eNOS

Researchers are working to understand how protein quality control works in cells.  From plants to animals, all living things depend on proteins to help their cells function properly. In certain cases, like when under stress in response to heat or toxins, some proteins within the cell condense into liquid-like droplets called condensates. This process is hypothesized to occur via phase separation and provides a quick way for the cell to assemble certain components. Research in Syracuse University Professor Carlos Castañeda's lab has recently shown that protein quality control (PQC) components are important for many of these condensates. Castañeda, associate professor of biology and chemistry in the College of Arts and Sciences (A&S), is among a team of researchers working to understand how protein quality control works in cells. Similar to the way computers use coding as a set of instructions, the PQC receives its instructions from polyubiquitin chains. Ubiquitin (Ub) is a regulatory protein found in all eukaryotic cells (cells containing a defined nucleus) and polyubiquitin is an assembly containing at least a few ubiquitin molecules. Under normal operating conditions, condensates form and react to a stressor, and then dissolve after the stress is relieved. But when this system becomes disrupted, it can result in protein clumping or aggregation, which can cause cells, especially those in the nervous system, to die. These abnormal protein aggregates are markers for neurological diseases like amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease. By understanding the circumstances that lead to dysregulation of the PQC, scientists hope this research will one day lead to a cure for such neurodegenerative diseases. In a paper published last year in EMBO Reports, Castañeda and his collaborators established a framework for how polyubiquitin chains regulate the formation and disassembly of a condensate made of Ubiquilin-2 (UBQLN2) proteins, whose dysregulation is implicated in ALS. Polyubiquitin is important for UBQLN2 function and the two bind noncovalently, meaning that the interaction between them is weaker than a covalent chemical bond. Following up on that work, the researchers wanted to further explore the specific conditions that affect assembly of these condensates. "While working on the EMBO Reports project, we started to see that more extended polyubiquitin chains favored phase separation (condensate formation) with UBQLN2," says Castañeda. "So, we wondered if this is always true. We decided to make even more extended chains." Through the Research Experience for Undergraduates (REU) program, Castañeda welcomed students Suzanne Enos and Antara Chaudhuri into his lab in 2021. The NSF-sponsored REU program brings undergraduate students from across the country to Syracuse's campus to participate in research over the summer. In Castañeda's lab, the team engineered and designed different types of polyubiquitin chains with variable lengths and topologies. Enos and Chaudhuri then helped test how well these chains phase separated with UBQLN2. After further design-work by Sarasi Galagedera, a former postdoctoral researcher in Castañeda's lab; Thuy Dao, a lab manager in the Department of Chemistry; and Jeremy Schmit, a professor of physics at Kansas State University, the team found there was a "sweet spot" -- or specific spacing between Ub units -- where condensate formation was optimized. Fittingly coined as "goldilocks," the group's findings were recently published in Proceedings of the National Academy of Sciences (PNAS). "We found that there is an arrangements of ubiquitin units in polyubiquitin that is 'just right' for condensates to form," says Castañeda. "Ubiquitin units that are too far apart or too close together don't favor condensate formation as much. Jeremy Schmit used theoretical modeling and polyphasic linkage concepts to generalize these experimental observations." Furthermore, they uncovered that polyubiquitin in excess causes the condensates to disassemble. "In a cell, you can imagine that concentrations of polyubiquitin, as well as the spacing between ubiquitin units within different types of polyubiquitin, can up- or down-regulate condensate formation. You essentially have multiple ways to tune condensate formation with just adding this one polyubiquitin molecule," notes Castañeda. While their research merely scratches the surface of how polyubiquitin chains can regulate phase separation of condensates, Castañeda says it offers proof that these chains will be a main regulator of droplets. Future studies will involve adapting their rules to an in vitro system that models PQC to prove and test their theories in living cells. "This work provides a principle that can be applied to understanding how biomolecular condensates are generally controlled and will have large implications for anyone studying the regulation of their favorite biomolecular condensate," says Castañeda. The team's work was funded by the following grants: NIH R01 GM136946 awarded to Carlos Castañeda; NSF REU grant awarded to the Department of Chemistry; and NIH R01 GM141235 awarded to Jeremy Schmit.

