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Autophagy Regulator Rufy 4 Promotes Osteoclastic Bone Resorption by Orchestrating Cytoskeletal Organization via Its RUN Domain. 自噬调节器 Rufy 4 通过其 RUN 域协调细胞骨架组织,促进破骨细胞的骨吸收
IF 8.3 2区 生物学 Q2 CELL BIOLOGY Pub Date : 2024-10-25 DOI: 10.3390/cells13211766
Eiko Sakai, Minoru Saito, Yu Koyanagi, Yoshitsugu Takayama, Fatima Farhana, Yu Yamaguchi, Takayuki Tsukuba

Rufy4, a protein belonging to the RUN and FYVE domain-containing protein family, participates in various cellular processes such as autophagy and intracellular trafficking. However, its role in osteoclast-mediated bone resorption remains uncertain. In this study, we investigated the expression and role of the Rufy4 gene in osteoclasts using small interfering RNA (siRNA) transfection and gene overexpression systems. Our findings revealed a significant increase in Rufy4 expression during osteoclast differentiation. Silencing Rufy4 enhanced osteoclast differentiation, intracellular cathepsin K levels, and formation of axial protrusive structures but suppressed bone resorption. Conversely, overexpressing wild-type Rufy4 in osteoclasts hindered differentiation while promoting podosome formation and bone resorption. Similarly, overexpression of a Rufy4 variant lacking the RUN domain mimics the effects of Rufy4 knockdown, significantly increasing intracellular cathepsin K levels, promoting osteoclastogenesis, and elongated axial protrusions formation, yet inhibiting bone resorption. These findings indicate that Rufy4 plays a critical role in osteoclast differentiation and bone resorption by regulating the cytoskeletal organization through its RUN domain. Our study provides new insights into the molecular mechanisms governing osteoclast activity and underscores Rufy4's potential as a novel therapeutic target for bone disorders characterized by excessive bone resorption.

Rufy4是一种属于含RUN和FYVE结构域蛋白家族的蛋白质,参与自噬和细胞内运输等多种细胞过程。然而,它在破骨细胞介导的骨吸收中的作用仍不确定。在这项研究中,我们利用小干扰 RNA(siRNA)转染和基因过表达系统研究了 Rufy4 基因在破骨细胞中的表达和作用。我们的研究结果表明,在破骨细胞分化过程中,Rufy4的表达量明显增加。沉默Rufy4可促进破骨细胞分化、细胞内猫嗜蛋白酶K水平和轴突结构的形成,但抑制骨吸收。相反,在破骨细胞中过表达野生型 Rufy4 会阻碍分化,同时促进荚膜的形成和骨吸收。同样,过表达缺乏RUN结构域的Rufy4变体也能模拟Rufy4敲除的效应,显著增加细胞内猫嗜蛋白酶K的水平,促进破骨细胞生成和拉长的轴突形成,但却抑制骨吸收。这些发现表明,Rufy4通过其RUN结构域调控细胞骨架组织,在破骨细胞分化和骨吸收过程中发挥着关键作用。我们的研究为破骨细胞活动的分子机制提供了新的见解,并强调了 Rufy4 作为以骨过度吸收为特征的骨疾病的新型治疗靶点的潜力。
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引用次数: 0
17β-Estradiol Stimulates Oxidative Stress Components and Thyroid Specific Genes in Porcine Thyroid Follicular Cells: Potential Differences Between Sexes. 17β-雌二醇刺激猪甲状腺滤泡细胞中的氧化应激成分和甲状腺特异基因:性别间的潜在差异。
IF 8.3 2区 生物学 Q2 CELL BIOLOGY Pub Date : 2024-10-25 DOI: 10.3390/cells13211769
Jan Stępniak, Małgorzata Karbownik-Lewińska

17β-estradiol plays a crucial role in regulating cellular processes in both reproductive and non-reproductive tissues, including the thyroid gland. It modulates oxidative stress and contributes to sexual dimorphism in thyroid diseases, with ROS production, particularly H2O2, generated by NOX/DUOX enzymes. This study aimed to investigate the effects of 17β-estradiol (10 nM or 100 nM) on the expression of NOX/DUOX, thyroid-specific genes, and endoplasmic reticulum (ER) stress-related genes in male and female porcine thyroid follicular cells. Expression of the studied genes was evaluated by RT-PCR before and after treatment with 17β-estradiol alone or with the addition of NOX4 inhibitor (GKT-136901). Additionally, the level of ROS was measured by flow cytometry analysis. Our results show that 17β-estradiol significantly upregulates thyroid-specific genes, particularly TPO, and stimulates NOX/DUOX expression, affecting the redox state of thyroid cells. It also stimulates ER stress-related genes such as CHOP. In conclusion, estrogen excess may contribute to thyroid disease development via such possible mechanisms as the upregulation of key thyroid-specific genes, particularly TPO, and of genes involved in the cellular response to ER stress, especially CHOP, as well as by the stimulation of the NOX/DUOX system with consequent ROS overproduction. These mechanisms may play a certain role in the higher prevalence of thyroid diseases in women.

