FBP1

引言端粒功能障碍与衰老过程错综复杂地联系在一起，并作为一个突出的癌症标志而突出。2024年3月27日，浙江大学吕志民及许大千共同通讯在Nature Chemical Biology在线发表题为“Fructose-1,6-bisphosphatase 1 dephosphorylates and inhibits TERT for tumor suppression”的研究论文，该研究证明端粒酶活性在癌症细胞和正常细胞中受到不同的调节，这取决于果糖-1,6-双磷酸酶1 (FBP1)的表达状态。在表达FBP1的细胞中，FBP1直接与端粒酶逆转录酶(TERT)的Ser227位点相互作用并使其去磷酸化。去磷酸化的TERT无法转位到细胞核中，导致小鼠端粒酶活性受到抑制，端粒长度减少，衰老加剧，肿瘤细胞增殖和生长受到抑制。脂质纳米颗粒介导的FBP1 mRNA递送抑制肝脏肿瘤生长。此外，在肾癌和肝癌标本中，FBP1表达水平与TERT pSer227水平呈负相关，并与患者预后不良相关。这些发现表明，FBP1通过其蛋白磷酸酶活性来控制细胞的不朽，并揭示了肿瘤细胞中由于FBP1表达下调或缺乏而产生的独特的端粒酶调节。端粒由重复的TTAGGG核苷酸序列组成，形成一个“帽状结构”，其功能是维持染色体的完整性。端粒在每一轮DNA复制过程中逐渐缩短，端粒功能的丧失导致有丝分裂期间染色体端到端融合，导致双中心染色体的形成和随后染色体断裂的后期桥接。生成的两个子细胞接受不均匀的染色单体，含有两个缺乏端粒的染色单体，导致有丝分裂后的断裂-融合桥(BFB)周期，最终导致正常细胞衰老和凋亡。在癌细胞中，端粒伸长和长度主要依赖于高度激活的端粒酶，在大多数人类癌症中，端粒酶合成端粒重复序列，在5-15%的癌症中，端粒选择性延长(ALT)。端粒酶的活性受端粒酶逆转录酶(TERT)的调控，TERT是端粒酶的催化蛋白亚基，在转录水平和翻译后水平均受调控。TERT磷酸化显著增加端粒酶活性。用于组装具有催化活性的端粒酶，细胞质TERT必须转运到细胞核中，这一过程需要AKT介导的TERT Ser227磷酸化才能将输入蛋白α与TERT8的核定位信号结合。然而，TERT磷酸化和随后的端粒功能在正常细胞和癌细胞中是否受到不同的调节，这在很大程度上是未知的。果糖-1,6-二磷酸酶1 (FBPase 1)是糖异生过程中的限速酶，可将果糖-1,6-二磷酸(F1,6BP)水解为果糖- 6-磷酸(F6P)。在哺乳动物中，两个独立的基因，FBP1和FBP2，分别编码肝脏和肌肉中FBPase的亚型。FBP1主要表达于肝脏和肾脏，在几乎100%的透明细胞肾细胞癌(ccRCC)中普遍缺失，在肝细胞癌(HCC)和其他类型的癌症中经常下调。它的缺乏促进了肝脏肿瘤的进展，并与较差的总生存率和较高的肿瘤复发率相关。研究表明，FBP1与HCC和ccRCC细胞中的缺氧诱导因子(HIFs)或增强子zeste同源物2 (EZH2)以酶活性不依赖的方式相互作用并抑制HIFs。此外，FBP1作为蛋白磷酸酶去磷酸化组蛋白H3来调节基因表达。然而，FBP1的酶活性是否具有直接调节肿瘤细胞增殖的兼职功能尚不清楚。、模式图（Credit: Nature Chemical Biology）该研究证明了FBP1通过FBP1的疏水残基N273与TERT相互作用。FBP1作为蛋白磷酸酶，使TERT的S227位点(pS227)去磷酸化，从而抑制TERT核易位和端粒酶活性，减少端粒长度，抑制肿瘤细胞的增殖和衰老。FBP1蛋白磷酸酶死亡突变体的表达促进了小鼠肿瘤的生长，同时TERT S227磷酸化水平和端粒长度增加。该研究揭示了FBP1蛋白磷酸酶活性对肿瘤和正常细胞中TERT活性差异调节的一个未知的关键机制。低FBP1表达与高TERT S227磷酸化水平以及较短的ccRCC和HCC患者生存期呈负相关，结合lnp为基础的FBP1 mRNA递送有效抑制肿瘤生长，强调了调节蛋白磷酸酶活性和FBP1表达以治疗人类癌症的潜力。原文链接https://www-nature-com.libproxy1.nus.edu.sg/articles/s41589-024-01597-2责编|探索君排版|探索君文章来源|“iNature”End往期精选围观一文读透细胞死亡(Cell Death) | 24年Cell重磅综述（长文收藏版）热文Nature | 破除传统：为何我们需要重新思考肿瘤的命名方式热文Nature | 2024年值得关注的七项技术热文Nature | 自身免疫性疾病能被治愈吗？科学家们终于看到了希望热文CRISPR技术进化史 | 24年Cell综述

引言在现代生物医学研究领域，生物发光成像技术凭借其非侵入性、无需外加光源以及高灵敏度的独特优势，已经成为研究生物体内各种生理和病理过程的重要工具。特别是随着1月22日一项研究成果（An improved pathway for autonomous bioluminescence imaging in eukaryotes）在Nat Methods （IF 48）杂志上的发表，科学家们通过对霓虹灯菌(Neonothopanus nambi)中的生物发光途径进行改良，成功实现了在植物、真菌和哺乳动物宿主中自主发光亮度的显著提升，这一成就无疑为生物发光成像技术的进一步发展和广泛应用提供了强有力的支持。自然界的生物发光现象一直是科学家们研究的热门话题。霓虹灯菌的发现为在非自发光的真核生物中引入自主发光的可能性提供了新的思路。然而，将这一现象应用于异源宿主时，由于原生真菌酶的性能限制，其发光亮度往往难以满足实验要求，这成为推广其应用的一大障碍。在这项研究中，科学家们成功优化了真核生物中的自主生物发光途径，显著提高了在植物、真菌和哺乳动物宿主中的生物发光亮度。