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Identification and Validation of Novel Biomarkers for Unstable Angina Using Olink Proteomics 不稳定心绞痛新标志物的蛋白质组学鉴定与验证。
IF 3.6 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-12-24 DOI: 10.1021/acs.jproteome.5c00831
Ting Cao, , , Wenli Li, , , Jungang Pang, , , Shaohua Tan, , and , Jun Liu*, 

Unstable angina (UA) represents a prevalent cardiovascular condition characterized by significant morbidity and mortality, necessitating the development of effective early diagnostic biomarkers. This study sought to identify plasma protein biomarkers associated with UA utilizing Olink proteomics. A cohort of 120 participants, comprising 60 individuals diagnosed with UA and 60 healthy controls, was recruited. A total of 92 inflammation-related plasma proteins were quantified through a proximity extension assay. In the discovery cohort, differential expression analysis revealed significant alterations in 49 proteins, which were predominantly enriched in cytokine-mediated signaling and MAPK pathways, as demonstrated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Least absolute shrinkage and selection operator regression and random forest methodologies prioritized three biomarkers─SPON2, PTX3, and GBGT1─that exhibited elevated levels in UA patients compared to controls. Receiver operating characteristic analysis demonstrated area under the curve values of 0.859, 0.937, and 0.900 for SPON2, PTX3, and GBGT1, respectively. A combined diagnostic model incorporating these three proteins achieved an AUC of 0.979 and demonstrated robust performance in an independent validation cohort. These results suggest that SPON2, PTX3, GBGT1, and their composite model hold promise as diagnostic biomarkers for UA.

不稳定型心绞痛(UA)是一种常见的心血管疾病,具有显著的发病率和死亡率,因此需要开发有效的早期诊断生物标志物。本研究试图利用Olink蛋白质组学鉴定与UA相关的血浆蛋白生物标志物。招募了120名参与者,包括60名诊断为UA的个体和60名健康对照者。通过近距离扩展试验,共量化了92种与炎症相关的血浆蛋白。在发现队列中,差异表达分析显示49个蛋白显著改变,这些蛋白主要富集于细胞因子介导的信号通路和MAPK通路,如基因本体和京都基因与基因组百科全书分析所证明的那样。最小绝对收缩、选择算子回归和随机森林方法优先考虑了三种生物标志物──SPON2、PTX3和GBGT1──与对照组相比,这三种标志物在UA患者中表现出较高的水平。受试者工作特征分析显示,SPON2、PTX3和GBGT1的曲线下面积分别为0.859、0.937和0.900。结合这三种蛋白的联合诊断模型的AUC为0.979,在独立验证队列中表现出稳健的性能。这些结果表明,SPON2、PTX3、GBGT1及其复合模型有望作为UA的诊断生物标志物。
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引用次数: 0
Penicillin–Streptomycin Treatment Rewires Core Metabolic and Ribosomal Programs in HepG2 Cells 青霉素-链霉素治疗重组HepG2细胞核心代谢和核糖体程序。
IF 3.6 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-12-24 DOI: 10.1021/acs.jproteome.5c00934
Cameron S. Movassaghi,  and , Jesse G. Meyer*, 

Antibiotics are routinely added to mammalian cell culture media to prevent bacterial growth. However, the use of antibiotics in a cell culture can confound downstream experimental results. While genomic and transcriptomic differences between cell cultures treated with and without antibiotics are well-documented, far fewer, if any, comprehensive proteomic comparisons on the use of antibiotics in cell culture have been performed. Here, we present a study on the proteome-wide differences of culturing HepG2 cells in antibiotic (i.e., penicillin/streptomycin) and nonantibiotic-containing media. Using a longitudinal and crossover treatment study design, we analyzed 119 samples across nine passages and four conditions. On average, 9,374 proteins were detected per sample, and we identified 383 proteins that were differentially abundant between conditions. These changes included ribosomal and mitochondrial proteins, demonstrating that off-target effects of antibiotics on mammalian cells occur at the protein level. Linear mixed-effect modeling suggested that the proteomic impact of antibiotic treatment is strongest in the first passage after treatment and stabilizes after approximately three passages. Furthermore, initiating antibiotic treatment induced a greater number of differentially abundant proteins than discontinuing treatment. Lastly, we compared our results to existing literature on the use of common antibiotics in mammalian cell culture. We identified proteins and pathways conserved across studies, omics layers, and cell types. We hope that this detailed proteomic survey of the ubiquitous pencillin–streptyomcin-treated HepG2 in vitro model will aid researchers in comparing cross-study or cross-condition results from antibiotic-treated mammalian cells and inform appropriate experimental designs for the use of antibiotics in cell culture.

