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Metabolic reprogramming contributes to radioprotection by protein kinase Cδ. 代谢重编程有助于蛋白激酶Cδ的辐射保护。
Pub Date : 2023-10-01 Epub Date: 2023-08-21 DOI: 10.1016/j.jbc.2023.105186
Angela M Ohm, Trisiani Affandi, Julie A Reisz, M Cecilia Caino, Angelo D'Alessandro, Mary E Reyland

Loss of protein kinase Cδ (PKCδ) activity renders cells resistant to DNA damaging agents, including irradiation; however, the mechanism(s) underlying resistance is poorly understood. Here, we have asked if metabolic reprogramming by PKCδ contributes to radioprotection. Analysis of global metabolomics showed that depletion of PKCδ affects metabolic pathways that control energy production and antioxidant, nucleotide, and amino acid biosynthesis. Increased NADPH and nucleotide production in PKCδ-depleted cells is associated with upregulation of the pentose phosphate pathway (PPP) as evidenced by increased activation of G6PD and an increase in the nucleotide precursor, 5-phosphoribosyl-1-pyrophosphate. Stable isotope tracing with U-[13C6] glucose showed reduced utilization of glucose for glycolysis in PKCδ-depleted cells and no increase in U-[13C6] glucose incorporation into purines or pyrimidines. In contrast, isotope tracing with [13C5, 15N2] glutamine showed increased utilization of glutamine for synthesis of nucleotides, glutathione, and tricarboxylic acid intermediates and increased incorporation of labeled glutamine into pyruvate and lactate. Using a glycolytic rate assay, we confirmed that anaerobic glycolysis is increased in PKCδ-depleted cells; this was accompanied by a reduction in oxidative phosphorylation, as assayed using a mitochondrial stress assay. Importantly, pretreatment of cells with specific inhibitors of the PPP or glutaminase prior to irradiation reversed radioprotection in PKCδ-depleted cells, indicating that these cells have acquired codependency on the PPP and glutamine for survival. Our studies demonstrate that metabolic reprogramming to increase utilization of glutamine and nucleotide synthesis contributes to radioprotection in the context of PKCδ inhibition.

蛋白激酶Cδ(PKCδ)活性的丧失使细胞对DNA损伤剂具有抵抗力,包括辐射;然而,人们对阻力背后的机制知之甚少。在这里,我们询问PKCδ的代谢重编程是否有助于辐射保护。全球代谢组学分析表明,PKCδ的缺失影响控制能量产生和抗氧化剂、核苷酸和氨基酸生物合成的代谢途径。PKCδ缺失细胞中NADPH和核苷酸产生的增加与戊糖磷酸途径(PPP)的上调有关,G6PD的激活增加和核苷酸前体5-磷酸核糖基-1-焦磷酸的增加证明了这一点。用U-[13C6]葡萄糖进行稳定同位素示踪显示,在PKCδ缺失的细胞中,葡萄糖用于糖酵解的利用率降低,而U-[13C6]葡萄糖掺入嘌呤或嘧啶的情况没有增加。相反,[13C5,15N2]谷氨酰胺的同位素示踪显示,谷氨酰胺在核苷酸、谷胱甘肽和三羧酸中间体合成中的利用率增加,标记的谷氨酰胺在丙酮酸和乳酸盐中的结合率增加。使用糖酵解速率测定,我们证实在PKCδ缺失的细胞中厌氧糖酵解增加;这伴随着氧化磷酸化的减少,如使用线粒体应激测定所测定的。重要的是,在照射前用PPP或谷氨酰胺酶的特异性抑制剂预处理细胞,逆转了PKCδ缺失细胞的放射保护作用,表明这些细胞已经获得了对PPP和谷氨酰胺的相互依赖性。我们的研究表明,在PKCδ抑制的情况下,代谢重编程以增加谷氨酰胺和核苷酸合成的利用有助于辐射保护。
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引用次数: 0
Porcine IKKε is involved in the STING-induced type I IFN antiviral response of the cytosolic DNA signaling pathway. 猪IKKε参与STING诱导的胞质DNA信号通路的I型IFN抗病毒反应。
Pub Date : 2023-10-01 Epub Date: 2023-09-01 DOI: 10.1016/j.jbc.2023.105213
Jia Luo, Qi Cao, Jiajia Zhang, Sen Jiang, Nengwen Xia, Shaohua Sun, Wanglong Zheng, Nanhua Chen, Francois Meurens, Jianzhong Zhu