通过对Neonothopanus nambi真菌中的生物发光途径组分进行定向进化和改造，研究团队开发出了两种优化的生物发光途径版本：FBP2和FBP3，它们在多种宿主中实现了比原始途径（FBP1）高出一个到两个数量级的发光亮度。研究中涉及对两个关键酶nnLuz（荧光素酶）和nnH3H（hispidin-3-羟化酶）的改造。对nnLuz的定向进化产生了含有三个氨基酸替换（T99P, T192S和A199P）的nnLuz_v3变体，这些替换增加了其在细菌和哺乳动物细胞中的稳定性和亮度。进一步的随机突变产生了nnLuz_v4，它包含了额外的四个氨基酸替换（I3S, N4T, F11L和I63T），在酵母中显示出改善的热稳定性和催化活性。nnH3H的定向进化确定了五个增强亮度的氨基酸替换（D37E, V181I, A183P, S323M和M385K）。通过组合这些突变，得到了进一步增强亮度的nnH3H_v2。此外，研究还发现，来自Mycena citricolor的hispidin合成酶mcitHispS在多种异源宿主中的表现优于nnHispS，尤其是在植物中。通过将改造后的酶组合在一起，形成了FBP2（包含nnHispS, nnH3H_v2, nnLuz_v4, nnCPH和NpgA）和FBP3（包含mcitHispS, nnH3H_v2, nnLuz_v4, nnCPH和NpgA）两种优化途径。在实验中，FBP2和FBP3在植物、酵母和哺乳动物细胞培养系统中表现出了明显的亮度优势。特别是在植物中，FBP2和FBP3不仅提高了发光亮度，而且在六个代表四个双子叶植物科的物种中实现了稳定的转基因表达，这些物种包括模式植物拟南芥（Arabidopsis thaliana）和烟草（Nicotiana benthamiana），以及快速生长的树种加拿大杨（Populus canadensis）、观赏植物矮牵牛（Petunia hybrida）和菊花（Chrysanthemum morifolium）。研究结果表明，FBP2和FBP3具有在植物中实现视频速率生物发光成像的潜力，即使在微弱的外部光照条件下也能使用消费级相机可靠地监测植物的生物发光。最明亮的组织——矮牵牛的花蕾甚至可以使用现代智能手机相机进行记录。转基因植株稳定表达生物发光途径不同版本的发光性：3周龄尼古丁树叶的平均亮度（Credit: Nat Methods）Methods速览定向进化与共识突变分析：对N. nambi的荧光素酶nnLuz和hispidin-3-羟化酶nnH3H进行定向进化和共识突变分析，通过筛选获得具有增强稳定性和亮度的nnLuz_v3和nnLuz_v4变体，以及提高亮度的nnH3H_v2变体。基因表达与瞬时转染：将改良后的生物发光基因构建到表达载体中，并通过瞬时转染的方式将其引入植物（如烟草Nicotiana benthamiana和拟南芥Arabidopsis thaliana）、酵母（如Pichia pastoris）和哺乳动物细胞（如人类HEK293细胞）中，以评估改良途径的生物发光性能。酶活性与热稳定性测试：在细菌、酵母和哺乳动物细胞中测试改良酶的活性和热稳定性，通过添加底物并使用荧光计或相机捕捉发光信号，从而评估酶的性能。农杆菌介导的植物转化：利用农杆菌（Agrobacterium tumefaciens）介导的转化方法，将改良的生物发光基因稳定整合到植物基因组中，创建能够稳定发光的转基因植物系。发光成像与数据分析：使用IVIS Spectrum CT等成像系统或普通数码相机，在无外部光源的条件下捕获植物、细胞或酵母的自主生物发光图像。通过图像处理软件对捕获的图像进行分析，定量评估发光信号的强度和分布。这一创新性研究成果的实现，极大地拓宽了生物发光成像技术在植物学、神经科学、发育生物学等多个学科领域的应用范围，同时也为今后的研究提供了新的方向。其完全基因编码的特性，使实验操作变得更加简便，大大降低了研究成本，提高了实验效率。未来，随着对该技术的进一步优化和改进，预期将在更广泛的生物体系中实现更高效、更稳定的自主发光，为生物医学研究乃至临床诊断提供更加强大的技术支撑。参考文献https://www-nature-com.libproxy1.nus.edu.sg/articles/s41592-023-02152-y责编|探索君排版|探索君End往期精选围观Nat Med | 黄荷凤院士/张静澜/徐晨明团队发表综合性无创产前筛查临床研究成果热文Science最新发布：全世界最前沿的125个科学问题热文不孕症治疗新方向！又一导致女性不孕的关键因素被发现热文Let-7家族miRNA或为癌症潜在免疫治疗靶点热文一项最新发现或可用于阿尔茨海默病药物开发

Researchers describe a key player in the defense mechanism that safeguards against excessive insulin in the body, which can be as harmful as too little. Just over a century has passed since the discovery of insulin, a time period during which the therapeutic powers of the hormone have broadened and refined. Insulin is an essential treatment for type 1 diabetes and often for type 2 diabetes, as well. Roughly 8.4 million Americans use insulin, according to the American