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Transcriptomic landscape of sex differences in obesity and type 2 diabetes in subcutaneous adipose tissue 皮下脂肪组织中肥胖和 2 型糖尿病性别差异的转录组图谱
Pub Date : 2024-09-19 DOI: 10.1101/2024.09.18.613678
Roxana Andreea Moldovan, Marta R Hidalgo, Helena Castane, Andrea Jimenez-Franco, Jorge Joven, Deborah J Burks, Amparo Galan, Francisco Garcia-Garcia
Obesity represents a significant risk factor in the development of type 2 diabetes (T2D), a chronic metabolic disorder characterized by elevated blood glucose levels. Significant sex differences have been identified in the prevalence, development, and pathophysiology of obesity and T2D; however, the underlying molecular mechanisms remain unclear. This study aims to identify sex-specific biomarkers in obesity and T2D and enhance our understanding of the underlying mechanisms associated with sex differences by integrating expression data. A systematic review, individual transcriptomic analysis, gene-level meta-analysis, and functional characterization were performed to achieve this aim. Eight studies and 236 subcutaneous adipose tissue samples were analyzed, identifying common and sex-specific biomarkers, many of which were previously associated with obesity or T2D.The obesity meta-analysis yielded nineteen differentially-expressed biomarkers from a sex-specific perspective (e.g., SPATA18, KREMEN1, NPY4R, and PRM3), while a comparison of the expression profiles between sexes in T2D prompted the identification and validation of specific transcriptomic signatures in males (SAMD9, NBPF3, LDHD, and EHD3) and females (RETN, HEY1, PLPP2, and PM20D2). At the functional level, we highlighted the fundamental role of the Wnt pathway in the development of obesity and T2D in females and the roles of more significant mitochondrial damage and free fatty acids in males. Overall, our sex-specific meta-analyses supported the detection of differentially expressed genes in males and females associated with the development of obesity and T2D, emphasizing the relevance of sex-based information in biomedical data and opening new avenues for research.
肥胖是罹患 2 型糖尿病(T2D)的重要风险因素,而 2 型糖尿病是一种以血糖水平升高为特征的慢性代谢性疾病。在肥胖和 T2D 的发病率、发展和病理生理学方面,已经发现了显著的性别差异;然而,其潜在的分子机制仍不清楚。本研究旨在确定肥胖和 T2D 的性别特异性生物标志物,并通过整合表达数据加深我们对性别差异相关内在机制的理解。为实现这一目标,我们进行了系统综述、个体转录组分析、基因水平荟萃分析和功能表征。分析了八项研究和 236 份皮下脂肪组织样本,确定了常见的和性别特异性的生物标志物,其中许多以前与肥胖或 T2D 相关、SPATA18、KREMEN1、NPY4R 和 PRM3),而通过比较 T2D 中不同性别的表达谱,我们发现并验证了男性(SAMD9、NBPF3、LDHD 和 EHD3)和女性(RETN、HEY1、PLPP2 和 PM20D2)的特定转录组特征。在功能层面,我们强调了 Wnt 通路在女性肥胖和 T2D 发病中的基本作用,以及线粒体损伤和游离脂肪酸在男性肥胖和 T2D 发病中的重要作用。总之,我们的性别特异性荟萃分析支持在男性和女性中检测到与肥胖和 T2D 发展相关的差异表达基因,强调了生物医学数据中基于性别的信息的相关性,并为研究开辟了新的途径。
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引用次数: 0
Medin Induces Pro-Inflammatory Activation of Human Brain Vascular Smooth Muscle Cells Medin 可诱导人脑血管平滑肌细胞的促炎性激活
Pub Date : 2024-09-18 DOI: 10.1101/2024.09.16.613366
Nina Karamanova, Kaleb T Morrow, Alana Maerivoet, Jillian Madine, Ming Li, Raymond Q Migrino
Background: Medin is one of the most common amyloidogenic proteins and accumulates in the vasculature with aging. Vascular medin accumulation is associated with Alzheimer's disease, vascular dementia and aortic aneurysms. Medin impairs smooth muscle-dependent vasodilation in isolated human brain cerebral arteries. The role of medin in vascular smooth muscle (VSMC) activation is unknown. We aim to evaluate the effects of medin on human brain VSMC activation. Methods: VSMCs were exposed to physiologic doses of medin (0.5, 1 and 5 