The cyclic GMP-AMP synthase and stimulator of interferon (IFN) genes (cGAS-STING) pathway serves as a crucial component of innate immune defense and exerts immense antiviral activity by inducing the expression of type I IFNs. Currently, STING-activated production of type I IFNs has been thought to be mediated only by TANK-binding kinase 1 (TBK1). Here, we identified that porcine IKKε (pIKKε) is also directly involved in STING-induced type I IFN expression and antiviral response by using IKKε-/- porcine macrophages. Similar to pTBK1, pIKKε interacts directly with pSTING on the C-terminal tail. Furthermore, the TBK1-binding motif of pSTING C-terminal tail is essential for its interaction with pIKKε, and within the TBK1-binding motif, the leucine (L) 373 is also critical for the interaction. On the other hand, both kinase domain and scaffold dimerization domain of pIKKε participate in the interactions with pSTING. Consistently, the reconstitution of pIKKε and its mutants in IKKε-/- porcine macrophages corroborated that IKKε and its kinase domain and scaffold dimerization domain are all involved in the STING signaling and antiviral function. Thus, our findings deepen the understanding of porcine cGAS-STING pathway, which lays a foundation for effective antiviral therapeutics against porcine viral diseases.

环状GMP-AMP合酶和干扰素(IFN)基因刺激因子(cGAS-STING)途径是先天免疫防御的关键组成部分,并通过诱导I型IFN的表达发挥巨大的抗病毒活性。目前,STING激活的I型IFN的产生被认为仅由TANK结合激酶1(TBK1)介导。在这里,我们发现猪IKKε(pIKKε)也直接参与STING诱导的I型IFN表达和使用IKKε-/-猪巨噬细胞的抗病毒反应。与pTBK1类似,pIKKε与C末端尾部的pSTING直接相互作用。此外,pSTING C末端尾部的TBK1结合基序对于其与pIKKε的相互作用是必需的,并且在TBK1结合基序中,亮氨酸(L)373对于相互作用也是关键的。另一方面,pIKKε的激酶结构域和支架二聚化结构域都参与了与pSTING的相互作用。一致地,pKKε及其突变体在IKKε-/-猪巨噬细胞中的重建证实了IKKε及其激酶结构域和支架二聚化结构域都参与STING信号传导和抗病毒功能。因此,我们的发现加深了对猪cGAS-STING通路的理解,为有效的抗病毒治疗猪病毒性疾病奠定了基础。
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引用次数: 0
Matriptase-2 regulates iron homeostasis primarily by setting the basal levels of hepatic hepcidin expression through a nonproteolytic mechanism. 基质蛋白酶-2主要通过非促细胞分裂机制调节肝铁调素表达的基础水平来调节铁稳态。
Pub Date : 2023-10-01 Epub Date: 2023-09-09 DOI: 10.1016/j.jbc.2023.105238
Caroline A Enns, Tyler Weiskopf, Richard H Zhang, Jeffrey Wu, Shall Jue, Makiko Kawaguchi, Hiroaki Kataoka, An-Sheng Zhang

