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Analysis of antibiotic response in Clinical Wound Pseudomonas aeruginosa isolates: Unveiling Proteome Dynamics of tobramycin tolerant phenotype. 临床伤口铜绿假单胞菌分离物的抗生素反应分析:揭示妥布霉素耐受表型的蛋白质组动力学。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-16 DOI: 10.1016/j.mcpro.2024.100861
Kasandra Buchholtz, Rosa Jersie-Christensen, Karen Angeliki Krogfelt, Biljana Mojsoska

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen, causing serious chronic infections. P. aeruginosa can adapt efficiently to antibiotic stressors via different genotypic or phenotypic strategies such as resistance and tolerance. The adaptation regulatory system is not always very well understood. In this study, we use shotgun proteomics to investigate the system-level response to tobramycin in two clinical wound P. aeruginosa isolates and PAO1. We profiled each strain for its antibiotic drug-tolerant phenotype using supra-minimum inhibitory concentrations (supra-MIC) of tobramycin and applied proteomics to investigate the protein expression profiles. The MIC revealed that all isolates were susceptible to tobramycin but at supra-MIC concentrations at stationary growth, a degree of tolerance was observed for the isolates. We identified around 40 % of the total proteins encoded by the P. aeruginosa genome and highlighted shared and unique protein signatures for all isolates. Comparative proteome profiling in the absence of antibiotic treatment showed divergent fingerprints, despite similarities in the growth behavior of the isolates. In the presence of tobramycin, the isolates shared a common response in the downregulation of proteins involved in the two-component system, whereas stress response proteins were present at higher levels. Our findings provide insight into the use of proteomic tools to dissect the system-level response in clinical isolates in the absence and presence of antibiotic stress.

铜绿假单胞菌(P. aeruginosa)是一种机会性人类病原体,可导致严重的慢性感染。铜绿假单胞菌可通过不同的基因型或表型策略(如耐药性和耐受性)有效地适应抗生素压力源。但人们对其适应调控系统并不十分了解。在本研究中,我们利用枪式蛋白质组学研究了两株临床伤口铜绿假单胞菌分离株和 PAO1 对托布霉素的系统级响应。我们使用超最低抑菌浓度(supra-MIC)的妥布霉素分析了每个菌株的抗生素耐药表型,并应用蛋白质组学研究了蛋白质表达谱。最低抑菌浓度显示,所有分离菌株都对妥布霉素敏感,但在静止生长的超最低抑菌浓度下,分离菌株出现了一定程度的耐药性。我们鉴定了铜绿微囊藻基因组编码的全部蛋白质中的约 40%,并强调了所有分离菌株共有和特有的蛋白质特征。尽管铜绿微囊藻分离物的生长行为相似,但在未使用抗生素的情况下进行的蛋白质组比较分析却显示出不同的指纹图谱。在使用妥布霉素的情况下,分离物的共同反应是参与双组分系统的蛋白质下调,而应激反应蛋白质的水平更高。我们的研究结果为利用蛋白质组学工具剖析临床分离物在无抗生素胁迫和有抗生素胁迫时的系统级反应提供了启示。
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引用次数: 0
Mapping Start Codons of Small Open Reading Frames by N-Terminomics Approach. 通过 N-端组学方法绘制小型开放阅读框的起始密码子。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-16 DOI: 10.1016/j.mcpro.2024.100860
Mingbo Peng, Tianjing Wang, Yujie Li, Zheng Zhang, Cuihong Wan

sORF-encoded peptides (SEPs) refer to proteins encoded by small open reading frames (sORFs) with a length of less than 100 amino acids, which play an important role in various life activities. Analysis of known SEPs showed that using non-canonical initiation codons of SEPs was more common. However, the current analysis of SEP sequences mainly relies on bioinformatics prediction, and most of them use AUG as the start site, which may not be completely correct for SEPs. Chemical labeling was used to systematically analyze the N-terminal sequences of SEPs to accurately define the start sites of SEPs. By comparison, we found that dimethylation and guanidinylation are more efficient than acetylation. The ACN precipitation and heating precipitation performed better in SEP enrichment. As an N-terminal peptide enrichment material, Hexadhexaldehyde was superior to CNBr-activated agarose and NHS-activated agarose. Combining these methods, we identified 128 SEPs with 131 N-terminal sequences. Among them, two-thirds are novel N-terminal sequences, and most of them start from the 11-31st amino acids of the original sequence. Partial novel N-termini were produced by proteolysis or signal peptide removal. Some SEPs' transcription start sites were corrected to be non-AUG start codons. One novel start codon was validated using GFP-tag vectors. These results demonstrated that the chemical labeling approaches would be beneficial for identifying the start codons of sORFs and the real N-terminal of their encoded peptides, which helps better understand the characterization of SEPs.