µM) without or with small molecule nuclear factor-κB (NFκB) inhibitor RO106-9920 (10 µM) for 20 hours. Polymerase chain reaction, Western blot/enzyme-linked immunosorbent assay were used to quantify gene and protein expressions/secretions, respectively, of pro-inflammatory factors (interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1) and structural and enzyme proteins associated with VSMC phenotypic transformation (smooth muscle actin alpha 2 (ACTA2), myosin heavy chain 11 (MYH11) and NADPH oxidase 4 (NOX4)). Results: Medin exposure increased VSMC gene expression and protein secretion of IL-6, IL-8 and MCP-1 (protein secretion 46.0±12.8x, 20.2±4.1x and 8.7±3.1x, respectively, medin 5 µM versus vehicle, all p<0.05). There was no change in gene or protein expressions of ACTA2, MYH11 and NOX4. Co-treatment with RO106-9920 reduced medin-induced increases in IL-6 and IL-8 and a trend towards reduced MCP-1 secretion. Conclusions: Medin induced pro-inflammatory activation of human brain VSMCs that is mediated, at least in part, by NF?B. Acute medin treatment did not alter structural proteins involved in VSMC phenotypic transformation. The findings support medin as a potential novel mediator of and therapeutic target for vascular aging pathology.
背景:Medin 是最常见的淀粉样蛋白之一,会随着年龄的增长在血管中积累。血管中的 Medin 积聚与阿尔茨海默病、血管性痴呆和主动脉瘤有关。Medin 会损害离体人脑脑动脉平滑肌依赖性血管扩张。medin在血管平滑肌(VSMC)活化中的作用尚不清楚。我们旨在评估 Medin 对人脑 VSMC 活化的影响。研究方法将 VSMC 暴露于生理剂量的 Medin(0.5、1 和 5 µM)下 20 小时,同时不使用或使用小分子核因子-κB(NFκB)抑制剂 RO106-9920(10 µM)。聚合酶链反应、Western 印迹/酶联免疫吸附试验分别用于定量分析促炎因子(白细胞介素(IL)-6、IL-8 和单核细胞趋化蛋白(MCP)-1)以及与 VSMC 表型转化相关的结构蛋白和酶蛋白(平滑肌肌动蛋白α2(ACTA2)、肌球蛋白重链 11(MYH11)和 NADPH 氧化酶 4(NOX4))的基因和蛋白表达/分泌。结果暴露于 Medin 会增加 VSMC 基因表达以及 IL-6、IL-8 和 MCP-1 的蛋白分泌(蛋白分泌量分别为 46.0±12.8x、20.2±4.1x 和 8.7±3.1x, Medin 5 µM 对车辆,所有 p<0.05)。ACTA2、MYH11 和 NOX4 的基因或蛋白表达没有变化。与 RO106-9920 联合用药可减少medin 诱导的 IL-6 和 IL-8 的增加,并有减少 MCP-1 分泌的趋势。结论Medin 可诱导人脑 VSMCs 的促炎性活化,这种活化至少部分是由 NF?急性 Medin 处理不会改变参与 VSMC 表型转化的结构蛋白。研究结果支持将 Medin 作为血管老化病理学的潜在新型介质和治疗靶点。
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引用次数: 0
U2AF regulates the translation and localization of nuclear-encoded mitochondrial mRNAs U2AF 调节核编码线粒体 mRNA 的翻译和定位
Pub Date : 2024-09-18 DOI: 10.1101/2024.09.18.613780
Gloria R Garcia, Murali Palangat, Josquin Moraly, Benjamin T. Donovan, Bixuan Wang, Ira Phadke, David Sturgill, Jiji Chen, Brittney Short, Gustavo A. Rivero, David M. Swoboda, Naomi Taylor, Kathy McGraw, Daniel R Larson
The mechanisms underlying molecular targeting to mitochondria remain enigmatic, yet this process is crucial for normal cellular function. The RNA binding proteins U2AF1/2 form a heterodimer (U2AF) that shuttles between the nucleus and cytoplasm, regulating splicing in the nucleus and translation in the cytoplasm. Our study identifies an unexpected role for U2AF in mitochondrial function. We demonstrate that U2AF interacts with nuclear-transcribed mitochondrial mRNAs and proteins, inhibits translation, localizes to the outer mitochondrial membrane, and regulates mRNA localization to mitochondria. Moreover, an oncogenic point-mutation in U2AF1(S34F) disrupts this regulation, leading to altered mitochondrial structure, increased translation, and OXPHOS-dependent metabolic rewiring, recapitulating changes observed in bone marrow progenitors from patients with myelodysplastic syndromes. These findings reveal a non-canonical role for U2AF, where it modulates multiple aspects of mitochondrial function by regulating the translation and mitochondrial targeting of nuclear-encoded mRNAs.