一氧化氮合酶家族将精氨酸转化为L瓜氨酸和一氧化氮，由三个独立基因nos1、nos2和nos3编码的三种异构体组成。这些异构体也是根据它们最初被发现的组织命名的。 一氧化氮合酶家族将精氨酸转化为L瓜氨酸和一氧化氮，由三个独立基因nos1、nos2和nos3编码的三种异构体组成。这些异构体也是根据它们最初被发现的组织命名的。NOS1或神经元型NOS(NNOS)是从神经元中分离出来的，NOS2是从单核细胞中分离出来的，由于受到促炎细胞因子的诱导而被称为巨噬细胞NOS或诱导型NOS(INOS)。NOS3是从内皮细胞(ECs)中分离出来的，定义为内皮型一氧化氮合酶(ENOS)。 图片来源：https://pubmed.ncbi.nlm.nih.gov/37658519/ 近日，来自海因里希·海涅大学的研究者们在Br J Pharmacol杂志上发表了题为“Red blood cell eNOS: a major player in regulating cardiovascular health.”的文章，该研究揭示了红细胞内皮型一氧化氮合酶是调节心血管健康的主要因素。 传统上，红细胞(RBC)被视为体内气体和营养物质的简单载体。红细胞在心血管系统中的一个重要的非规范功能是调节一氧化氮(NO)代谢。结果表明，在低氧条件下，红细胞可清除NO，转运NO代谢产物，产生NO，从而诱导缺氧性血管扩张。红细胞还表达内皮型一氧化氮合酶(ENOS)。 然而，它的生理意义多年来一直存在争议。本文对体内RBC eNOS信号转导的实验研究作一综述。这些数据表明，RBC eNOS信号调节细胞内NO产生和NOheme水平，以及细胞外旁分泌NO代谢产物信号，这有助于调节外周血管阻力、血压和心脏保护。此外，本文还探讨了RBC eNOS潜在的细胞内分子机制及其在心血管健康和疾病中的意义。 越来越多的证据表明，eNOS上游和下游信号在成熟红细胞和红系细胞分化中的作用 图片来源：https://pubmed.ncbi.nlm.nih.gov/37658519/ 综上所述，需要进一步的研究来全面了解Arg1/eNOS/NO-GC通路在健康和疾病中复杂的细胞功能和调控机制。通过解决这些知识差距，研究者可以促进eNOS信号、NO生物利用度和血液和其他细胞/组织中的生理过程之间复杂相互作用的理解，最终可能有助于改进治疗和临床结果。（生物谷 Bioon.com） 参考文献 Anthea LoBue et al. Red blood cell eNOS: a major player in regulating cardiovascular health. Br J Pharmacol. 2023 Sep 1. doi: 10.1111/bph.16230.

冠心病是动脉粥样硬化导致冠状动脉狭窄和闭塞，引起心肌缺血、梗死的高死亡率心血管疾病。血管新生构建侧支循环对于心肌组织的血运重建十分重要。然而，冠心病状态下，多种危险因素持续损伤血管的功能，导致一系列促 冠心病是动脉粥样硬化导致冠状动脉狭窄和闭塞，引起心肌缺血、梗死的高死亡率心血管疾病。血管新生构建侧支循环对于心肌组织的血运重建十分重要。然而，冠心病状态下，多种危险因素持续损伤血管的功能，导致一系列促进血管生长的措施没有疗效，其中一个关键因素是高密度脂蛋白(HDL)的功能障碍。正常HDL (nHDL)具有诱导血管新生的功能，但在冠心病等状态下，HDL的组分发生改变，导致诱导血管新生的功能丧失，甚至损伤血管新生，这种HDL称为失功能HDL(dHDL)。目前，对于dHDL为什么丧失诱导血管新生功能的机制尚未完全清楚。 非编码RNA（Non-coding RNA）是指不编码蛋白质的RNA。其中包括微小RNA（miRNA），长链非编码RNA (lncRNA)等。这些RNA的共同特点是都能从基因组上转录而来，但是不翻译成蛋白，在RNA 水平上能行使各自的生物学功能了。其中lncRNA是一类大于200nt的非编码RNA，它的数量极其庞大，功能复杂多样，可调节血管细胞的增殖、衰老、凋亡等细胞活动，影响血管的生长和功能。我院区景松教授团队前期研究发现nHDL和dHDL诱导血管内皮细胞的lncRNA表达谱存在明显差异。然而，这些lncRNAs在nHDL和dHDL调节血管新生中的作用尚未清楚。