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease. It plays a key role in iron homeostasis by suppressing the iron-regulatory hormone, hepcidin. Lack of functional MT2 results in an inappropriately high hepcidin and iron-refractory iron-deficiency anemia. Mt2 cleaves multiple components of the hepcidin-induction pathway in vitro. It is inhibited by the membrane-anchored serine protease inhibitor, Hai-2. Earlier in vivo studies show that Mt2 can suppress hepcidin expression independently of its proteolytic activity. In this study, our data indicate that hepatic Mt2 was a limiting factor in suppressing hepcidin. Studies in Tmprss6-/- mice revealed that increases in dietary iron to ∼0.5% were sufficient to overcome the high hepcidin barrier and to correct iron-deficiency anemia. Interestingly, the increased iron in Tmprss6-/- mice was able to further upregulate hepcidin expression to a similar magnitude as in wild-type mice. These results suggest that a lack of Mt2 does not impact the iron induction of hepcidin. Additional studies of wild-type Mt2 and the proteolytic-dead form, fMt2S762A, indicated that the function of Mt2 is to lower the basal levels of hepcidin expression in a manner that primarily relies on its nonproteolytic role. This idea is supported by the studies in mice with the hepatocyte-specific ablation of Hai-2, which showed a marginal impact on iron homeostasis and no significant effects on iron regulation of hepcidin. Together, these observations suggest that the function of Mt2 is to set the basal levels of hepcidin expression and that this process is primarily accomplished through a nonproteolytic mechanism.

基质蛋白酶-2(MT2)由TMPRSS6编码,是一种膜锚定的丝氨酸蛋白酶。它通过抑制铁调节激素铁调素在铁稳态中发挥关键作用。缺乏功能性MT2会导致铁调素过高和铁难治性缺铁性贫血。Mt2在体外切割铁调素诱导途径的多种成分。它被膜锚定丝氨酸蛋白酶抑制剂Hai-2抑制。早期的体内研究表明,Mt2可以抑制铁调素的表达,而不依赖于其蛋白水解活性。在本研究中,我们的数据表明,肝脏Mt2是抑制铁调素的限制因素。对Tmprss6-/-小鼠的研究表明,将膳食铁含量增加至-0.5%就足以克服高铁调素屏障并纠正缺铁性贫血。有趣的是,Tmprss6-/-小鼠体内铁的增加能够进一步上调铁调素的表达,其幅度与野生型小鼠相似。这些结果表明Mt2的缺乏并不影响铁调素的铁诱导。对野生型Mt2和蛋白水解死亡形式fMt2S762A的额外研究表明,Mt2的功能是以主要依赖于其非促蛋白水解作用的方式降低铁调素表达的基础水平。这一观点得到了肝细胞特异性切除Hai-2的小鼠研究的支持,该研究显示对铁稳态的影响很小,对铁调素的铁调节没有显著影响。总之,这些观察结果表明,Mt2的功能是设定铁调素表达的基础水平,而这一过程主要是通过非蛋白水解机制实现的。
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引用次数: 0
Synergistic computational and experimental studies of a phosphoglycosyl transferase membrane/ligand ensemble. 磷酸糖基转移酶膜/配体系综的协同计算和实验研究。
Pub Date : 2023-10-01 Epub Date: 2023-08-25 DOI: 10.1016/j.jbc.2023.105194
Ayan Majumder, Nemanja Vuksanovic, Leah C Ray, Hannah M Bernstein, Karen N Allen, Barbara Imperiali, John E Straub

Complex glycans serve essential functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane-associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates of specific isoprene unit geometry, such as undecaprenol phosphate (UndP) in bacteria. How these enzymes and substrates interact within a lipid bilayer needs further investigation. Here, we focus on a small enzyme, PglC from Campylobacter, structurally characterized for the first time in 2018 as a detergent-solubilized construct. PglC is a monotopic phosphoglycosyl transferase that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high-level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results provided a high-level view of the membrane-embedded PglC/UndP complex. The findings suggested that it is advantageous for the polyprenol phosphate to adopt a conformation in the same leaflet where the monotopic membrane protein resides as opposed to additionally disrupting the opposing leaflet of the bilayer. Further, the analysis showed that electrostatic steering acts as a major driving force contributing to the recognition and binding of both UndP and the soluble nucleotide sugar substrate. Iterative computational and experimental mutagenesis support a specific interaction of UndP with phosphoglycosyl transferase cationic residues and suggest a role for critical conformational transitions in substrate binding and specificity.

