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

什么是已知的：二硫化物还原燃料酶！支持体内平衡和对抗氧化损伤， 导致神经退化、炎性疾病和癌症。NADPH提供还原力， 大多数合成代谢和细胞保护还原反应，但只有两种酶可以使用NADPH来还原 胞质二硫化物：硫氧还蛋白还原酶-1（TrxR 1）和谷胱甘肽还原酶（Gsr）1。TrxR 1和Gsr都具有 活性位点，主要抑制亲电毒素和氧化剂2，3。在合作PI施密特的实验室里， TrxR 1/Gsr缺失的肝脏发现了二硫还原酶系统的意外稳健性，包括 NADPH非依赖性途径，使用蛋氨酸（Met）的催化剂来维持氧化还原稳态4。 重要的是，这种途径也被认为是在氧化或亲电应激下维持正常细胞5。相遇并 Cys是在蛋白质中发现的2种硫（S）-氨基酸，但是由Met或Cys合成的含S分子， 包括S-腺苷-Met（SAM）、谷胱甘肽（GSH）、CoA等，在氧化还原，排毒， 能量学、生物合成、调节和其他过程。Co-PI DeNicola一直在研究改变的 S-氨基酸代谢在维持某些癌症6.这些研究揭示了一些癌症是如何利用 改变的S-氨基酸氧化还原代谢，这可能揭示靶向癌症特异性的易感性。 未解决的问题：目前还不清楚其他代谢活动，包括那些直接利用 Met或Cys，以及更多的外周系统，其（i）为这些途径提供资源;（ii）依赖于 或（iii）在某些条件下可能与这些途径竞争底物， 帮助细胞在压力下存活我们假设，转换为MET依赖涉及重新调整 不同的代谢途径。这项工作意义重大，因为更好地了解这些过程将 揭示可以治疗靶向的过程，以特异性地增加关键的 在氧化或毒性应激下的细胞，或特别是增加癌症中致病细胞的脆弱性， 炎症性疾病。此次重新提交的新初步调查证明了我们的执行能力 在整个小鼠和小鼠来源的肝类器官中进行的稳定同位素通量标记研究。 建议内容：在这个修订后的多机构合作项目中，我们将定义代谢途径 当肝细胞从NADPH依赖性二硫键还原转换为非依赖性二硫键还原时发生的重组。 我们提出了3个具体目标：目标1，定义NADPH与Met燃料二硫键还原酶的稳态 影响S-代谢优先化。目的2，定义丝氨酸代谢的重新布线如何支持Met燃料 二硫还原酶稳态目的3，测试Met依赖性存活是否增加活性， 依赖肝甲基转移酶。 预期结果，价值：该项目将帮助我们了解肝脏代谢的全球变化 发生在对肝脏严重氧化或亲电应激的反应中，以及这如何有助于维持健康。

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.