分子靶向线粒体的机制仍是一个谜,但这一过程对细胞的正常功能至关重要。RNA 结合蛋白 U2AF1/2 形成一个异源二聚体(U2AF),在细胞核和细胞质之间穿梭,调节细胞核中的剪接和细胞质中的翻译。我们的研究发现了 U2AF 在线粒体功能中的意外作用。我们证明了 U2AF 与核转录的线粒体 mRNA 和蛋白质相互作用,抑制翻译,定位到线粒体外膜,并调节 mRNA 在线粒体中的定位。此外,U2AF1(S34F)的致癌点突变破坏了这种调控,导致线粒体结构改变、翻译增加和依赖 OXPHOS 的新陈代谢重新布线,再现了在骨髓增生异常综合征患者骨髓祖细胞中观察到的变化。这些发现揭示了 U2AF 的非典型作用,它通过调节核编码 mRNA 的翻译和线粒体靶向来调节线粒体功能的多个方面。
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引用次数: 0
Core splicing architecture and early spliceosomal recognition determine microexon sensitivity to SRRM3/4 核心剪接结构和早期剪接体识别决定了微外显子对 SRRM3/4 的敏感性
Pub Date : 2024-09-18 DOI: 10.1101/2024.09.17.613571
Sophie Bonnal, Simon Bajew, Rosa Martinez Corral, Manuel Irimia
Microexons are essential for proper functioning of neurons and pancreatic endocrine cells, where their inclusion depends on the splicing factors SRRM3/4. However, in pancreatic cells, lower expression of these regulators limits inclusion to only the most sensitive subset among all neuronal microexons. Although various cis-acting elements can contribute to microexon regulation, how they determine this differential dose response and high or low sensitivity to SRRM3/4 remains unknown. Here, Massively Parallel Splicing Assays probing 28,535 variants show that sensitivity to SRRM4 is conserved across vertebrates and support a regulatory model whereby high or low microexon sensitivity is largely determined by an interplay between core splicing architecture and length constraints. This conclusion is further supported by distinct spliceosome activities in the absence of SRRM3/4 and by a mathematical model that assumes that the two types of microexons differ only in their efficiency to recruit early spliceosomal components.