17β-雌二醇在调节包括甲状腺在内的生殖和非生殖组织的细胞过程中发挥着至关重要的作用。它能调节氧化应激,并导致甲状腺疾病中的性双态性,由NOX/DUOX酶产生ROS,尤其是H2O2。本研究旨在探讨17β-雌二醇(10 nM或100 nM)对雌雄猪甲状腺滤泡细胞中NOX/DUOX、甲状腺特异性基因和内质网(ER)应激相关基因表达的影响。在单独使用 17β-estradiol 或添加 NOX4 抑制剂(GKT-136901)处理前后,通过 RT-PCR 对所研究基因的表达进行了评估。此外,还通过流式细胞术分析测量了 ROS 的水平。我们的研究结果表明,17β-雌二醇能显著上调甲状腺特异性基因,尤其是TPO,并刺激NOX/DUOX的表达,从而影响甲状腺细胞的氧化还原状态。它还会刺激 ER 应激相关基因,如 CHOP。总之,雌激素过量可能通过以下机制导致甲状腺疾病的发生:上调关键的甲状腺特异性基因,尤其是TPO;上调参与细胞对ER应激反应的基因,尤其是CHOP;刺激NOX/DUOX系统,从而导致ROS过量产生。这些机制可能是女性甲状腺疾病发病率较高的原因之一。
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引用次数: 0
Acute Chikungunya Infection Induces Vascular Dysfunction by Directly Disrupting Redox Signaling in Endothelial Cells. 急性基孔肯雅病毒感染通过直接破坏内皮细胞的氧化还原信号诱发血管功能障碍
IF 8.3 2区 生物学 Q2 CELL BIOLOGY Pub Date : 2024-10-25 DOI: 10.3390/cells13211770
José Teles de Oliveira-Neto, Juliano de P Souza, Daniel Rodrigues, Mirele R Machado, Juliano V Alves, Paula R Barros, Alecsander F Bressan, Josiane F Silva, Tiago J Costa, Rafael M Costa, Daniella Bonaventura, Eurico de Arruda-Neto, Rita C Tostes, Emiliana P Abrão

Chikungunya virus (CHIKV) infection is characterized by febrile illness, severe joint pain, myalgia, and cardiovascular complications. Given that CHIKV stimulates reactive oxygen species (ROS) and pro- and anti-inflammatory cytokines, events that disrupt vascular homeostasis, we hypothesized that CHIKV induces arterial dysfunction by directly impacting redox-related mechanisms in vascular cells. Wild-type (WT) and iNOS knockout (iNOS-/-) mice were administered either CHIKV (1.0 × 106 PFU/µL) or Mock vehicle via the intracaudal route. In vivo, CHIKV infection induced vascular dysfunction (assessed by a wire myograph), decreased systolic blood pressure (tail-cuff plethysmography), increased IL-6 and IFN-γ, but not TNF-α levels (determined by ELISA), and increased protein content by Western blot. Marked contractile hyporesponsiveness to phenylephrine was observed 48 h post-infection, which was restored by endothelium removal. L-NAME, 1400W, Tiron, and iNOS gene deletion prevented phenylephrine hyporesponsiveness. CHIKV infection increased vascular nitrite concentration (Griess reaction) and superoxide anion (O2•-) generation (lucigenin chemiluminescence), and decreased hydrogen peroxide (H2O2, by Amplex Red) levels 48 h post-infection, alongside increased TBARS levels. In vitro, CHIKV infected endothelial cells (EA.hy926) and upregulated ICAM-1 and iNOS protein expression (determined by Western blot). These data support the conclusion that CHIKV-induced alterations in vascular ROS/NF-kB/iNOS/NO signaling potentially contribute to cardiovascular events associated with Chikungunya infection.

基孔肯雅病毒(CHIKV)感染的特点是发热、严重关节痛、肌痛和心血管并发症。鉴于基孔肯雅病毒会刺激活性氧(ROS)以及促炎和抗炎细胞因子,从而破坏血管稳态,我们假设基孔肯雅病毒会直接影响血管细胞中的氧化还原相关机制,从而诱发动脉功能障碍。通过心内途径给野生型(WT)和 iNOS 基因敲除(iNOS-/-)小鼠注射 CHIKV(1.0 × 106 PFU/µL)或 Mock 车辆。在体内,CHIKV 感染会诱发血管功能障碍(通过线性肌电图评估)、收缩压下降(尾袖式压力计)、IL-6 和 IFN-γ 水平升高,但 TNF-α 水平不升高(通过 ELISA 检测),并通过 Western 印迹检测蛋白质含量升高。感染后 48 小时,观察到对苯肾上腺素的收缩反应明显减弱,去除内皮后即可恢复。L-NAME、1400W、Tiron 和 iNOS 基因缺失可防止苯肾上腺素的低反应性。CHIKV 感染后 48 小时,血管亚硝酸盐浓度(Griess 反应)和超氧阴离子(O2--)生成(荧光素化学发光)增加,过氧化氢(H2O2,通过 Amplex Red)水平降低,同时 TBARS 水平增加。在体外,CHIKV 感染内皮细胞(EA.hy926)并上调 ICAM-1 和 iNOS 蛋白表达(通过 Western 印迹测定)。这些数据支持这样的结论,即 CHIKV 诱导的血管 ROS/NF-kB/iNOS/NO 信号改变可能会导致与基孔肯雅病毒感染相关的心血管事件。
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引用次数: 0
Aptamer-Hytac Chimeras for Targeted Degradation of SARS-CoV-2 Spike-1. 用于靶向降解 SARS-CoV-2 Spike-1 的 Aptamer-Hytac Chimeras。
IF 8.3 2区 生物学 Q2 CELL BIOLOGY Pub Date : 2024-10-25 DOI: 10.3390/cells13211767
Carme Fàbrega, Núria Gallisà-Suñé, Alice Zuin, Juan Sebastián Ruíz, Bernat Coll-Martínez, Gemma Fabriàs, Ramon Eritja, Bernat Crosas