sORF编码肽(SEPs)是指由长度小于100个氨基酸的小开放阅读框(sORFs)编码的蛋白质,它们在各种生命活动中发挥着重要作用。对已知 SEP 的分析表明,使用 SEP 的非规范起始密码子较为常见。然而,目前对SEP序列的分析主要依赖于生物信息学预测,且大多使用AUG作为起始位点,这对于SEP来说可能并不完全正确。我们采用化学标记法系统分析了SEPs的N端序列,以准确界定SEPs的起始位点。通过比较,我们发现二甲基化和鸟苷酸化比乙酰化更有效。ACN 沉淀和加热沉淀的 SEP 富集效果更好。作为 N 端多肽富集材料,六甲醛优于 CNBr 活化的琼脂糖和 NHS 活化的琼脂糖。结合这些方法,我们共鉴定出 128 个 SEPs,131 个 N 端序列。其中,三分之二是新的 N 端序列,它们大多从原始序列的第 11-31 个氨基酸开始。部分新型 N 端是通过蛋白水解或信号肽去除产生的。一些 SEP 的转录起始位点被修正为非 AUG 起始密码子。使用 GFP 标记载体对一个新的起始密码子进行了验证。这些结果表明,化学标记方法有助于鉴定 sORFs 的起始密码子及其编码肽的真正 N-末端,从而有助于更好地理解 SEPs 的特征。
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引用次数: 0
Integrative Omics Reveals the Metabolic Patterns During Oocyte Growth. 综合全息研究揭示了卵母细胞生长过程中的代谢模式。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-15 DOI: 10.1016/j.mcpro.2024.100862
Xiang Zhang, Juan Ge, Yue Wang, Minjian Chen, Xuejiang Guo, Shuai Zhu, Hui Wang, Qiang Wang

Well-controlled metabolism is associated with high-quality oocytes and optimal development of a healthy embryo. However, the metabolic framework that controls mammalian oocyte growth remains unknown. In the present study, we comprehensively depict the temporal metabolic dynamics of mouse oocytes during in vivo growth through the integrated analysis of metabolomics and proteomics. Many novel metabolic features are discovered during this process. Of note, glycolysis is enhanced, and oxidative phosphorylation capacity is reduced in the growing oocytes, presenting a Warburg-like metabolic program. For nucleotide biosynthesis, the salvage pathway is markedly activated during oocyte growth, whereas the de novo pathway is evidently suppressed. Fatty acid synthesis and channeling into phosphoinositides are specifically elevated in oocytes accompanying primordial follicle activation; nevertheless, fatty acid oxidation is reduced in these oocytes simultaneously. Our data establish the metabolic landscape during in vivo oocyte growth and serve as a broad resource for probing mammalian oocyte metabolism.

控制良好的新陈代谢与卵母细胞的高质量和健康胚胎的最佳发育有关。然而,控制哺乳动物卵母细胞生长的代谢框架仍然未知。在本研究中,我们通过代谢组学和蛋白质组学的综合分析,全面描述了小鼠卵母细胞在体内生长过程中的时间代谢动态。在这一过程中,我们发现了许多新的代谢特征。值得注意的是,生长中的卵母细胞糖酵解能力增强,氧化磷酸化能力降低,呈现出类似沃伯格的代谢程序。在核苷酸生物合成方面,卵母细胞生长过程中,挽救途径被明显激活,而新生途径则明显受到抑制。伴随原始卵泡激活的卵母细胞中脂肪酸合成和转化为磷酸肌酸的途径特别增加;然而,这些卵母细胞中的脂肪酸氧化同时减少。我们的数据确定了体内卵母细胞生长过程中的新陈代谢状况,为探究哺乳动物卵母细胞的新陈代谢提供了广泛的资源。
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引用次数: 0
Receptor Kinase Signaling of BRI1 and SIRK1 Is Tightly Balanced by Their Interactomes as Revealed From Domain-Swap Chimaera in AE-MS Approaches. AE-MS方法中的结构域交换嵌合体揭示了BRI1和SIRK1的受体激酶信号通过它们的相互作用组紧密平衡。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-15 DOI: 10.1016/j.mcpro.2024.100857
Lin Xi, Xuna Wu, Jiahui Wang, Zhaoxia Zhang, Mingjie He, Zeeshan Zeeshan, Thorsten Stefan, Waltraud X Schulze