抗生素通常被添加到哺乳动物细胞培养基中以防止细菌生长。然而,在细胞培养中使用抗生素会混淆下游实验结果。虽然使用抗生素和不使用抗生素的细胞培养物之间的基因组和转录组差异有充分的文献记载,但在细胞培养物中使用抗生素的全面蛋白质组学比较却很少,如果有的话。在此,我们研究了在抗生素(即青霉素/链霉素)和不含抗生素的培养基中培养HepG2细胞的蛋白质组差异。采用纵向和交叉治疗研究设计,我们分析了9个通道和4种条件下的119个样本。平均每个样品检测到9374种蛋白质,我们确定了383种蛋白质在不同条件下的丰度差异。这些变化包括核糖体和线粒体蛋白,表明抗生素对哺乳动物细胞的脱靶效应发生在蛋白质水平上。线性混合效应模型表明,抗生素治疗的蛋白质组学影响在治疗后的第一代最强,并在大约三代后稳定。此外,开始抗生素治疗比停止治疗诱导了更多的差异丰富的蛋白质。最后,我们将我们的结果与现有的关于在哺乳动物细胞培养中使用常见抗生素的文献进行了比较。我们确定了在研究、组学层和细胞类型中保守的蛋白质和途径。我们希望对普遍存在的铅笔链霉素处理的HepG2体外模型进行详细的蛋白质组学调查,将有助于研究人员比较抗生素处理的哺乳动物细胞的交叉研究或交叉条件结果,并为在细胞培养中使用抗生素提供适当的实验设计。
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引用次数: 0
Proteome-Wide Multipoint Internal Calibration Curves for Evaluating Peptide-Level Linearity in Relative Quantitative Proteomics 相对定量蛋白质组学中多肽水平线性评价的蛋白质组多点内部校准曲线。
IF 3.6 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-12-23 DOI: 10.1021/acs.jproteome.5c00179
Cristina Chiva, , , Zahra Elhamraoui, , , Julia Morales-Sanfrutos, , , Olga Pastor, , and , Eduard Sabidó*, 

Mass spectrometry (MS)-based proteomics is known for its high accuracy in quantifying peptides and proteins using various calibration strategies including internal and external calibration curves. While external multipoint calibration curves are created from serial dilutions, they often fail to account for sample-specific matrix effects. In contrast, internal calibration curves account for the sample matrix but face scalability and cost challenges for whole proteome analyses. In this manuscript, we present a novel TMT-based multipoint internal calibration curve strategy, which enables the generation of internal calibration curves for all peptides identified within a proteome in a single experiment to assess their linearity prior relative quantification. We applied this strategy to human ovarian cancer cells to evaluate the linear quantitative responses of all of the identified peptides and reveal the significant proteome changes associated with cisplatin treatment.