复杂聚糖在所有生命系统中都具有重要功能。这些复杂的拜占庭生物分子中的许多是通过生物合成途径组装的,其中组成酶是膜相关的。逐步组装过程的一个标志性特征是具有特定异戊二烯单元几何形状的不同寻常的线性长链聚丙烯醇磷酸酯连接底物的重要性,例如细菌中的十一碳烯醇磷酸酯(UndP)。这些酶和底物如何在脂质双层中相互作用还需要进一步研究。在这里,我们关注的是一种来自弯曲杆菌的小酶PglC,它在2018年首次在结构上被表征为一种可溶解洗涤剂的构建体。PglC是一种单主题磷酸糖基转移酶,它体现了整个酶超家族的功能核心结构,并催化糖蛋白组装途径中的第一个膜结合步骤。酶的大小是重要的,因为它能够进行高水平的计算,并且对于膜蛋白来说,实验分析相对容易。我们的整体计算和实验结果提供了膜嵌入的PglC/UndP复合物的高级视图。研究结果表明,与额外破坏双层的相对小叶相比,聚丙醛磷酸酯在单主题膜蛋白所在的同一小叶中采用构象是有利的。此外,分析表明,静电操纵是有助于识别和结合UndP和可溶性核苷酸糖底物的主要驱动力。迭代计算和实验诱变支持UndP与磷酸糖基转移酶阳离子残基的特异性相互作用,并表明关键构象转变在底物结合和特异性中的作用。
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引用次数: 0
APOLLO, a testis-specific Drosophila ortholog of importin-4, mediates the loading of protamine-like protein Mst77F into sperm chromatin. APOLLO是一种重要蛋白-4的睾丸特异性果蝇直系同源物,介导精蛋白样蛋白Mst77F进入精子染色质。
Pub Date : 2023-10-01 Epub Date: 2023-09-02 DOI: 10.1016/j.jbc.2023.105212
Alexander V Emelyanov, Daniel Barcenilla-Merino, Benjamin Loppin, Dmitry V Fyodorov

DNA in sperm is packed with small, charged proteins termed SNBPs (sperm nuclear basic proteins), including mammalian and Drosophila protamines. During spermiogenesis, somatic-type chromatin is taken apart and replaced with sperm chromatin in a multistep process leading to an extraordinary condensation of the genome. During fertilization, the ova face a similarly challenging task of SNBP eviction and reassembly of nucleosome-based chromatin. Despite its importance for the animal life cycle, sperm chromatin metabolism, including the biochemical machinery mediating the mutual replacement of histones and SNBPs, remains poorly studied. In Drosophila, Mst77F is one of the first SNBPs loaded into the spermatid nuclei. It persists in mature spermatozoa and is essential for sperm compaction and male fertility. Here, by using in vitro biochemical assays, we identify chaperones that can mediate the eviction and loading of Mst77F on DNA, thus facilitating the interconversions of chromatin forms in the male gamete. Unlike NAP1 and TAP/p32 chaperones that disassemble Mst77F-DNA complexes, ARTEMIS and APOLLO, orthologs of mammalian importin-4 (IPO4), mediate the deposition of Mst77F on DNA or oligonucleosome templates, accompanied by the dissociation of histone-DNA complexes. In vivo, a mutation of testis-specific Apollo brings about a defect of Mst77F loading, abnormal sperm morphology, and male infertility. We identify IPO4 ortholog APOLLO as a critical component of sperm chromatin assembly apparatus in Drosophila. We discover that in addition to recognized roles in protein traffic, a nuclear transport receptor (IPO4) can function directly in chromatin remodeling as a dual, histone- and SNBP-specific, chaperone.