微外显子对神经元和胰腺内分泌细胞的正常功能至关重要,在神经元和胰腺内分泌细胞中,微外显子的包含取决于剪接因子 SRRM3/4。然而,在胰腺细胞中,这些调节因子的较低表达限制了所有神经元微外显子中最敏感的子集的包含。虽然各种顺式作用元件都可能有助于微外显子的调控,但它们如何决定这种不同的剂量反应以及对 SRRM3/4 的高或低敏感性仍是未知数。在这里,对 28,535 个变体进行的大规模平行剪接分析表明,脊椎动物对 SRRM4 的敏感性是一致的,并支持一种调控模型,即微外显子敏感性的高低主要由核心剪接结构和长度限制之间的相互作用决定。在没有 SRRM3/4 的情况下,不同的剪接体活动以及假定两种微外显子仅在招募早期剪接体成分的效率上有所不同的数学模型进一步支持了这一结论。
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引用次数: 0
CRISPR/Cas9-induced breaks are insufficient to break linkage drag surrounding the ToMV locus of Solanum lycopersicum CRISPR/Cas9诱导的断裂不足以打破番茄茄属植物ToMV基因座周围的连接阻力
Pub Date : 2024-09-18 DOI: 10.1101/2024.09.17.613470
Jillis Grubben, Gerard Bijsterbosch, Burak Aktürk, Richard G. F. Visser, Henk Schouten
Despite the success of CRISPR/Cas9 in inducing DNA double-strand breaks (DSBs) for genome editing, achieving targeted recombination in somatic cells remains challenging, particularly at recombination cold spots like the Tomato Mosaic Virus (ToMV) resistance locus in Solanum lycopersicum. We investigated the potential of CRISPR/Cas9-induced targeted recombination in somatic cells to overcome linkage drag surrounding the ToMV locus. We employed two strategies: first, inducing DSBs in both alleles of F1 tomato seedlings to promote non-homologous end joining (NHEJ) and homology-directed repair (HDR); second, targeting a single allele in a heterozygous background to induce HDR in seedlings. CRISPR/Cas9 activity was confirmed in F1 seedlings by detecting NHEJ-mediated mutations at the target sites in ToMV. We developed a bioinformatics pipeline to identify targeted recombinants by analyzing single nucleotide polymorphisms (SNPs) between parental haplotypes, allowing precise tracking of SNP variations. A two-dimensional pooling strategy was employed to distinguish genuine recombination events from PCR artifacts. Despite these advances and the active CRISPR/Cas9 system in F1 progeny, no increase in recombination frequency was observed compared to wild-type plants. We extended our research to protoplasts to assess whether CRISPR/Cas9 could induce targeted recombination under different cellular conditions at the same locus. Consistent with our findings in F1 plants, we observed no increase in recombinant patterns compared to wild-type controls in protoplasts. Our findings suggest that CRISPR/Cas9-induced DSBs are insufficient to break the genetic linkage at the ToMV locus on chromosome 9 in recombination cold spots within somatic cells.
尽管 CRISPR/Cas9 在诱导 DNA 双链断裂(DSB)以进行基因组编辑方面取得了成功,但在体细胞中实现靶向重组仍具有挑战性,尤其是在重组冷点,如番茄花叶病毒(ToMV)在番茄中的抗性基因座。我们研究了 CRISPR/Cas9 诱导的体细胞定向重组克服 ToMV 基因座周围连锁阻力的潜力。我们采用了两种策略:第一,在 F1 番茄幼苗的两个等位基因中诱导 DSB,以促进非同源末端连接(NHEJ)和同源定向修复(HDR);第二,在杂合背景中靶向单个等位基因,以诱导幼苗中的 HDR。通过检测 ToMV 目标位点上 NHEJ 介导的突变,在 F1 幼苗中证实了 CRISPR/Cas9 的活性。我们开发了一个生物信息学管道,通过分析亲本单倍型之间的单核苷酸多态性(SNP)来识别目标重组子,从而精确追踪 SNP 变异。该研究采用了一种二维汇集策略,以区分真正的重组事件和 PCR 伪影。尽管取得了这些进展,F1 后代中的 CRISPR/Cas9 系统也很活跃,但与野生型植物相比,重组频率并没有增加。我们将研究扩展到原生质体,以评估 CRISPR/Cas9 能否在不同细胞条件下诱导同一基因座的定向重组。与 F1 植物的研究结果一致,我们在原生质体中观察到的重组模式与野生型对照相比没有增加。我们的研究结果表明,CRISPR/Cas9诱导的DSB不足以打破体细胞内重组冷点中9号染色体上ToMV基因座的遗传连接。
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引用次数: 0
Enhancing lysosome function via mTOR/TFEB activation reduces lipofuscin-like granules in early Age-related Macular Degeneration 通过激活 mTOR/TFEB 增强溶酶体功能,减少早期老年性黄斑变性的脂褐素样颗粒
Pub Date : 2024-09-17 DOI: 10.1101/2024.09.17.613413
Ana S Falcao, Mafalda Lopes da Silva, Pedro Antas, Cristina Escrevente, Margarida Pedro, Rita Coelho, Ines S Ferreira, Ines P Santos, Thomas C. Ciossek, Paul Nicklin, Sandra Tenreiro, Miguel C. Seabra