基于质谱(MS)的蛋白质组学以其在定量多肽和蛋白质方面的高精度而闻名,使用各种校准策略,包括内部和外部校准曲线。虽然外部多点校准曲线是由连续稀释创建的,但它们通常无法解释样品特异性矩阵效应。相比之下,内部校准曲线解释了样品矩阵,但面临整个蛋白质组分析的可扩展性和成本挑战。在本文中,我们提出了一种新颖的基于tmt的多点内部校准曲线策略,该策略能够在单个实验中生成蛋白质组中鉴定的所有肽的内部校准曲线,以评估其线性度之前的相对定量。我们将这一策略应用于人卵巢癌细胞,以评估所有已鉴定肽的线性定量反应,并揭示与顺铂治疗相关的显著蛋白质组变化。
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引用次数: 0
Toward Proteomic-Based Prediction of Ex Vivo Platinum Sensitivity in Ovarian Cancer Ascitic Cellular Aggregates: A Pilot Study 基于蛋白质组学的卵巢癌腹水细胞聚集体体外铂敏感性预测:一项初步研究。
IF 3.6 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-12-23 DOI: 10.1021/acs.jproteome.5c00771
Jack Scanlan, , , Parul Mittal, , , Noor A. Lokman, , , Martin K. Oehler, , , Peter Hoffmann, , and , Manuela Klingler-Hoffmann*, 

The accumulation of malignant ascites in the peritoneal cavity is a hallmark of high-grade serous ovarian cancer (HGSC). This fluid contains three-dimensional multicellular aggregates known as spheroids, which contribute to chemoresistance and are an accessible source of tumor material for proteomic-based biomarker discovery studies. Although heterogeneous ascitic spheroids can be generated from primary cell suspensions for ex vivo applications, they suffer from long generation times and reduced biological relevance. Here, we compare their ex vivo chemotherapy responses and proteomes to native spheroids that are collected directly from HGSC ascites, with the aim of assessing their suitability for proteomic-based chemoresponse prediction strategies that yield results within a clinically relevant time frame. We demonstrate that the chemoresponses of native spheroids better correlate with patients’ clinical treatment responses in 4 of 5 cases and that their proteomes uniquely segregate according to ex vivo carboplatin response along the first component. This pilot study suggests key proteins and biological pathways that may facilitate a global proteomic-based screening strategy for personalized HGSC treatment, with particular emphasis on extracellular matrix proteins. As such, native spheroids have the potential to progress the personalized treatment of HGSC patients with malignant ascites.

恶性腹水在腹腔内的积累是高级别浆液性卵巢癌(HGSC)的标志。这种液体含有三维多细胞聚集体,称为球状体,有助于化学耐药,是基于蛋白质组学的生物标志物发现研究的肿瘤材料的可获得来源。虽然异质腹水球可以从原代细胞悬浮液中产生,用于体外应用,但它们的产生时间长,生物学相关性降低。在这里,我们将它们的体外化疗反应和蛋白质组学与直接从HGSC腹水中收集的天然球状体进行比较,目的是评估它们在临床相关时间框架内产生结果的基于蛋白质组学的化学反应预测策略的适用性。我们证明,在5个病例中,有4个天然球体的化学反应与患者的临床治疗反应有更好的相关性,并且它们的蛋白质组根据体外卡铂反应沿第一个成分独特地分离。这项初步研究提出了关键的蛋白质和生物学途径,可以促进基于蛋白质组学的个性化HGSC治疗的全球筛选策略,特别强调细胞外基质蛋白。因此,原生球体有可能推进HGSC恶性腹水患者的个性化治疗。
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引用次数: 0
TAG-PL: A Universal Proximity Labeling Platform for Mapping Mitochondrial Proteomes and Organelle Interactions under Stress TAG-PL:在胁迫下绘制线粒体蛋白质组和细胞器相互作用的通用接近标记平台。
IF 3.6 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-12-23 DOI: 10.1021/acs.jproteome.5c00855
Enming Miao, , , Xuyang Yue, , , Zhixiong Tao, , , Hongqiang Qin*, , and , Mingliang Ye*, 