精子中的DNA充满了小的带电蛋白质,称为SNBP(精子核碱性蛋白),包括哺乳动物和果蝇的鱼精蛋白。在精子生成过程中,体细胞型染色质在一个多步骤的过程中被分离并替换为精子染色质,从而导致基因组的异常浓缩。在受精过程中,卵子面临着同样具有挑战性的任务,即SNBP驱逐和基于核小体的染色质的重组。尽管精子染色质代谢对动物生命周期很重要,但它,包括介导组蛋白和SNBP相互替换的生化机制,仍然研究不足。在果蝇中,Mst77F是第一批装载到精子细胞核中的SNBP之一。它在成熟精子中持续存在,对精子压实和男性生育能力至关重要。在这里,通过使用体外生化分析,我们鉴定了可以介导Mst77F在DNA上的排出和装载的伴侣蛋白,从而促进雄配子中染色质形式的相互转化。与分解Mst77F DNA复合物的NAP1和TAP/p32伴侣不同,哺乳动物导入蛋白-4(IPO4)的直系同源物ARTEMIS和APOLLO介导Mst77F在DNA或寡核小体模板上的沉积,并伴随组蛋白DNA复合物解离。在体内,睾丸特异性Apollo的突变导致Mst77F负荷缺陷、精子形态异常和男性不育。我们确定IPO4同源APOLLO是果蝇精子染色质组装器的关键组成部分。我们发现,除了在蛋白质运输中的公认作用外,核运输受体(IPO4)还可以作为组蛋白和SNBP特异性的双重伴侣直接在染色质重塑中发挥作用。
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引用次数: 0
Differential regulation of tetramerization of the AMPA receptor glutamate-gated ion channel by auxiliary subunits. 辅助亚基对AMPA受体谷氨酸门控离子通道四聚作用的差异调节。
Pub Date : 2023-10-01 Epub Date: 2023-09-09 DOI: 10.1016/j.jbc.2023.105227
Noele Certain, Quan Gan, Joseph Bennett, Helen Hsieh, Lonnie P Wollmuth

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) auxiliary subunits are specialized, nontransient binding partners of AMPARs that modulate AMPAR channel gating properties and pharmacology, as well as their biogenesis and trafficking. The most well-characterized families of auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs), cornichon homologs (CNIHs), and the more recently discovered GSG1-L. These auxiliary subunits can promote or reduce surface expression of AMPARs (composed of GluA1-4 subunits) in neurons, thereby impacting their functional role in membrane signaling. Here, we show that CNIH-2 enhances the tetramerization of WT and mutant AMPARs, presumably by increasing the overall stability of the tetrameric complex, an effect that is mainly mediated by interactions with the transmembrane domain of the receptor. We also find CNIH-2 and CNIH-3 show receptor subunit-specific actions in this regard with CNIH-2 enhancing both GluA1 and GluA2 tetramerization, whereas CNIH-3 only weakly enhances GluA1 tetramerization. These results are consistent with the proposed role of CNIHs as endoplasmic reticulum cargo transporters for AMPARs. In contrast, TARP γ-2, TARP γ-8, and GSG1-L have no or negligible effect on AMPAR tetramerization. On the other hand, TARP γ-2 can enhance receptor tetramerization but only when directly fused with the receptor at a maximal stoichiometry. Notably, surface expression of functional AMPARs was enhanced by CNIH-2 to a greater extent than TARP γ-2, suggesting that this distinction aids in maturation and membrane expression. These experiments define a functional distinction between CNIHs and other auxiliary subunits in the regulation of AMPAR biogenesis.

