Metabolic basis of the NADPH-independent disulfide reductase system in mouse liver

已知：二硫化物还原驱动的酶S！支持体内平衡和对抗氧化损伤 会导致神经退化、炎症性疾病和癌症。NADPH为 大多数合成代谢和细胞保护还原反应，但只有两种酶能使用NADPH还原 胞质二硫化物：硫氧还蛋白还原酶-1(TrxR1)和谷胱甘肽还原酶(GSR)1。TrxR1和GSR都有 主要被亲电毒素和氧化剂抑制的活性部位2，3。在Co-Pi Schmidt的实验室中，小鼠 利用TrxR1/GSR-空肝脏发现了二硫化物还原酶系统中意想不到的健壮性，包括 NADPH非依赖性途径，利用蛋氨酸(Met)的分解代谢来维持氧化还原动态平衡。 重要的是，这一途径也被认为在氧化或亲电应激下维持正常细胞。 半胱氨酸是蛋白质中发现的两种含硫(S)-氨基酸，但含有S的分子是由蛋氨酸或半胱氨酸合成的， 包括S-腺苷蛋氨酸(SAM)、谷胱甘肽(GSH)、辅酶A(CoA)等，在氧化还原、脱毒、 能量学、生物合成、调节和其他过程。联席PI DeNicola一直在研究Alternate的角色 S-氨基酸代谢在某些癌症中的作用6。这些研究揭示了一些癌症是如何利用 改变S-氨基酸氧化还原代谢，可能揭示靶向癌症特异性易感性。 悬而未决的问题：尚不清楚其他代谢活动，包括那些直接利用 MET或Cys，以及更多的外围系统，它们或者(I)向这些通路提供资源；(Ii)依赖于 在这些途径上；或(Iii)在某些情况下，可能与这些途径竞争底物， 重新排列以帮助细胞在压力下生存。我们假设，向Met依赖的转换需要重新调整 不同的新陈代谢途径。这项工作意义重大，因为对这些过程的更好理解将 发现可作为治疗目标的流程，以明确提高关键 氧化或毒性应激下的细胞，或特别增加致病细胞在癌症或 炎症性疾病。重新提交的新的初步调查表明，我们有能力履行 在整个小鼠和小鼠衍生的肝脏器官中的稳定同位素通量标记研究。 建议：在这个修订的多机构合作项目中，我们将定义代谢途径 当肝细胞从NADPH依赖的还原转变为非依赖的二硫化物还原时发生的重新排列。 我们提出了三个具体的目标：目标1，确定NADPH与Met燃料的二硫键还原酶如何保持动态平衡 影响S的新陈代谢优先顺序。目标2，定义丝氨酸代谢的重新连接如何支持蛋氨酸燃料 二硫化物还原酶动态平衡。目标3，测试蛋氨酸依赖生存是否增加了活性和 对肝脏甲基转移酶的依赖。 预期结果，价值：这个项目将帮助我们了解全球肝脏代谢的变化 在肝脏中发生严重的氧化或亲电应激，以及这如何帮助维持健康。

What is known: Disulfide reduction-fueled enzymes s!upport homeostasis and combat oxidative damage that contributes to neurodegeneration, inflammatory diseases, and cancer. NADPH provides the reducing power for most anabolic and cytoprotective reduction reactions, yet only two enzymes can use NADPH to reduce cytosolic disulfides: thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr) 1. Both TrxR1 and Gsr have active sites that are dominantly inhibited by electrophilic toxins and oxidants 2, 3. In Co-PI Schmidt’s lab, mice with TrxR1/Gsr-null livers uncovered unexpected robustness in the disulfide reductase systems, including an NADPH-independent pathway that uses catabolism of methionine (Met) to sustain redox homeostasis 4. Importantly, this pathway is also thought to sustain normal cells under oxidative or electrophilic stress 5. Met and Cys are the 2 sulfur (S)-amino acids found in proteins, but S-containing molecules synthesized from Met or Cys, including S-adenosyl-Met (SAM), glutathione (GSH), CoA, and others, are also important in redox, detox, energetics, biosynthesis, regulation, and other processes. Co-PI DeNicola has been studying the roles of altered S-amino acid metabolism in sustaining some cancers6. These studies are revealing how some cancers use altered S-amino acid redox metabolism, which could uncover targetable cancer-specific susceptibilities. Unresolved questions: It remains unknown how other metabolic activities, including those that directly utilize Met or Cys, as well as more peripheral systems that either (i) supply resources to these pathways; (ii) depend upon these pathways; or (iii) might, in some conditions, compete with these pathways for substrates, are realigned to help cells survive stress. We hypothesize that conversion to Met-dependence involves realignment of diverse metabolic pathways. This work is significant because a better understanding of these processes will uncover processes that can be therapeutically targeted to either specifically increase the robustness of critical cells under oxidative or toxic stress, or specifically increase the vulnerability of pathogenic cells in cancer or inflammatory diseases. New preliminary investigations in this resubmission demonstrate our ability to perform stable isotope flux labeling studies in whole mice and in mouse-derived hepatic organoids. What is proposed: In this revised multi-institution collaborative project, we will define the metabolic pathway realignments that occur when hepatocytes switch from NADPH-dependent to -independent disulfide reduction. We propose 3 Specific Aims: Aim 1, Define how NADPH- versus Met-fueled disulfide reductase homeostasis influences S-metabolism prioritization. Aim 2, Define how re-wiring of serine metabolism supports Met-fueled disulfide reductase homeostasis. Aim 3, Test whether Met-dependent survival increases the activity and dependence on liver methyltransferases. Anticipated outcomes, value: This project will help us understand how global shifts in hepatic metabolism occurs in response to severe oxidative or electrophilic stress in liver, and how this helps sustain health.