The development of novel tools to tackle viral processes has become a central focus in global health, during the COVID-19 pandemic. The spike protein is currently one of the main SARS-CoV-2 targets, owing to its key roles in infectivity and virion formation. In this context, exploring innovative strategies to block the activity of essential factors of SARS-CoV-2, such as spike proteins, will strengthen the capacity to respond to current and future threats. In the present work, we developed and tested novel bispecific molecules that encompass: (i) oligonucleotide aptamers S901 and S702, which bind to the spike protein through its S1 domain, and (ii) hydrophobic tags, such as adamantane and tert-butyl-carbamate-based ligands. Hydrophobic tags have the capacity to trigger the degradation of targets recruited in the context of a proteolytic chimera by activating quality control pathways. We observed that S901-adamantyl conjugates promote the degradation of the S1 spike domain, stably expressed in human cells by genomic insertion. These results highlight the suitability of aptamers as target-recognition molecules and the robustness of protein quality control pathways triggered by hydrophobic signals, and place aptamer-Hytacs as promising tools for counteracting coronavirus progression in human cells.

在 COVID-19 大流行期间,开发解决病毒过程的新型工具已成为全球健康领域的核心重点。尖峰蛋白是目前 SARS-CoV-2 的主要靶标之一,因为它在感染性和病毒形成中起着关键作用。在这种情况下,探索阻断尖峰蛋白等 SARS-CoV-2 重要因子活性的创新策略,将增强应对当前和未来威胁的能力。在本研究中,我们开发并测试了新型双特异性分子,其中包括:(i) 寡核苷酸适配体 S901 和 S702,它们通过尖峰蛋白的 S1 结构域与尖峰蛋白结合;(ii) 疏水性标签,如金刚烷和氨基甲酸叔丁酯配体。疏水性标签能够通过激活质量控制途径,触发在蛋白水解嵌合体背景下招募的目标降解。我们观察到,S901-金刚烷基共轭物促进了通过基因组插入在人体细胞中稳定表达的 S1 穗状结构域的降解。这些结果凸显了适配体作为目标识别分子的适用性和疏水信号触发的蛋白质质量控制途径的稳健性,并将适配体-Hytacs作为对抗冠状病毒在人体细胞中发展的有前途的工具。
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引用次数: 0
Renal Inflammation, Oxidative Stress, and Metabolic Abnormalities During the Initial Stages of Hypertension in Spontaneously Hypertensive Rats. 自发性高血压大鼠高血压初期的肾脏炎症、氧化应激和代谢异常
IF 8.3 2区 生物学 Q2 CELL BIOLOGY Pub Date : 2024-10-25 DOI: 10.3390/cells13211771
Paweł Wojtacha, Ewelina Bogdańska-Chomczyk, Mariusz Krzysztof Majewski, Kazimierz Obremski, Michał Stanisław Majewski, Anna Kozłowska

Background: Hypertension is a major cause of mortality worldwide. The kidneys play a crucial role in regulating blood pressure and fluid volume. The relationship between the kidneys and hypertension is complex, involving factors such as the renin-angiotensin system, oxidative stress, and inflammation. This study aims to assess the levels of inflammatory markers, oxidative stress, and metabolic factors in the kidneys, focusing on their potential role in early renal damage and their association with the development of hypertension. Methods: This study was designed to compare the levels of selected inflammatory markers, e.g., interleukins, tumor necrosis factor-α (TNF-α), transforming growth factor, and serine/threonine-protein (mTOR); oxidative stress markers such as malondialdehyde, sulfhydryl group, and glucose (GLC); and metabolic markers among other enzymes, such as alanine transaminase (ALT), aspartate transaminase (AST), hexokinase II (HK-II), and hypoxia-inducible factor-1α (HIF-1α), as well as creatinine in the kidneys of spontaneously hypertensive rats (SHR/NCrl, n = 12) and Wistar Kyoto rats (WKY/NCrl, n = 12). Both juvenile (5 weeks old) and maturing (10 weeks old) specimens were examined using spectrophotometric methods, e.g., ELISA. Results: Juvenile SHRs exhibited reduced renal levels of all studied cytokines and chemokines, with lower oxidative stress and deficits in the mTOR and HK-II levels compared to the age-matched WKYs. Maturing SHRs showed increased renal levels of interleukin-1β (IL-1β), IL-6, IL-18, and TNF-α, alongside elevated carbonyl stress and increased HIF-1α as opposed to their control peers. The levels of all other studied markers were normalized in these animals, except for ALT (increased), ALP, and GLC (both reduced). Conclusions: This study underscores the significant impact of inflammatory, oxidative stress, and metabolic marker changes on renal function. Juvenile SHRs display lower marker levels, indicating an immature immune response and potential subclinical kidney damage that may contribute to hypertension development. In contrast, mature SHRs exhibit chronic inflammation, oxidative dysregulation, and metabolic disturbances, suggesting cellular damage. These changes create a feedback loop that worsens kidney function and accelerates hypertension progression, highlighting the kidneys' crucial role in both initiating and exacerbating this condition.