α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体(AMPAR)辅助亚基是AMPAR的特异性、非瞬时结合伴侣,调节AMPAR通道门控特性和药理学,以及它们的生物发生和运输。最具特征的辅助亚基家族是跨膜AMPAR调节蛋白(TARP)、角蛋白同源物(CNIH)和最近发现的GSG1-L。这些辅助亚基可以促进或减少神经元中AMPAR(由GluA1-4亚基组成)的表面表达,从而影响其在膜信号传导中的功能作用。在这里,我们发现CNIH-2增强了WT和突变AMPAR的四聚体,可能是通过增加四聚体复合物的整体稳定性,这种作用主要由与受体跨膜结构域的相互作用介导的。我们还发现CNIH-2和CNIH-3在这方面表现出受体亚基特异性作用,CNIH-2增强GluA1和GluA2四聚,而CNIH-3仅微弱地增强GluAl四聚。这些结果与CNIH作为AMPAR的内质网货物转运蛋白的作用一致。相反,TARPγ-2、TARPγ-8和GSG1-L对AMPAR四聚没有影响或可以忽略不计。另一方面,TARPγ-2可以增强受体四聚,但只有当以最大化学计量直接与受体融合时。值得注意的是,与TARPγ-2相比,CNIH-2在更大程度上增强了功能性AMPAR的表面表达,这表明这种差异有助于成熟和膜表达。这些实验确定了CNIH和其他辅助亚基在调节AMPAR生物发生中的功能区别。
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引用次数: 0
Preservation of circadian rhythm in hepatocellular cancer. 肝细胞癌症昼夜节律的保护。
Pub Date : 2023-10-01 Epub Date: 2023-09-14 DOI: 10.1016/j.jbc.2023.105251
Yanyan Yang, Ashraf N Abdo, Hiroaki Kawara, Christopher P Selby, Aziz Sancar

Circadian rhythms are controlled at the cellular level by a molecular clock consisting of several genes/proteins engaged in a transcription-translation-degradation feedback loop. These core clock proteins regulate thousands of tissue-specific genes. Regarding circadian control in neoplastic tissues, reports to date have demonstrated anomalous circadian function in tumor models and cultured tumor cells. We have extended these studies by analyzing circadian rhythmicity genome-wide in a mouse model of liver cancer, in which mice treated with diethylnitrosamine at 15 days develop liver tumors by 6 months. We injected tumor-bearing and control tumor-free mice with cisplatin every 2 h over a 24-h cycle; 2 h after each injection mice were sacrificed and gene expression was measured by XR-Seq (excision repair sequencing) assay. Rhythmic expression of several core clock genes was observed in both healthy liver and tumor, with clock genes in tumor exhibiting typically robust amplitudes and a modest phase advance. Interestingly, although normal hepatic cells and hepatoma cancer cells expressed a comparable number of genes with circadian rhythmicity (clock-controlled genes), there was only about 10% overlap between the rhythmic genes in normal and cancerous cells. "Rhythmic in tumor only" genes exhibited peak expression times mainly in daytime hours, in contrast to the more common pre-dawn and pre-dusk expression times seen in healthy livers. Differential expression of genes in tumors and healthy livers across time may present an opportunity for more efficient anticancer drug treatment as a function of treatment time.

昼夜节律在细胞水平上由分子钟控制,分子钟由参与转录-翻译-降解反馈回路的几个基因/蛋白质组成。这些核心时钟蛋白调节数千个组织特异性基因。关于肿瘤组织的昼夜节律控制,迄今为止的报道已经证明肿瘤模型和培养的肿瘤细胞的昼夜节律功能异常。我们通过分析癌症小鼠模型中的昼夜节律全基因组来扩展这些研究,在该模型中,用二乙基亚硝胺治疗15天的小鼠在6个月后发展为肝肿瘤。我们在24小时的周期内每2小时给荷瘤和对照无瘤小鼠注射一次顺铂;每次注射后2小时处死小鼠,并通过XR-Seq(切除修复测序)测定法测量基因表达。在健康肝脏和肿瘤中都观察到几个核心时钟基因的节律性表达,肿瘤中的时钟基因表现出典型的稳健振幅和适度的相位提前。有趣的是,尽管正常肝细胞和肝癌癌症细胞表达了相当数量的具有昼夜节律性的基因(时钟控制基因),但在正常细胞和癌细胞中,节律基因之间只有大约10%的重叠。与健康肝脏中更常见的黎明前和黄昏前表达时间相比,“仅在肿瘤中有节律”基因的峰值表达时间主要在白天。随着时间的推移,肿瘤和健康肝脏中基因的差异表达可能为更有效的抗癌药物治疗提供了机会。
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引用次数: 0
A novel phosphorylation site involved in dissociating RAF kinase from the scaffolding protein 14-3-3 and disrupting RAF dimerization. 一种新的磷酸化位点,参与从支架蛋白14-3-3中解离RAF激酶并破坏RAF二聚化。
Pub Date : 2023-10-01 Epub Date: 2023-08-23 DOI: 10.1016/j.jbc.2023.105188
Alison Yu, Duc Nguyen, Thomas Joseph Nguyen, Zhihong Wang

