WT: MATa；sah1-1: MATa sah1-1；SSG1-1: MATa SSG1-1 在SAH1突變下，要造成Met的短缺， 所以要找一個有沒有可以抑制生長延緩的突變然後要看是不是跟lifespan regulation有關的 The features of sah1-1 mutant: Slow-growth Accumulations of SAM and SAH A shortened lifespan

YHR032W Ethionine resistance The AdoMet accumulation WT: MATa intragenic suppressors(內源性抑制因子)是恢復突變基因產物功能的相同基因內的第二突變 1628-1634 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 毛細管電泳主要是利用外加高電壓下，分析物在緩衝夜中解離成不同帶電性質的形式，藉由不同的帶電性質在電場中受吸引或排斥的力量產生 遷移速率的差異，而造成分離的現象 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 SSG1-1單突變體都顯示出更長的CLS，這表明Ssg1-1蛋白可能在長壽中起作用。因為長壽命的突變體偶爾更耐氧化和熱應激 一般認為會延長壽命的基因突變，會對環境具有較好抗逆性 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 SDC, 25℃ YPD, 55℃ WT: MATa sah1-1: MATa sah1-1 sah1-1 SSG1-1: MATa sah1-1 SSG1-1 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 因為SSG1-1細胞的AdoMet水平升高，所以我們確定了SSG1-1的CLS延伸是否被AdoMet合成所介導的在SAM1和SAM2編碼AdoMet合成酶中刪除的菌株中 作者想要知道SSG1-1這株具有較多SAM SAM accumulation 的原因是什麼? 是因為SSG1會造成SAM1/2合成的更多?或者是有其他機制會使得他收集累積更多的SAM?

Deleted AdoMet production by AdoMet synthetases decreased CLS SSG1-1有較高的SAM量是由SAM1/SAM2合成酶介導合成的 但是SSG1-1的lifespan 不是因為sam1,2影響的 看到了delete sam1/2會使WT LS 增加，所以問說單獨對WT的sam1/2 o/v看會不會影響 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 不論是deletion 或 overexpression WT sam1/2都會影響LS 但是WT deleted sam1/2的不太影響SAM/SAH濃度，反而在o/v中發現有高濃度的SAM產生 如果能合成更多的SAM會來影響LS 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 只針對WT 用1mM SAH時細胞內SAM 和 SAH 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 這些實驗知道SAM的合成會使壽命延長 那作者就去檢查了這些合成SAM的原料來源 Stimulating SAM synthesis could promote longevity.

Methionine metabolic cycle Methionine Synthase

SSG1-1 cells required methionine or glucose for longevity ATP AdoMet + 我們發現用0.05％葡萄糖進行微量處理，但可重複增加WT的最大CLS Met 20 mg/l: the medium contains 20mg/l Methionine. Met 0 mg/l: the medium without Methionine. 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 The supernatant Ion-exchange chromatography amino acid analyzer Boiled cell to extract intracellular ATP The lysed sample with ATP assay buffer Measured fluorescence LUMITESTER Centrifuged Extracted by Methanol, centrifuged 藉由ATP的含量影響LS，作者想到過去以調控ATP濃度的實驗會影響LS 就是施予0.5%葡萄糖，會明顯延長酵母壽命 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 DR使壽命延長的研究中，使用0.5% CR的策略是確定能夠使酵母壽命延長 表明通用途徑可能是CR與其之間長壽的調控機制的基礎SSG1-1介導機制 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-methylmethionine-homocysteine methyltransferase YHR092C HXT4 4.07 High-affinity glucose transporter 通過接種YPD培養基開始培養以獲得約0.05 的起始OD 660nm。當OD 660nm達到1.0 時收穫酵母細胞（WT和SSG1-1菌株）。 為了研究刺激AdoMet合成介導的壽命的基礎 與CR（0.5％葡萄糖）相關的高親和力葡萄糖轉運蛋白HXT基因和葡萄糖代謝基因 這些數據提供了進一步的證據，證明SSG1-1和CR共享共同的途徑來延長壽命 Cell lysate Labeled with cDNA Hybridized on the Gene Chip Yeast Genome mRNA Extracted by hot phenol, DNase  mRNA purification kit SSG1-1 cell WT

The AMP-activated protein kinase (AMPK) ↑AMP:ATP 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. 葡萄糖代謝路徑

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 通過刺激AdoMet合成來激活Snf1，通過放大ATP有助於維持AdoMet水平 SSG1-1 snf1Δ: MATa SSG1-1 snf1Δ::kanMX6

SNF1 might have been required for extending the CLS of the SSG1-1 cells 過去有在其他酵母菌中發現在低葡萄糖濃度下培養時會含有較多的SAM 且在Snf1也是在體內呈低葡萄糖濃度下時會被磷酸化 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