背景:高血压是全球死亡的主要原因。肾脏在调节血压和液体容量方面起着至关重要的作用。肾脏与高血压之间的关系非常复杂,涉及肾素-血管紧张素系统、氧化应激和炎症等因素。本研究旨在评估肾脏中炎症标志物、氧化应激和代谢因素的水平,重点研究它们在早期肾损伤中的潜在作用及其与高血压发病的关系。研究方法本研究旨在比较某些炎症标志物的水平,如白细胞介素、肿瘤坏死因子-α(TNF-α)、转化生长因子和丝氨酸/苏氨酸蛋白(mTOR);氧化应激标记物,如丙二醛、巯基和葡萄糖(GLC);自发性高血压大鼠(SHR/NCrl,n = 12)和 Wistar Kyoto 大鼠(WKY/NCrl,n = 12)肾脏中的代谢标记物和其他酶,如丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)、己糖激酶 II(HK-II)和低氧诱导因子-1α(HIF-1α)以及肌酐。采用分光光度法(如 ELISA)对幼年大鼠(5 周大)和成熟大鼠(10 周大)的标本进行了检测。结果:与年龄匹配的 WKYs 相比,幼年 SHR 的肾脏中所有研究细胞因子和趋化因子的水平都有所降低,氧化应激和 mTOR 及 HK-II 的水平也有所下降。与对照组相比,成熟的 SHR 肾脏白细胞介素-1β(IL-1β)、IL-6、IL-18 和 TNF-α 水平升高,同时羰基应激升高,HIF-1α 水平升高。除了谷丙转氨酶(升高)、谷草转氨酶(ALP)和谷草转氨酶(GLC)(均降低)外,这些动物体内所有其他研究指标的水平均恢复正常。结论:本研究强调了炎症、氧化应激和代谢标记物变化对肾功能的重大影响。幼年 SHR 的标记物水平较低,表明其免疫反应尚未成熟,可能存在亚临床肾损伤,这可能会导致高血压的发生。与此相反,成熟的 SHR 表现出慢性炎症、氧化失调和代谢紊乱,表明存在细胞损伤。这些变化形成了一个反馈回路,使肾功能恶化,加速了高血压的发展,凸显了肾脏在引发和加剧高血压中的关键作用。
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引用次数: 0
Structure-Function Correlation in Cobalt-Induced Brain Toxicity. 钴诱发脑中毒的结构-功能相关性
IF 8.3 2区 生物学 Q2 CELL BIOLOGY Pub Date : 2024-10-24 DOI: 10.3390/cells13211765
Basel Obied, Stephen Richard, Alon Zahavi, Dror Fixler, Olga Girshevitz, Nitza Goldenberg-Cohen

Cobalt toxicity is difficult to detect and therefore often underdiagnosed. The aim of this study was to explore the pathophysiology of cobalt-induced oxidative stress in the brain and its impact on structure and function. Thirty-five wild-type C57B16 mice received intraperitoneal cobalt chloride injections: a single high dose with evaluations at 24, 48, and 72 h (n = 5, each) or daily low doses for 28 (n = 5) or 56 days (n = 15). A part of the 56-day group also received minocycline (n = 5), while 10 mice served as controls. Behavioral changes were evaluated, and cobalt levels in tissues were measured with particle-induced X-ray emission. Brain sections underwent magnetic resonance imaging (MRI), electron microscopy, and histological, immunohistochemical, and molecular analyses. High-dose cobalt caused transient illness, whereas chronic daily low-dose administration led to long-term elevations in cobalt levels accompanied by brain inflammation. Significant neurodegeneration was evidenced by demyelination, increased blood-brain barrier permeability, and mitochondrial dysfunction. Treated mice exhibited extended latency periods in the Morris water maze test and heightened anxiety in the open field test. Minocycline partially mitigated brain injury. The observed signs of neurodegeneration were dose- and time-dependent. The neurotoxicity after acute exposure was reversible, but the neurological and functional changes following chronic cobalt administration were not.