Rapidly accelerated fibrosarcoma (ARAF, BRAF, CRAF) kinase is central to the MAPK pathway (RAS-RAF-MEK-ERK). Inactive RAF kinase is believed to be monomeric, autoinhibited, and cytosolic, while activated RAF is recruited to the membrane via RAS-GTP, leading to the relief of autoinhibition, phosphorylation of key regulatory sites, and dimerization of RAF protomers. Although it is well known that active and inactive BRAF have differential phosphorylation sites that play a crucial role in regulating BRAF, key details are still missing. In this study, we report the characterization of a novel phosphorylation site, BRAFS732 (equivalent in CRAFS624), located in proximity to the C-terminus binding motif for the 14-3-3 scaffolding protein. At the C terminus, 14-3-3 binds to BRAFpS729 (CRAFpS621) and enhances RAF dimerization. We conducted mutational analysis of BRAFS732A/E and CRAFS624A/E and revealed that the phosphomimetic S→E mutant decreases 14-3-3 association and RAF dimerization. In normal cell signaling, dimerized RAF phosphorylates MEK1/2, which is observed in the phospho-deficient S→A mutant. Our results suggest that phosphorylation and dephosphorylation of this site fine-tune the association of 14-3-3 and RAF dimerization, ultimately impacting MEK phosphorylation. We further characterized the BRAF homodimer and BRAF:CRAF heterodimer and identified a correlation between phosphorylation of this site with drug sensitivity. Our work reveals a novel negative regulatory role for phosphorylation of BRAFS732 and CRAFS624 in decreasing 14-3-3 association, dimerization, and MEK phosphorylation. These findings provide insight into the regulation of the MAPK pathway and may have implications for cancers driven by mutations in the pathway.

快速加速纤维肉瘤(ARAF、BRAF、CRAF)激酶是MAPK通路(RAS-RAF-MEK-ERK)的核心。非活性RAF激酶被认为是单体的、自动抑制的和胞质的,而活化的RAF通过RAS-GTP募集到膜上,导致自动抑制的缓解、关键调控位点的磷酸化和RAF原聚体的二聚化。尽管众所周知,活性和非活性BRAF具有差异磷酸化位点,在调节BRAF中起着至关重要的作用,但关键细节仍然缺失。在本研究中,我们报道了一个新的磷酸化位点BRAFS732(相当于CRAFS624)的特征,该位点位于14-3-3支架蛋白的C末端结合基序附近。在C末端,14-3-3与BRAFpS729(CRAFpS621)结合并增强RAF二聚化。我们对BRAFS732A/E和CRAFS624A/E进行了突变分析,发现→E突变体降低14-3-3缔合和RAF二聚化。在正常细胞信号传导中,二聚化的RAF磷酸化MEK1/2,这在磷酸缺乏的S中观察到→突变体。我们的结果表明,该位点的磷酸化和去磷酸化微调了14-3-3和RAF二聚化的结合,最终影响MEK磷酸化。我们进一步鉴定了BRAF同源二聚体和BRAF:CRAF异二聚体,并确定了该位点的磷酸化与药物敏感性之间的相关性。我们的工作揭示了BRAFS732和CRAFS624磷酸化在减少14-3-3结合、二聚化和MEK磷酸化中的新的负调控作用。这些发现提供了对MAPK途径调控的深入了解,并可能对由该途径突变驱动的癌症具有启示。
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引用次数: 0
Reply to Record et al. "The role of PMP22 T118M in Charcot-Marie-Tooth disease remains unsolved". 回复记录等。“PMP22 T118M在Charcot-Marie Tooth病中的作用尚未解决”。
Pub Date : 2023-10-01 Epub Date: 2023-09-14 DOI: 10.1016/j.jbc.2023.105181
Katherine M Stefanski, Geoffrey C Li, Justin T Marinko, Bruce D Carter, David C Samuels, Charles R Sanders
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引用次数: 0
SUMOylation of Smad2 mediates TGF-β-regulated endothelial-mesenchymal transition. Smad2的SUMO化介导TGF-β调节的内皮-间充质转化。
Pub Date : 2023-10-01 Epub Date: 2023-09-09 DOI: 10.1016/j.jbc.2023.105244
Qi Su, Xu Chen, Xing Ling, Danqing Li, Xiang Ren, Yang Zhao, Yanyan Yang, Yuhang Liu, Anqi He, Xinjie Zhu, Xinyi Yang, Wenbin Lu, Hongmei Wu, Yitao Qi