Mitochondrial dysfunction induces numerous diseases, yet current proximity labeling methods require gene transfection and membrane potential-sensitive probes, limiting their use in hard-to-transfect cells and disease models. We developed TAG-PL (Tailored Antibody-Guided Proximity Labeling), a transfection-free approach for in-depth mapping of the mitochondrial proteome, achieving >90% specificity and identifying >450 mitochondrial proteins─more than the coverage of existing nontransfection methods. Applied to heat-stressed macrophages, TAG-PL revealed dynamic mitochondrial proteome remodeling, including antioxidant responses and metabolic shifts during heat stress. Notably, we discovered physical interactions between stress granules and mitochondria, identifying 10 interaction mediators (including MSRA and UBA1). These findings establish stress granules as regulatory hubs for organelle dynamics and immune responses. TAG-PL’s high performance and broad applicability across diverse sample types, particularly immune cells and tissues, make it a powerful tool for dissecting mitochondrial function in disease models without genetic manipulation.

线粒体功能障碍可诱发多种疾病,但目前的接近标记方法需要基因转染和膜电位敏感探针,这限制了它们在难以转染的细胞和疾病模型中的应用。我们开发了TAG-PL(量身定制的抗体引导接近标记),这是一种无需转染的方法,用于深入绘制线粒体蛋白质组图,实现了bbb90 %的特异性,并鉴定了>450个线粒体蛋白,比现有的非转染方法的覆盖率更高。应用于热应激巨噬细胞,TAG-PL揭示了热应激过程中线粒体蛋白质组的动态重塑,包括抗氧化反应和代谢变化。值得注意的是,我们发现了应激颗粒和线粒体之间的物理相互作用,确定了10种相互作用介质(包括MSRA和UBA1)。这些发现确立了应激颗粒作为细胞器动力学和免疫反应的调节枢纽。TAG-PL的高性能和在不同样品类型,特别是免疫细胞和组织中的广泛适用性,使其成为无需基因操作的疾病模型中解剖线粒体功能的强大工具。
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引用次数: 0
Temporal Profiling of Peroxynitrite-Mediated Protein Nitration and Phosphorylation Using Proteomics Analysis 利用蛋白质组学分析过氧亚硝酸盐介导的蛋白质硝化和磷酸化的时间谱。
IF 3.6 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-12-22 DOI: 10.1021/acs.jproteome.5c00417
Yating Yao*, , , Shuang Wang, , , Jingyi Li, , , Xiaohua Wang, , and , Jinghua Yang*, 

Peroxynitrite is capable of inducing protein nitration, which alters protein structure and interferes with signaling pathways dependent on phosphorylation. This study aims to investigate the effects of peroxynitrite-driven nitration and phosphorylation on proteins in HEK293T cells. Through label-free quantitative mass spectrometry, we successfully identified over 5,000 proteins from 0.5 μg of cell extracts collected at five different time intervals (0, 2, 15, 30, and 60 min) after exposure to peroxynitrite. We found that protein expression profiles from 2 to 60 min were distinct from those at 0 min. LC-MS/MS was also applied to quantify and map the nitration and phosphorylation sites. Analysis of protein nitration and phosphorylation revealed distinct temporal profiles, suggesting that nitration may contribute to the altered phosphorylation of the same protein or its interacting proteins. This study provides valuable insights for further mechanistic studies in peroxynitrite-related pathways.

过氧亚硝酸盐能够诱导蛋白质硝化,从而改变蛋白质结构并干扰依赖于磷酸化的信号通路。本研究旨在探讨过氧亚硝酸盐驱动的硝化和磷酸化对HEK293T细胞蛋白的影响。通过无标记定量质谱法,我们成功地从0.5 μg的细胞提取物中鉴定出5000多种蛋白质,这些细胞提取物在暴露于过氧亚硝酸盐后的5个不同时间间隔(0、2、15、30和60分钟)采集。我们发现,2 - 60分钟的蛋白表达谱与0分钟的蛋白表达谱不同。LC-MS/MS还用于定量和绘制硝化和磷酸化位点。蛋白质硝化和磷酸化的分析揭示了不同的时间谱,表明硝化可能有助于改变相同蛋白质或其相互作用蛋白的磷酸化。该研究为过氧亚硝酸盐相关途径的进一步机制研究提供了有价值的见解。
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引用次数: 0
Comparative Analysis of Lysine-Specific Peptidases for Optimizing Proteomics Workflows 优化蛋白质组学工作流程的赖氨酸特异性肽酶的比较分析。
IF 3.6 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-12-22 DOI: 10.1021/acs.jproteome.5c00872
Leander Roeland van der Hoeven, , , Maico Lechner, , , Cristina Hernandez-Rollan, , , Tanveer Singh Batth, , and , Jesper V. Olsen*, 