钴中毒很难检测,因此往往诊断不足。本研究旨在探讨钴诱导的大脑氧化应激的病理生理学及其对大脑结构和功能的影响。35 只野生型 C57B16 小鼠接受了氯化钴腹腔注射:单次高剂量,并在 24、48 和 72 小时进行评估(每组 5 只);或每日低剂量,持续 28 天(每组 5 只)或 56 天(每组 15 只)。56 天组的部分小鼠也接受了米诺环素(n = 5),另有 10 只小鼠作为对照组。对小鼠的行为变化进行评估,并用粒子诱导 X 射线发射法测量组织中的钴含量。对脑部切片进行了磁共振成像(MRI)、电子显微镜以及组织学、免疫组化和分子分析。高剂量钴会导致一过性疾病,而每天长期低剂量给药会导致钴含量长期升高,并伴有脑部炎症。严重的神经变性表现为脱髓鞘、血脑屏障通透性增加和线粒体功能障碍。经治疗的小鼠在莫里斯水迷宫测试中表现出更长的潜伏期,并在开阔地测试中表现出更强的焦虑。米诺环素部分缓解了脑损伤。观察到的神经变性迹象与剂量和时间有关。急性暴露后的神经毒性是可逆的,但长期服用钴后的神经和功能变化则不可逆。
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引用次数: 0
Comparative Analysis of Plasma Protein Dynamics in Women with ST-Elevation Myocardial Infarction and Takotsubo Syndrome. ST段抬高型心肌梗死和塔克次氏综合征女性患者血浆蛋白动态对比分析
IF 8.3 2区 生物学 Q2 CELL BIOLOGY Pub Date : 2024-10-24 DOI: 10.3390/cells13211764
Shafaat Hussain, Sandeep Jha, Evelin Berger, Linnea Molander, Valentyna Sevastianova, Zahra Sheybani, Aaron Shekka Espinosa, Ahmed Elmahdy, Amin Al-Awar, Yalda Kakaei, Mana Kalani, Ermir Zulfaj, Amirali Nejat, Abhishek Jha, Tetiana Pylova, Maryna Krasnikova, Erik Axel Andersson, Elmir Omerovic, Björn Redfors

Background: ST-elevation myocardial infarction (STEMI) and Takotsubo syndrome (TS) are two distinct cardiac conditions that both result in sudden loss of cardiac dysfunction and that are difficult to distinguish clinically. This study compared plasma protein changes in 24 women with STEMI and 12 women with TS in the acute phase (days 0-3 post symptom onset) and the stabilization phase (days 7, 14, and 30) to examine the molecular differences between these conditions.

Methods: Plasma proteins from STEMI and TS patients were extracted during the acute and stabilization phases and analyzed via quantitative proteomics. Differential expression and functional significance were assessed. Data are accessible on ProteomeXchange, ID PXD051367.

Results: During the acute phase, STEMI patients showed higher levels of myocardial inflammation and tissue damage proteins compared to TS patients, along with reduced tissue repair and anti-inflammatory proteins. In the stabilization phase, STEMI patients exhibited ongoing inflammation and disrupted lipid metabolism. Notably, ADIPOQ was consistently downregulated in STEMI patients in both phases. When comparing the acute to the stabilization phase, STEMI patients showed increased inflammatory proteins and decreased structural proteins. Conversely, TS patients showed increased proteins involved in inflammation and the regulatory response to counter excessive inflammation. Consistent protein changes between the acute and stabilization phases in both conditions, such as SAA2, CRP, SAA1, LBP, FGL1, AGT, MAN1A1, APOA4, COMP, and PCOLCE, suggest shared underlying pathophysiological mechanisms.

Conclusions: This study presents protein changes in women with STEMI or TS and identifies ADIPOQ, SAA2, CRP, SAA1, LBP, FGL1, AGT, MAN1A1, APOA4, COMP, and PCOLCE as candidates for further exploration in both therapeutic and diagnostic contexts.

背景:ST段抬高型心肌梗死(STEMI)和Takotsubo综合征(TS)是两种不同的心脏疾病,它们都会导致心脏功能突然丧失,在临床上很难区分。本研究比较了 24 名 STEMI 女性患者和 12 名 TS 女性患者在急性期(症状出现后第 0-3 天)和稳定期(第 7、14 和 30 天)的血浆蛋白变化,以研究这两种疾病的分子差异:方法:提取 STEMI 和 TS 患者在急性期和稳定期的血浆蛋白质,并通过定量蛋白质组学进行分析。评估差异表达和功能意义。数据可在 ProteomeXchange 上查阅,编号为 PXD051367:结果:在急性期,STEMI 患者的心肌炎症和组织损伤蛋白水平高于 TS 患者,同时组织修复和抗炎蛋白水平降低。在稳定期,STEMI 患者表现出持续的炎症和紊乱的脂质代谢。值得注意的是,STEMI 患者在这两个阶段的 ADIPOQ 均持续下调。急性期与稳定期相比,STEMI 患者的炎症蛋白增加,结构蛋白减少。相反,TS 患者体内参与炎症和对抗过度炎症的调节反应的蛋白质增加。两种情况下急性期和稳定期的蛋白质变化一致,如SAA2、CRP、SAA1、LBP、FGL1、AGT、MAN1A1、APOA4、COMP和PCOLCE,这表明潜在的病理生理机制是相同的:本研究揭示了 STEMI 或 TS 女性患者的蛋白质变化,并确定了 ADIPOQ、SAA2、CRP、SAA1、LBP、FGL1、AGT、MAN1A1、APOA4、COMP 和 PCOLCE 作为在治疗和诊断方面进一步探索的候选蛋白。
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引用次数: 0
Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model. 在小鼠模型中敲除 Prominin-1 会导致 RPE 退化。
IF 8.3 2区 生物学 Q2 CELL BIOLOGY Pub Date : 2024-10-24 DOI: 10.3390/cells13211761
Sujoy Bhattacharya, Tzushan Sharon Yang, Bretton P Nabit, Evan S Krystofiak, Tonia S Rex, Edward Chaum