Age-related macular degeneration (AMD) is the most common blinding disease in the western world and is currently incurable. Although the exact causes of AMD are not clear, the primary origin of pathology appears to be in the retinal pigment epithelium (RPE). RPE is responsible for the daily digestion of photoreceptor outer segments (POS), which imposes a heavy continuous burden on the lysosomal network. POS feeding assay in vitro suggested that the accumulation of autofluorescence granules (AFG), similar to lipofuscin in vivo, derives from lysosomal dysfunction. Here we show that synchronous phagocytosis of POS leads to early transient mTOR activation followed by inhibition in late phagosome maturation. One of its substrates, the transcription factor EB (TFEB) increases during phagosome maturation albeit mostly in its inactive phosphorylated form. We questioned whether induction of the mTOR/TFEB axis could improve digestion of POS and hence reduce AFG load. Treatment of POS-fed cells with rapamycin, an mTORC1 inhibitor after the appearance of AFG results in 30% reduction of AFG load. This effect is dependent on active lysosomal enzymes and induction of active dephosphorylated TFEB with consequent activation of GADD34 and lysosomal biogenesis. As a proof of concept, we show that overexpressing a constitutively active form of unphosphorylated TFEB dramatically reduces POS-dependent AFG accumulation. Overall, this study suggests that viral or pharmacological approaches activating the mTOR/TFEB axis in the RPE could be beneficial as cell-protective treatment of early/intermediate cases of AMD, acting to delay progression of the disease.
老年性黄斑变性(AMD)是西方世界最常见的致盲疾病,目前还无法治愈。虽然老年黄斑变性的确切病因尚不清楚,但病变的主要起源似乎是视网膜色素上皮(RPE)。RPE 负责每天消化感光体外节段(POS),这给溶酶体网络带来了沉重的持续负担。体外喂食光感受器外节的实验表明,自发荧光颗粒(AFG)的积累与体内的脂褐素相似,源于溶酶体功能障碍。在这里,我们发现同步吞噬 POS 会导致早期短暂的 mTOR 激活,然后在后期吞噬体成熟过程中受到抑制。mTOR的底物之一,转录因子EB(TFEB)在吞噬体成熟过程中会增加,尽管大部分是以非活性磷酸化形式增加。我们想知道,诱导 mTOR/TFEB 轴是否能改善对 POS 的消化,从而减少 AFG 的负荷。在出现 AFG 后,用雷帕霉素(一种 mTORC1 抑制剂)处理喂养 POS 的细胞,可使 AFG 负荷减少 30%。这种效果依赖于活跃的溶酶体酶和诱导活跃的去磷酸化 TFEB,从而激活 GADD34 和溶酶体生物生成。作为概念的证明,我们发现,过表达构成性活性的去磷酸化 TFEB 能显著减少 POS 依赖性 AFG 的积累。总之,这项研究表明,通过病毒或药物方法激活 RPE 中的 mTOR/TFEB 轴,可作为早期/中期 AMD 病例的细胞保护治疗方法,起到延缓疾病进展的作用。
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引用次数: 0
pytom-match-pick: a tophat-transform constraint for automated classification in template matching pytom-match-pick:用于模板匹配自动分类的拓扑变换约束条件
Pub Date : 2024-09-17 DOI: 10.1101/2024.09.17.613497
Marten L. Chaillet, Sander Roet, Remco C. Veltkamp, Friedrich Förster
Template matching (TM) in cryo-electron tomography (cryo-ET) enables in situ detection and localization of known macromolecules. However, TM faces challenges such as interfering features with a high signal-to-noise ratio and the need for manual curation of results. To address these challenges, we introduce pytom-match-pick, a GPU-accelerated, open-source command line interface for enhanced TM in cryo-ET. Using pytom-match-pick, we first quantify the effects of point spread function (PSF) weighting and show that a tilt-weighted PSF outperforms a binary wedge with a single defocus estimate. We also assess previously introduced background normalization methods for classification performance. This indicates that phase randomization is more effective than spectrum whitening in reducing false positives. Furthermore, a novel application of the tophat transform on score maps, combined with a dual-constraint thresholding strategy, reduces false positives and improves precision. We benchmarked pytom-match-pick on public datasets, demonstrating improved classification and localization of macromolecules like ribosomal subunits and proteasomes that led to fewer artifacts in subtomogram averages. This tool promises to advance visual proteomics by improving the efficiency and accuracy of macromolecule detection in cellular contexts.