This study presents a comparative analysis of three LysC endopeptidase homologues from Achromobacter lyticus (A. lyticus),Pseudomonas aeruginosa and Lysobacter enzymogenes for mass spectrometry-based proteomics. Utilizing a protein aggregation capture workflow with HeLa cell lysates, we assessed the enzymes’ cleavage specificity, digestion efficiency, and performance across various experimental conditions. Results showed that while all three LysC homologues exhibited high cleavage specificity at lysine residues, A. lyticus LysC outperformed the two others with superior peptide identification, digestion efficiency, and protein coverage, especially at shorter digestion times. Our experiments using a combination ofA. lyticusLysC and trypsin demonstrated the importance of employing LysC for significantly minimizing missed cleavage rates in tryptic digests, especially with regard to lysine-containing peptides. This study underscores A. lyticus LysC’s potential as an optimal choice for enhancing mass spectrometry-based proteomics.

本研究对来自lyticus无色杆菌(a . lyticus)、铜绿假单胞菌(Pseudomonas aeruginosa)和Lysobacter momogenes的三种LysC内肽酶同源物进行了质谱分析。利用HeLa细胞裂解物的蛋白质聚集捕获工作流程,我们评估了酶在不同实验条件下的裂解特异性、消化效率和性能。结果表明,虽然这三种LysC同源物在赖氨酸残基上都表现出很高的裂解特异性,但A. lyticus LysC在肽识别、消化效率和蛋白质覆盖方面优于其他两种,尤其是在更短的消化时间内。我们的实验使用a的组合。lyticusLysC和胰蛋白酶证明了使用LysC在胰蛋白酶消化中显著减少遗漏裂解率的重要性,特别是关于含有赖氨酸的肽。这项研究强调了A. lyticus LysC作为增强质谱蛋白质组学的最佳选择的潜力。
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引用次数: 0
Physiological Consequences of Overexpression of a Twin-Arginine Translocase in Bacillus subtilis Revealed by 14N/15N Labeling 14N/15N标记揭示枯草芽孢杆菌双精氨酸转位酶过表达的生理后果
IF 3.6 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-12-22 DOI: 10.1021/acs.jproteome.5c00787
Margarita Bernal-Cabas, , , Minia Antelo-Varela, , , Bimal Prajapati, , , Tobias Schilling, , , Marina López-Álvarez, , , Stefano Grasso, , , Sandra Maaβ, , , Andreas Otto, , , Girbe Buist, , , Dörte Becher, , and , Jan Maarten van Dijl*, 

Bacillus subtilis is a Gram-positive bacterium widely used in biotechnology due to its efficient secretion systems. Among these, the twin-arginine (Tat) pathway facilitates the export of fully folded cofactor-containing proteins across the cytoplasmic membrane. The TatAyCy translocase, which is expressed constitutively, is key to this process. Previous studies showed that this translocase not only consists of the TatAy and TatCy subunits, but that it also recruits the LiaH protein upon overexpression. Presumably, the recruitment of LiaH represents an intrinsic protective mechanism against the potentially detrimental effects of facilitating the membrane passage of large, fully folded proteins. However, to date the full spectrum of physiological consequences of protein translocation via TatAyCy has remained elusive. In this study, we employed a 14N/15N metabolic labeling approach combined with subcellular fractionation to quantitatively analyze proteomic changes in the cytoplasm, membrane, and extracellular milieu upon TatAyCy overexpression. Our findings show that high-level TatAyCy expression leads to a prolonged vegetative state and disrupts key cellular processes, including genetic competence, motility, chemotaxis, and biofilm formation. Notably, arginine metabolism emerges as a central factor in the cellular adaptation to TatAyCy-induced stress.