Diabetes Association. One hundred years of research have greatly advanced medical and biochemical understanding of how insulin works and what happens when it is lacking, but the reverse, how potentially fatal insulin hyper-responsiveness is prevented, has remained a persistent mystery. In a new study, published in the April 20, 2023 online edition of Cell Metabolism, a team of scientists at the University of California San Diego School of Medicine, with colleagues elsewhere, describe a key player in the defense mechanism that safeguards us against excessive insulin in the body. "Although insulin is one of the most essential hormones, whose insufficiency can result in death, too much insulin can also be deadly," said senior study author Michael Karin, PhD, Distinguished Professor of Pharmacology and Pathology at UC San Diego School of Medicine. "While our body finely tunes insulin production, patients who are treated with insulin or drugs that stimulate insulin secretion often experience hypoglycemia, a condition that if gone unrecognized and untreated can result in seizures, coma and even death, which collectively define a condition called insulin shock." Hypoglycemia (low blood sugar) is a significant cause of death among persons with diabetes. In the new study, Karin, first author Li Gu, PhD, a postdoctoral scholar in Karin's lab, and colleagues describe "the body's natural defense or safety valve" that reduces the risk of insulin shock. That valve is a metabolic enzyme called fructose-1,6-bisphosphate phosphatase or FBP1, which acts to control gluconeogenesis, a process in which the liver synthesizes glucose (the primary source of energy used by cells and tissues) during sleep and secretes it to maintain steady supply of glucose in the bloodstream. Some antidiabetic drugs, such as metformin, inhibit gluconeogenesis but without apparent ill effect. Children born with a rare, genetic disorder in which they do not produce sufficient FBP1 can also remain healthy and live long lives. But in other cases, when the body is starved for glucose or carbohydrates, an FBP1 deficiency can result in severe hypoglycemia. Without a glucose infusion, convulsions, coma and possibly death can ensue. Compounding and confounding the problem, FPB1 deficiency combined with glucose starvation produces adverse effects unrelated to gluconeogenesis, such as an enlarged, fatty liver, mild liver damage and elevated blood lipids or fats. To