There are currently no effective treatments for retinal pigment epithelial (RPE) cell loss in atrophic AMD (aAMD). However, our research on Prominin-1 (Prom1), a known structural protein in photoreceptors (PRs), has revealed its distinct role in RPE and offers promising insights. While pathogenic Prom1 mutations have been linked to macular diseases with RPE atrophy, the broader physiological impact of dysfunctional Prom1 in RPE loss is unclear. We have shown that Prom1 plays a crucial role in regulating autophagy and cellular homeostasis in human and mouse RPE (mRPE) cells in vitro. Nevertheless, a comprehensive understanding of its in vivo expression and function in mRPE remains to be elucidated. To characterize Prom1 expression in RPE in situ, we used RNAscope assays and immunogold electron microscopy (EM). Our use of chromogenic and fluorescent RNAscope assays in albino and C57BL/6J mouse retinal sections has revealed Prom1 mRNA expression in perinuclear regions in mRPE in situ. Immunogold EM imaging showed Prom1 expression in RPE cytoplasm and mitochondria. To confirm Prom1 expression in RPE, we interrogated human RPE single-cell RNA-sequencing datasets using an online resource, Spectacle. Our analysis showed Prom1 expression in human RPE. To investigate Prom1's function in RPE homeostasis, we performed RPE-specific Prom1 knockdown (KD) using subretinal injections of AAV2/1.CMV.saCas9.U6.Prom1gRNA in male and female mice. Our data show that RPE-specific Prom1-KD in vivo resulted in abnormal RPE morphology, subretinal fluid accumulation, and secondary PR loss. These changes were associated with patchy RPE cell death and reduced a-wave amplitude, indicating retinal degeneration. Our findings underscore the central role of Prom1 in cell-autonomous mRPE homeostasis. The implications of Prom1-KD causing aAMD-like RPE defects and retinal degeneration in a mouse model are significant and could lead to novel treatments for aAMD.

对于萎缩性黄斑变性症(AAMD)中视网膜色素上皮(RPE)细胞的缺失,目前还没有有效的治疗方法。然而,我们对光感受器(PRs)中的一种已知结构蛋白 Prominin-1 (Prom1) 的研究揭示了它在 RPE 中的独特作用,并提供了很有前景的见解。虽然致病性 Prom1 基因突变与 RPE 萎缩的黄斑疾病有关,但 Prom1 功能障碍对 RPE 损失的广泛生理影响尚不清楚。我们已经证明,Prom1 在体外调节人和小鼠 RPE(mRPE)细胞的自噬和细胞稳态方面起着至关重要的作用。然而,对其在 mRPE 中的体内表达和功能的全面了解仍有待阐明。为了鉴定 Prom1 在 RPE 中的原位表达,我们使用了 RNAscope 检测法和免疫金电子显微镜(EM)。我们在白化小鼠和 C57BL/6J 小鼠视网膜切片中使用了色原和荧光 RNAscope 检测法,发现 Prom1 mRNA 在 mRPE 核周区域原位表达。免疫金电磁成像显示 Prom1 在 RPE 细胞质和线粒体中表达。为了证实 Prom1 在 RPE 中的表达,我们利用在线资源 Spectacle 对人类 RPE 单细胞 RNA 序列数据集进行了分析。我们的分析显示 Prom1 在人类 RPE 中表达。为了研究 Prom1 在 RPE 稳态中的功能,我们在雄性和雌性小鼠视网膜下注射了 AAV2/1.CMV.saCas9.U6.Prom1gRNA,进行了 RPE 特异性 Prom1 基因敲除(KD)。我们的数据显示,体内 RPE 特异性 Prom1-KD 导致 RPE 形态异常、视网膜下积液和继发性 PR 缺失。这些变化与成片的 RPE 细胞死亡和 a 波振幅降低有关,表明视网膜发生了变性。我们的发现强调了 Prom1 在细胞自主的 mRPE 平衡中的核心作用。Prom1-KD 在小鼠模型中导致类似于 aAMD 的 RPE 缺陷和视网膜变性,其意义重大,可为 aAMD 带来新的治疗方法。
{"title":"Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.","authors":"Sujoy Bhattacharya, Tzushan Sharon Yang, Bretton P Nabit, Evan S Krystofiak, Tonia S Rex, Edward Chaum","doi":"10.3390/cells13211761","DOIUrl":"10.3390/cells13211761","url":null,"abstract":"<p><p>There are currently no effective treatments for retinal pigment epithelial (RPE) cell loss in atrophic AMD (aAMD). However, our research on <i>Prominin-1</i> (<i>Prom1</i>), a known structural protein in photoreceptors (PRs), has revealed its distinct role in RPE and offers promising insights. While pathogenic <i>Prom1</i> mutations have been linked to macular diseases with RPE atrophy, the broader physiological impact of dysfunctional <i>Prom1</i> in RPE loss is unclear. We have shown that <i>Prom1</i> plays a <i>crucial</i> role in regulating autophagy and cellular homeostasis in <i>human</i> and <i>mouse</i> RPE (mRPE) cells in vitro. Nevertheless, a comprehensive understanding of its in vivo expression and function in mRPE remains to be elucidated. To characterize <i>Prom1</i> expression in RPE in situ, we used RNAscope assays and immunogold electron microscopy (EM). Our use of chromogenic and fluorescent RNAscope assays in albino and C57BL/6J <i>mouse</i> retinal sections has revealed <i>Prom1</i> mRNA expression in perinuclear regions in mRPE in situ. Immunogold EM imaging showed <i>Prom1</i> expression in RPE cytoplasm and mitochondria. To confirm <i>Prom1</i> expression in RPE, we interrogated <i>human</i> RPE single-cell RNA-sequencing datasets using an online resource, Spectacle. Our analysis showed <i>Prom1</i> expression in <i>human</i> RPE. To investigate <i>Prom1</i>'s function in RPE homeostasis, we performed RPE-specific <i>Prom1</i> knockdown (KD) using subretinal injections of AAV2/1.CMV.saCas9.U6.<i>Prom1</i>gRNA in male and female <i>mice</i>. Our data show that RPE-specific <i>Prom1</i>-KD in vivo resulted in abnormal RPE morphology, subretinal fluid accumulation, and secondary PR loss. These changes were associated with patchy RPE cell death and reduced a-wave amplitude, indicating retinal degeneration. Our findings underscore the central role of <i>Prom1</i> in cell-autonomous mRPE homeostasis. The implications of <i>Prom1</i>-KD causing aAMD-like RPE defects and retinal degeneration in a <i>mouse</i> model are significant and could lead to novel treatments for aAMD.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":"13 21","pages":""},"PeriodicalIF":8.3,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11545618/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142603341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Voltage-Gated Ion Channel Compensatory Effect in DEE: Implications for Future Therapies. DEE 中的电压门控离子通道补偿效应:对未来疗法的启示
IF 8.3 2区 生物学 Q2 CELL BIOLOGY Pub Date : 2024-10-24 DOI: 10.3390/cells13211763
Khadijeh Shabani, Johannes Krupp, Emilie Lemesre, Nicolas Lévy, Helene Tran