枯草芽孢杆菌是一种革兰氏阳性菌,因其高效的分泌系统而被广泛应用于生物技术。其中,双精氨酸(Tat)途径促进了含有完全折叠的辅因子的蛋白质穿过细胞质膜的输出。组成性表达的TatAyCy转位酶是这一过程的关键。先前的研究表明,这种转位酶不仅由TatAy和TatCy亚基组成,而且在过表达时也会招募LiaH蛋白。据推测,LiaH的募集代表了一种内在的保护机制,以防止促进大的、完全折叠的蛋白质通过膜的潜在有害影响。然而,迄今为止,通过TatAyCy进行的蛋白质易位的全部生理后果仍然是难以捉摸的。在本研究中,我们采用14N/15N代谢标记法结合亚细胞分离,定量分析了TatAyCy过表达后细胞质、膜和细胞外环境的蛋白质组学变化。我们的研究结果表明,高水平的TatAyCy表达会导致植物状态延长,并破坏关键的细胞过程,包括遗传能力、运动性、趋化性和生物膜的形成。值得注意的是,精氨酸代谢是细胞适应tataycy诱导的应激的核心因素。
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引用次数: 0
Redox Stoichiometry at the Single-Residue Level Using Mass Spectrometry Reveals Dynamic Methionine Sulfoxide Speciation in Actin and Calmodulin during Brain Aging 单残留水平的质谱氧化还原化学计量揭示了脑老化过程中肌动蛋白和钙调蛋白中蛋氨酸亚砜的动态形成。
IF 3.6 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-12-22 DOI: 10.1021/acs.jproteome.5c00715
Jiwoo Hwang, , , Madeleine F. Clore, , , Filipa Blasco Tavares Pereira Lopes, , , Marzieh Ayati, , , Daniela Schlatzer, , , Xin Qi, , , Rihua Wang, , , Mehmet Koyuturk, , and , Mark R. Chance*, 

Methionine oxidation to methionine sulfoxide (MSox) is often viewed as a nonspecific modification from reactive oxygen species. However, oxidation at specific methionine sites, such as Met44/47 in actin and Met77 in calmodulin, can be reversed by methionine sulfoxide reductase (Msr) and other enzyme families. This study uses liquid chromatography coupled with mass spectrometry to comprehensively investigate actin and calmodulin-based MSox speciation within the mouse hippocampus in an Alzheimer’s disease (AD) model (5XFAD), reflecting neuroinflammation and oxidative stress. Concurrent detection of both oxidized and unmodified peptides enabled direct calculation of absolute oxidation stoichiometry and protein-normalized % occupancy ─an analytical dimension seldom attainable for most post-translational modification studies. Our results indicate age-dependent but not AD-dependent redox dynamics. In actin, D-loop Met44/47 declined from ∼9 to ∼5% between 3 and 6 months and then rose to ∼14% by 9 months, while H-loop Met269 remained stable at ∼5% MSox. In calmodulin, linker Met77 climbed steadily with age (but not AD), whereas C-lobe Met145/146 fell sharply from 20 to ∼8% MSox from 3 to 9 months. These findings highlight dynamic, age-related methionine oxidation patterns in actin and calmodulin within the mouse hippocampus, likely relevant to brain development and aging.