低温电子断层成像(cryo-ET)中的模板匹配(TM)技术可对已知大分子进行原位检测和定位。然而,模板匹配面临着高信噪比干扰特征以及需要人工整理结果等挑战。为了应对这些挑战,我们引入了pytom-match-pick,这是一个GPU加速的开源命令行界面,用于在低温电子显微镜中增强TM。利用 pytom-match-pick,我们首先量化了点扩散函数(PSF)加权的效果,并证明倾斜加权 PSF 优于具有单一离焦估计值的二元楔形。我们还对之前引入的背景归一化方法的分类性能进行了评估。结果表明,在减少误报方面,相位随机化比光谱增白更有效。此外,在分数图上应用新颖的 tophat 变换,并结合双约束阈值策略,可以减少误报并提高精确度。我们在公共数据集上对 pytom-match-pick 进行了基准测试,结果表明它改进了核糖体亚基和蛋白酶体等大分子的分类和定位,从而减少了子图平均值中的伪影。该工具有望提高细胞环境中大分子检测的效率和准确性,从而推动可视蛋白质组学的发展。
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引用次数: 0
Structural insight into the nuclear transportation mechanism of PPARg by Transportin-1 从结构上洞察运输蛋白-1对PPARg的核运输机制
Pub Date : 2024-09-17 DOI: 10.1101/2024.09.17.612794
Sachiko TOMA-FUKAI, Yutaro Nakamura, Akihiro Kawamoto, Hikaru Shimizu, Koki Hayama, Ruri Kojima, Kanami Yoshimura, Masaki Ishii, Mika Hirose, Toshiaki Teratani, Shinya Ohata, Takayuki Kato, Hironari Kamikubo, Toshimasa Itoh, Kengo Tomita, Toshiyuki Shimizu
The spatial and temporal control of protein is essential for normal cellular function. Proteins working in the nucleus have nuclear localization signal (NLS) sequences and are escorted into the nucleus by cognate nuclear transport receptors. A wealth of experimental data about NLS has been accumulated, and nuclear transportation mechanisms are established at the biochemical and structural levels. The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various biological responses. We recently reported that the transportation of PPARg is mediated by Transportin-1, but PPARg lacks a typical NLS sequence recognized by Transportin-1. Moreover, the recognition mechanism remains largely unknown. In this study, we determined the Cryo-EM structure of PPARg in complex with Transportin-1 and revealed that Transportin-1 gripped the folded DNA binding domain and the Hinge region of PPARg, indicating that PPARg recognizes a folded domain with an extended region as a nuclear localization signal, not a canonical unstructured signal sequence, confirmed by the mutation analyses in vitro and in cultured cells. Our study is the first snapshot structure working in nuclear transportation, not in transcription, of PPARg.