Developmental and Epileptic Encephalopathies (DEEs) represent a clinically and genetically heterogeneous group of rare and severe epilepsies. DEEs commonly begin early in infancy with frequent seizures of various types associated with intellectual disability and leading to a neurodevelopmental delay or regression. Disease-causing genomic variants have been identified in numerous genes and are implicated in over 100 types of DEEs. In this context, genes encoding voltage-gated ion channels (VGCs) play a significant role, and part of the large phenotypic variability observed in DEE patients carrying VGC mutations could be explained by the presence of genetic modifier alleles that can compensate for these mutations. This review will focus on the current knowledge of the compensatory effect of DEE-associated voltage-gated ion channels and their therapeutic implications in DEE. We will enter into detailed considerations regarding the sodium channels SCN1A, SCN2A, and SCN8A; the potassium channels KCNA1, KCNQ2, and KCNT1; and the calcium channels CACNA1A and CACNA1G.

发育性癫痫性脑病(DEEs)是一组临床和遗传异质性的罕见严重癫痫。发育性和癫痫性脑病通常起病于婴儿早期,各种类型的癫痫频繁发作,伴有智力障碍,导致神经发育迟缓或倒退。目前已在许多基因中发现了致病基因组变异,与 100 多种 DEEs 有关联。在这种情况下,编码电压门控离子通道(VGC)的基因发挥了重要作用,而在携带 VGC 突变的 DEE 患者身上观察到的巨大表型变异,部分原因可能是存在可补偿这些突变的遗传修饰等位基因。本综述将重点介绍目前对 DEE 相关电压门控离子通道的代偿效应及其对 DEE 的治疗意义的了解。我们将详细讨论钠通道 SCN1A、SCN2A 和 SCN8A;钾通道 KCNA1、KCNQ2 和 KCNT1;以及钙通道 CACNA1A 和 CACNA1G。
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引用次数: 0
p70S6K as a Potential Anti-COVID-19 Target: Insights from Wet Bench and In Silico Studies. p70S6K 作为潜在的抗 COVID-19 靶点:湿式工作台和硅学研究的启示。
IF 8.3 2区 生物学 Q2 CELL BIOLOGY Pub Date : 2024-10-24 DOI: 10.3390/cells13211760
Sharon Shechter, Rajat Kumar Pal, Fabio Trovato, Or Rozen, Matthew J Gage, Dorit Avni