Endothelial-mesenchymal transition (EndoMT) is a complex biological process in which endothelial cells are transformed into mesenchymal cells, and dysregulated EndoMT causes a variety of pathological processes. Transforming growth factor beta (TGF-β) signaling effectively induces the EndoMT process in endothelial cells, and Smad2 is the critical protein of the TGF-β signaling pathway. However, whether small ubiquitin-like modifier modification (SUMOylation) is involved in EndoMT remains unclear. Here, we show that Smad2 is predominantly modified by SUMO1 at two major SUMOylation sites with PIAS2α as the primary E3 ligase, whereas SENP1 (sentrin/SUMO-specific protease 1) mediates the deSUMOylation of Smad2. In addition, we identified that SUMOylation significantly enhances the transcriptional activity and protein stability of Smad2, regulating the expression of downstream target genes. SUMOylation increases the phosphorylation of Smad2 and the formation of the Smad2-Smad4 complex, thus promoting the nuclear translocation of Smad2. Ultimately, the wildtype, but not SUMOylation site mutant Smad2 facilitated the EndoMT process. More importantly, TGF-β enhances the nuclear translocation of Smad2 by enhancing its SUMOylation and promoting the EndoMT process. These results demonstrate that SUMOylation of Smad2 plays a critical role in the TGF-β-mediated EndoMT process, providing a new theoretical basis for the treatment and potential drug targets of EndoMT-related clinical diseases.

内皮-间充质转化(EndoMT)是一个复杂的生物学过程,内皮细胞转化为间充质细胞,而失调的EndoMT会导致多种病理过程。转化生长因子β(TGF-β)信号传导有效诱导内皮细胞的EndoMT过程,Smad2是TGF-β信号传导途径的关键蛋白。然而,小泛素样修饰因子修饰(SUMOylation)是否参与EndoMT仍不清楚。在这里,我们发现Smad2主要被SUMO1在两个主要的SUMO化位点修饰,PIAS2α作为主要的E3连接酶,而SENP1(sentrin/SUMO特异性蛋白酶1)介导Smad2的去SUMO化。此外,我们发现SUMO化显著增强Smad2的转录活性和蛋白质稳定性,调节下游靶基因的表达。SUMO化增加Smad2的磷酸化和Smad2-Smad4复合物的形成,从而促进Smad2的核转位。最终,野生型而非SUMO化位点突变体Smad2促进了EndoMT过程。更重要的是,TGF-β通过增强其SUMO化和促进EndoMT过程来增强Smad2的核转位。这些结果表明,Smad2的SUMO化在TGF-β介导的EndoMT过程中起着关键作用,为EndoMT相关临床疾病的治疗和潜在药物靶点提供了新的理论基础。
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The Journal of Biological Chemistry
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