蛋白质的空间和时间控制对细胞的正常功能至关重要。在细胞核中工作的蛋白质具有核定位信号(NLS)序列,并由同源的核运输受体护送进入细胞核。目前已积累了大量有关 NLS 的实验数据,并在生化和结构水平上建立了核运输机制。过氧化物酶体增殖激活受体(PPARs)是配体依赖性转录因子,可控制各种生物反应。我们最近报道 PPARg 的运输是由运输蛋白-1 介导的,但 PPARg 缺乏运输蛋白-1 识别的典型 NLS 序列。此外,其识别机制在很大程度上仍然未知。本研究测定了 PPARg 与转运蛋白-1 复合物的冷冻电镜结构,发现转运蛋白-1 抓住了 PPARg 的折叠 DNA 结合结构域和铰链区,表明 PPARg 识别的核定位信号是带有扩展区的折叠结构域,而不是典型的非结构化信号序列,体外和培养细胞的突变分析也证实了这一点。我们的研究是 PPARg 核运输而非转录过程中的首个结构快照。
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引用次数: 0
Molecular dynamics simulations of human cohesin subunits identify DNA binding sites and their potential roles in DNA loop extrusion 人类凝聚素亚基的分子动力学模拟确定了 DNA 结合位点及其在 DNA 环挤压中的潜在作用
Pub Date : 2024-09-17 DOI: 10.1101/2024.09.17.613402
Chenyang Gu, Shoji Takada, Giovani B. Brandani, Tsuyoshi Terakawa
The SMC complex cohesin mediates interphase chromatin structural formation in eukaryotic cells through DNA loop extrusion. Here, we sought to investigate its mechanism using molecular dynamics simulations. To achieve this, we first constructed the amino-acid-residue-resolution structural models of the cohesin subunits, SMC1, SMC3, STAG1, and NIPBL. By simulating these subunits with double-stranded DNA molecules, we predicted DNA binding patches on each subunit and quantified the affinities of these patches to DNA using their dissociation rate constants as a proxy. Then, we constructed the structural model of the whole cohesin complex and mapped the predicted high-affinity DNA binding patches on the structure. From the spatial relations of the predicted patches, we identified that multiple patches on the SMC1, SMC3, STAG1, and NIPBL subunits form a DNA clamping patch group. The simulations of the whole complex with double-stranded DNA molecules suggest that this patch group facilitates DNA bending and helps capture a DNA segment in the cohesin ring formed by the SMC1 and SMC3 subunits. In previous studies, these have been identified as critical steps in DNA loop extrusion. Therefore, this study provides experimentally testable predictions of DNA binding sites implicated in previously proposed DNA loop extrusion mechanisms and highlights the essential roles of the accessory subunits STAG1 and NIPBL in the mechanism.
在真核细胞中,SMC 复合物凝聚素通过 DNA 环挤压介导染色质间期结构的形成。在此,我们试图利用分子动力学模拟研究其机制。为此,我们首先构建了SMC1、SMC3、STAG1和NIPBL等凝聚素亚基的氨基酸分辨率结构模型。通过用双链DNA分子模拟这些亚基,我们预测了每个亚基上的DNA结合斑块,并以这些斑块的解离率常数为代表,量化了这些斑块与DNA的亲和力。然后,我们构建了整个凝聚蛋白复合物的结构模型,并在该结构上绘制了预测的高亲和性 DNA 结合斑块。从预测的补丁的空间关系中,我们发现SMC1、SMC3、STAG1和NIPBL亚基上的多个补丁组成了一个DNA夹持补丁组。用双链 DNA 分子模拟整个复合体的结果表明,这个贴片组有利于 DNA 弯曲,并有助于在 SMC1 和 SMC3 亚基形成的凝聚素环中捕获 DNA 片段。在以前的研究中,这些已被确定为 DNA 环挤出的关键步骤。因此,本研究对之前提出的 DNA 环挤出机制中涉及的 DNA 结合位点进行了可实验检验的预测,并强调了附属亚基 STAG1 和 NIPBL 在该机制中的重要作用。
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引用次数: 0
Decoding polyubiquitin regulation of KV7. 1 functional expression with engineered linkage-selective deubiquitinases 解码多泛素对 KV7.1 功能表达的调控1 的功能性表达
Pub Date : 2024-09-17 DOI: 10.1101/2024.09.17.613539
Sri Karthika Shanmugam, Scott A Kanner, Xinle M Zou, Enoch Amarh, Papiya Choudhury, Rajesh SONI, Robert S Kass, Henry M Colecraft