The onset of SARS-CoV-2 infection in 2019 sparked a global COVID-19 pandemic. This infection is marked by a significant rise in both viral and host kinase activity. Our primary objective was to identify a pivotal host kinase essential for COVID-19 infection and the associated phenomenon of the cytokine storm, which may lead to long-term COVID-19 complications irrespective of viral genetic variations. To achieve this, our study tracked kinase phosphorylation dynamics in RAW264.7 macrophages following SPIKE transfection over time. Among the kinases surveyed, p70S6K (RPS6KB1) exhibited a 3.5-fold increase in phosphorylation at S418. This significant change prompted the selection of p70S6K for in silico investigation, utilizing its structure bound to M2698 (PDB: 7N93). M2698, an oral dual Akt/p70S6K inhibitor with an IC50 of 1.1 nM, exhibited psychosis side effects in phase I clinical trials, potentially linked to its interaction with Akt2. Our secondary objective was to discover a small-molecule analogue of M2698 that exhibits a distinct binding preference for p70S6K over Akt2 through computational modeling and analysis. The in silico part of our project began with validating the prediction accuracy of the docking algorithm, followed by an OCA analysis pinpointing specific atoms on M2698 that could be modified to enhance selectivity. Subsequently, our investigation led to the identification of an analog of M2698, designated as S34, that showed a superior docking score towards p70S6K compared to Akt2. To further assess the stability of S34 in its protein-ligand (PL) complexes with p70S6K and Akt2, MD simulations were conducted. These simulations suggest that S34, on average, forms two hydrogen bond interactions with p70S6K, whereas it only forms one hydrogen bond interaction with Akt2. This difference in hydrogen bond interactions likely contributed to the observed larger root mean square deviation (RMSD) of 0.3 nm in the S34-Akt2 complex, compared to 0.1 nm in the S34-p70S6K complex. Additionally, we calculated free binding energy to predict the strength of the binding interactions of S34 to p70S6K and Akt2, which showed ~2-fold favorable binding affinity of S34 in the p70S6K binding pocket compared to that in the Akt2 binding pocket. These observations may suggest that the S34-p70S6K complex is more stable than the S34-Akt2 complex. Our work focused on identifying a host kinase target and predicting the binding affinity of a novel small molecule to accelerate the development of effective treatments. The wet bench results specifically highlight p70S6K as a compelling anti-COVID-19 target. Meanwhile, our in silico investigations address the known off-target effects associated with M2698 by identifying a close analog called S34. In conclusion, this study presents novel and intriguing findings that could potentially lead to clinical applications with further investigations.

2019 年开始的 SARS-CoV-2 感染引发了全球 COVID-19 大流行。这种感染的特点是病毒和宿主激酶活性显著上升。我们的主要目标是确定一种对 COVID-19 感染和相关的细胞因子风暴现象至关重要的宿主激酶,无论病毒基因如何变异,这种激酶都可能导致 COVID-19 的长期并发症。为此,我们的研究跟踪了 SPIKE 转染后 RAW264.7 巨噬细胞中激酶磷酸化的动态变化。在调查的激酶中,p70S6K(RPS6KB1)在 S418 处的磷酸化增加了 3.5 倍。这一重大变化促使我们利用 p70S6K 与 M2698(PDB:7N93)结合的结构,选择 p70S6K 进行硅学研究。M2698是一种口服Akt/p70S6K双重抑制剂,IC50为1.1 nM,在I期临床试验中表现出精神错乱的副作用,这可能与其与Akt2的相互作用有关。我们的次要目标是通过计算建模和分析,发现 M2698 的小分子类似物,该类似物对 p70S6K 而非 Akt2 有明显的结合偏好。我们项目的硅学部分首先验证了对接算法的预测准确性,然后进行了 OCA 分析,找出了 M2698 上可以修改以提高选择性的特定原子。随后,我们的研究发现了 M2698 的类似物 S34,它与 p70S6K 的对接得分高于 Akt2。为了进一步评估 S34 与 p70S6K 和 Akt2 的蛋白配体(PL)复合物的稳定性,我们进行了 MD 模拟。这些模拟表明,S34 平均与 p70S6K 形成两个氢键相互作用,而与 Akt2 只形成一个氢键相互作用。这种氢键相互作用的差异很可能导致观察到的 S34-Akt2 复合物的均方根偏差(RMSD)较大,为 0.3 nm,而 S34-p70S6K 复合物的均方根偏差为 0.1 nm。此外,我们还计算了自由结合能,以预测 S34 与 p70S6K 和 Akt2 的结合相互作用强度,结果显示 S34 在 p70S6K 结合口袋中的结合亲和力比在 Akt2 结合口袋中的结合亲和力高出约 2 倍。这些观察结果可能表明,S34-p70S6K 复合物比 S34-Akt2 复合物更稳定。我们的工作重点是确定宿主激酶靶点并预测新型小分子的结合亲和力,以加速开发有效的治疗方法。湿法研究结果特别强调了 p70S6K 是一个引人注目的抗 COVID-19 靶点。与此同时,我们的硅学研究还发现了一种名为 S34 的近似物,从而解决了与 M2698 相关的已知脱靶效应。总之,这项研究提出了新颖而有趣的发现,通过进一步研究,有可能将其应用于临床。
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引用次数: 0
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