蛋氨酸氧化生成蛋氨酸亚砜(Methionine亚砜,MSox)通常被认为是活性氧的非特异性修饰。然而,特定蛋氨酸位点的氧化,如肌动蛋白中的Met44/47和钙调蛋白中的Met77,可以被蛋氨酸亚砜还原酶(Msr)和其他酶家族逆转。本研究采用液相色谱联用质谱法全面研究了阿尔茨海默病(AD)模型(5XFAD)小鼠海马内肌动蛋白和钙调蛋白为基础的MSox物种形成,反映了神经炎症和氧化应激。同时检测氧化和未修饰肽,可以直接计算绝对氧化化学计量学和蛋白质归一化占用率──这是大多数翻译后修饰研究很少能达到的分析维度。我们的研究结果表明,氧化还原动力学与年龄有关,而与ad无关。在肌动蛋白中,D-loop Met44/47在3 - 6个月间从~ 9%下降到~ 5%,然后在9个月时上升到~ 14%,而H-loop Met269保持稳定在~ 5%的MSox。在钙调素中,连接子Met77随着年龄的增长而稳步上升(但不是AD),而c -叶Met145/146从3到9个月从20%急剧下降到8% MSox。这些发现强调了小鼠海马内肌动蛋白和钙调蛋白中动态的、与年龄相关的蛋氨酸氧化模式,可能与大脑发育和衰老有关。
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引用次数: 0
Proximity Labeling Reveals RNA-Binding Proteins Associating with the Human Mitochondrial Import Receptor TOMM20 接近标记揭示与人类线粒体输入受体TOMM20相关的rna结合蛋白。
IF 3.6 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-12-22 DOI: 10.1021/acs.jproteome.5c00905
Saira Akram, , , Katharina I. Zittlau, , , Karan Sharma, , , Julia C. Fitzgerald, , , Nisha Rafiq, , , Boris Maček, , and , Ralf-Peter Jansen*, 

The import of most mitochondrial proteins requires that their precursor proteins be bound by the peripheral receptor proteins TOM20, TOM22, and TOM70. Budding yeast TOM20 and TOM70 have been extensively studied regarding their interaction partners and recognized substrates; however, little data is available for metazoan cells. Using APEX2-based proximity labeling, we created association profiles for human TOMM20 and TOMM70 in HeLa cells. We focused particularly on their interactions with RNA-binding proteins (RBPs) because there is evidence of RNA association with the mitochondrial outer membrane (MOM) and of local translation at the mitochondrial surface, however, these processes are poorly understood. Our results demonstrate that several RBPs and translation factors preferentially associate with TOMM20 rather than TOMM70. These include SYNJ2BP, a previously identified membrane-bound RBP that binds and protects mRNA encoding mitochondrial proteins. Inhibiting translation with puromycin increased the association of these RBPs with TOMM20 compared to TOMM70. This suggests that TOMM20, but not TOMM70, may play a role in maintaining cellular homeostasis during translation stress by retaining protective RBPs and translation-related proteins at the MOM.

大多数线粒体蛋白的输入需要其前体蛋白与外周受体蛋白TOM20、TOM22和TOM70结合。出芽酵母TOM20和TOM70已被广泛研究其相互作用伙伴和公认的底物;然而,关于后生动物细胞的数据很少。使用基于apex2的接近标记,我们在HeLa细胞中创建了人类TOMM20和TOMM70的关联图谱。我们特别关注它们与RNA结合蛋白(rbp)的相互作用,因为有证据表明RNA与线粒体外膜(MOM)和线粒体表面的局部翻译相关,然而,这些过程知之甚少。我们的研究结果表明,一些rbp和翻译因子优先与TOMM20而不是TOMM70相关。其中包括SYNJ2BP,这是一种先前鉴定的膜结合RBP,它结合并保护编码线粒体蛋白的mRNA。与TOMM70相比,用嘌呤霉素抑制翻译增加了这些rbp与TOMM20的关联。这表明TOMM20,而不是TOMM70,可能通过在MOM处保留保护性rbp和翻译相关蛋白,在翻译胁迫期间维持细胞稳态中发挥作用。
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引用次数: 0
期刊
Journal of Proteome Research
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