better understand the roles of FBP1, researchers created a mouse model with liver specific FBP1 deficiency, accurately mimicking the human condition. Like FBP1-deficient children, the mice appeared normal and healthy until fasted, which quickly resulted in the severe hypoglycemia and the liver abnormalities and hyperlipidemia described above. Gu and her colleagues discovered that FBP1 had multiple roles. Beyond playing a part in the conversion of fructose to glucose, FBP1 had a second non-enzymatic but critical function: It inhibited the protein kinase AKT, which is the primary conduit of insulin activity. "Basically, FBP1 keeps AKT in check and guards against insulin hyper-responsiveness, hypoglycemic shock and acute fatty liver disease," said first author Gu. Working with Yahui Zhu, a vising scientist from Chongqing University in China and second author of the study, Gu developed a peptide (a string of amino acids) derived from FBP1 that disrupted the association of FBP1 with AKT and another protein that inactivates AKT. "This peptide works like an insulin mimetic, activating AKT," said Karin. "When injected into mice that have been rendered insulin resistant, a highly common pre-diabetic condition, due to prolonged consumption of high-fat diet, the peptide (nicknamed E7) can reverse insulin resistance and restore normal glycemic control." Karin said the researchers would like to further develop E7 as a clinically useful alternative to insulin "because we have every reason to believe that it is unlikely to cause insulin shock." Co-authors include: Kosuke Watari, Maiya Lee, Junlai Liu, Sofia Perez, Melinda Thai, Joshua E. Mayfield, Bichen Zhang, Karina Cunha e Rocha, Alexander C. Jones, Igor H. Wierzbicki, Xiao Liu, Alexandra C. Newton, Tatiana Kisseleva, Wei Ying, David J. Gonzalez and Alan R. Saltiel, all at UC San Diego; Fuming Li, University of Pennsylvania and Fudan University, China; Laura C. Kim and M. Celeste Simon, University of Pennsylvania; Jun Hee Lee, University of Michigan. Funding for this research came, in part, from the National Institutes of Health (grants R01DK120714, R01CA234128, R01DK133448, P01CA104838, R35CA197602, R01DK117551, R01DK125820, R01DK76906, P30DK063491, R21HD107516, R00DK115998, R01DK125560 AND R35GM122523), the UC San Diego Graduate Training Program in Cellular and Molecular Pharmacology (GM007752) and the National Science Foundation Graduate Research Fellowship (#DGE-1650112).