Protein posttranslational modification with distinct polyubiquitin linkage chains is a critical component of the ubiquitin code that universally regulates protein expression and function to control biology. Functional consequences of diverse polyubiquitin linkages on proteins are mostly unknown, with progress hindered by a lack of methods to specifically tune polyubiquitin linkages on individual proteins in live cells. Here, we bridge this gap by exploiting deubiquitinases (DUBs) with preferences for hydrolyzing different polyubiquitin linkages: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K29/K33; and USP21 - non-specific. We developed a suite of engineered deubiquitinases (enDUBs) comprised of DUB catalytic domains fused to a GFP-targeted nanobody and used them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. Mass spectrometry of YFP-KCNQ1 expressed in HEK293 cells indicated channel polyubiquitination with K48 (72%) and K63 (24%) linkages being dominant. NEDD4-2 and ITCH both decreased KCNQ1 functional expression but with distinctive polyubiquitination signatures. All enDUBs reduced KCNQ1 ubiquitination but yielded unique effects on channel expression, surface density, ionic currents, and subcellular localization. The pattern of outcomes indicates K11, K29/K33, and K63 chains mediate net KCNQ1-YFP intracellular retention, but achieved in different ways: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling. The pattern of enDUB effects on KCNQ1-YFP differed in cardiomyocytes, emphasizing ubiquitin code mutability. Surprisingly, enDUB-O4 decreased KCNQ1-YFP surface density suggesting a role for K48 in forward trafficking. Lastly, linkage-selective enDUBs displayed varying capabilities to rescue distinct trafficking-deficient long QT syndrome type 1 mutations. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 functional expression, demonstrate ubiquitin code plasticity, and introduce linkage-selective enDUBs as a potent tool to help demystify the polyubiquitin code.
蛋白质通过不同的多泛素连接链进行翻译后修饰是泛素密码的重要组成部分,泛素密码普遍调节蛋白质的表达和功能,从而控制生物学。由于缺乏在活细胞中特异性调节单个蛋白质上多泛素连接的方法,研究进展受阻。在这里,我们利用对水解不同多泛素连接具有偏好的去泛素酶(DUBs)来弥补这一差距:OTUD1--K63;OTUD4--K48;Cezanne--K11;TRABID--K29/K33;USP21--非特异性。我们开发了一套由 DUB 催化结构域与 GFP 靶向纳米抗体融合而成的工程化去泛素化酶(enDUBs),并用它们来研究离子通道 YFP-KCNQ1 的多泛素连接调控。在 HEK293 细胞中表达的 YFP-KCNQ1 的质谱分析表明,通道多泛素化以 K48(72%)和 K63(24%)连接为主。NEDD4-2 和 ITCH 都会降低 KCNQ1 的功能表达,但具有独特的多泛素化特征。所有 enDUBs 都减少了 KCNQ1 泛素化,但对通道表达、表面密度、离子电流和亚细胞定位产生了独特的影响。结果表明,K11、K29/K33 和 K63 链介导了 KCNQ1-YFP 在细胞内的净保留,但实现的方式不同:K11 促进 ER 保留/降解,增强内吞,减少再循环;K29/K33 促进 ER 保留/降解;K63 增强内吞,减少再循环。enDUB对KCNQ1-YFP的影响模式在心肌细胞中有所不同,这强调了泛素编码的可变性。令人惊讶的是,enDUB-O4降低了KCNQ1-YFP的表面密度,这表明K48在前向转运中的作用。最后,连接选择性 enDUB 在挽救不同的转运缺陷长 QT 综合征 1 型突变方面表现出不同的能力。这些结果揭示了不同的多泛素链控制着 KCNQ1 功能表达的不同方面,证明了泛素密码的可塑性,并将连接选择性 enDUBs 作为一种有效的工具来帮助揭开多泛素密码的神秘面纱。
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bioRxiv - Molecular Biology
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