2023年8月14日，中山大学附属第一医院区景松教授团队在Signal Transduction and Targeted Therapy 在线发表了题为“High-density lipoprotein regulates angiogenesis by long non-coding RNA HDRACA”的研究论文，首次发现nHDL通过抑制血管内皮细胞的lncRNA HDRACA表达来促进血管新生，而在冠心病状态下，dHDL对HDRACA的抑制明显减弱，导致dHDL丧失促进血管新生的功能。 团队首先通过对lncRNA测序结果分析筛选，发现HDRACA可调节血管内皮细胞的增殖和管状形成，并确定了HDRACA是定位于内皮细胞浆中的长度553nt的无蛋白编码能力的lncRNA。进一步实验证实，nHDL可降低血管内皮细胞中的HDRACA表达，而dHDL对HDRACA表达的降低作用明显减弱。通过一系列分子实验发现，nHDL携带的S1P可与血管内皮细胞的S1P1相互作用诱导WWP2泛素连接酶磷酸化激活，催化HDRACA的转录因子KLF5泛素化降解，抑制HDRACA的转录。而dHDL携带的S1P含量明显减少，导致dHDL对HDRACA表达的抑制作用明显减弱。进一步实验证实nHDL抑制血管内皮细胞的HDRACA表达可促进血管新生，而dHDL对HDRACA表达的抑制明显减弱，导致它无法有效地促进血管新生。团队还发现HDRACA可以结合重要的血管新生调节蛋白RAIN，抑制RAIN和Vigilin的相互作用，进一步促进Vigilin诱导PCNA的mRNA不稳定降解，从而抑制PCNA的表达。nHDL通过抑制HDRACA表达影响RAIN-Vigilin-PCNA相互作用，促进PCNA的表达而促进血管新生。而dHDL无法有效地抑制HDRACA的表达，导致PCNA的表达减少，无法促进血管新生。 该研究阐明了nHDL通过抑制lncRNA HDRACA表达促进血管新生，dHDL不能有效抑制lncRNA HDRACA表达而丧失诱导血管新生的作用和机制，为冠心病的血管新生治疗提供新的研究方向和靶点。 HDL通过调控lncRNA HDRACA影响血管新生 此外，近年研究发现，HDL不但包含多种蛋白质和脂质，而且还携带非编码RNA，区景松教授团队发现，dHDL携带miRNA-24-3p明显nHDL多，nHDL可以通过HDL的经典受体SRB1抑制血管内皮细胞miRNA-24-3p的表达，从而使vinculin表达增多，激活PI3K-AKT-eNOS/ERK信号通路促进血管新生，但dHDL可以通过SRB1将携带的miRNA-24-3p导入血管内皮细胞，从而使vinculin表达下降，抑制PI3K-AKT-eNOS/ERK信号通路损伤血管新生。研究发现以题为“Angiogenic and antiangiogenic mechanisms of high density lipoprotein from healthy subjects and coronary artery diseases patients发表在期刊Redox Biology上。 最近，区景松教授团队还发现HDL可以通过调控不同的miRNA影响血管新生。nHDL通过SRB1抑制另一个miR-181a-5p表达，使ATG5表达上调促进自噬，增强eNOS表达，增加内皮一氧化氮（NO）产生，诱导血管新生。 dHDL通过SRB1增强miR-181a-5p表达，抑制ATG5表达从而抑制自噬，下调eNOS表达，刺激氧自由基产生，损伤血管新生。通过过表达ATG5增加自噬可以恢复高胆固醇血症的血管新生。研究发现以文章题为：“High-density lipoprotein regulates angiogenesis by affecting autophagy via miRNA-181a-5p”于2023年6月20日在线发表在期刊Science China-Life Sciences上。 综上，区景松教授团队近年来的研究系统地阐明了高密度脂蛋白通过调控非编码RNA影响血管新生的新机制, 为缺血性心脏病的血管新生治疗提供新的理论基础和治疗靶点。 该研究得到国家重点研发计划项目、国家杰出青年科学基金、国家自然科学基金重大研究计划重点项目、国家自然科学基金重点项目、科技部国际重大合作项目等的联合资助。