At the plasma membrane, in response to biotic and abiotic cues, specific ligands initiate the formation of receptor kinase heterodimers, which regulate the activities of plasma membrane proteins and initiate signaling cascades to the nucleus. In this study, we utilized affinity enrichment mass spectrometry to investigate the stimulus-dependent interactomes of LRR receptor kinases in response to their respective ligands, with an emphasis on exploring structural influences and potential cross-talk events at the plasma membrane. BRI1 and SIRK1 were chosen as receptor kinases with distinct coreceptor preference. By using interactome characteristic of domain-swap chimera following a gradient boosting learning algorithm trained on SIRK1 and BRI1 interactomes, we attribute contributions of extracellular domain, transmembrane domain, juxtamembrane domain, and kinase domain of respective ligand-binding receptors to their interaction with their coreceptors and substrates. Our results revealed juxtamembrane domain as major structural element defining the specific substrate recruitment for BRI1 and extracellular domain for SIRK1. Furthermore, the learning algorithm enabled us to predict the phenotypic outcomes of chimeric receptors based on different domain combinations, which was verified by dedicated experiments. As a result, our work reveals a tightly controlled balance of signaling cascade activation dependent on ligand-binding receptors domains and the internal ligand status of the plant. Moreover, our study shows the robust utility of machine learning classification as a quantitative metric for studying dynamic interactomes, dissecting the contribution of specific domains and predicting their phenotypic outcome.

在质膜上,针对生物和非生物线索,特定配体会启动受体激酶异二聚体的形成,从而调节质膜蛋白的活性并启动通向细胞核的信号级联。在这项研究中,我们利用亲和富集质谱法(AE-MS)研究了 LRR 受体激酶对各自配体的刺激依赖性相互作用组,重点是探索质膜上的结构影响和潜在的交叉对话事件。BRI1 和 SIRK1 被选为具有不同核心受体偏好的受体激酶。通过使用SIRK1和BRI1相互作用组训练的梯度提升学习算法,利用结构域交换嵌合体的相互作用组特征,我们确定了各自配体结合受体的胞外结构域、跨膜结构域、并膜结构域和激酶结构域对它们与核心受体和底物相互作用的贡献。我们的研究结果表明,并膜结构域是决定 BRI1 和 SIRK1 特异性底物招募的主要结构元素,而细胞外结构域则是决定 SIRK1 特异性底物招募的主要结构元素。此外,学习算法使我们能够根据不同的结构域组合预测嵌合受体的表型结果,这一点已通过专门的实验得到验证。因此,我们的工作揭示了信号级联激活的严格控制平衡取决于配体结合受体结构域和植物内部配体状态。此外,我们的研究还表明,机器学习分类作为一种定量指标,在研究动态相互作用组、剖析特定结构域的贡献以及预测其表型结果方面具有强大的实用性。
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引用次数: 0
From Fringe to the Mainstream: How ETD MS brought O-GlcNAc to the masses. 从边缘到主流:ETD MS 如何让大众认识 O-GlcNAc。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-14 DOI: 10.1016/j.mcpro.2024.100859
Namrata D Udeshi, Gerald W Hart, Chad Slawson

O-GlcNAcylation was identified in the 1980s by Torres and Hart (1) and modifies thousands of cellular proteins, yet the regulatory role of O-GlcNAc is still poorly understood compared to the abundance of mechanistic information known for other cycling post-translational modifications like phosphorylation. Many challenges are associated with studying O-GlcNAcylation and are tied to the technical hurdles with analysis by mass spectrometry. Over the years, many research groups have developed important methods to study O-GlcNAcylation revealing its role in the cell, and this perspective aims to review the challenges and innovations around O-GlcNAc research and chronicle the work by Donald F. Hunt and his laboratory, particularly in development of ETD and its application to this field of research.

O-GlcNAcylation 于 20 世纪 80 年代由 Torres 和 Hart 发现(1),可修饰数千种细胞蛋白质,但与磷酸化等其他循环翻译后修饰的大量机理信息相比,人们对 O-GlcNAc 的调控作用仍然知之甚少。研究 O-GlcNAcylation 面临许多挑战,其中包括质谱分析的技术障碍。多年来,许多研究小组开发出了研究 O-GlcNAcylation 的重要方法,揭示了它在细胞中的作用。本视角旨在回顾围绕 O-GlcNAc 研究的挑战和创新,并记录 Donald F. Hunt 及其实验室的工作,特别是开发 ETD 及其在该研究领域的应用。
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引用次数: 0
Proteomic Characterization of 1000 Human and Murine Neutrophils Freshly Isolated From Blood and Sites of Sterile Inflammation. 从血液和无菌炎症部位新鲜分离的 1000 个人类和鼠类中性粒细胞的蛋白质组特征。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-11 DOI: 10.1016/j.mcpro.2024.100858
Susmita Ghosh, Ali Ata Tuz, Martin Stenzel, Vikramjeet Singh, Mathis Richter, Oliver Soehnlein, Emanuel Lange, Robert Heyer, Zülal Cibir, Alexander Beer, Marcel Jung, Dennis Nagel, Dirk M Hermann, Anja Hasenberg, Anika Grüneboom, Albert Sickmann, Matthias Gunzer

Neutrophils are indispensable for defense against pathogens. Injured tissue-infiltrated neutrophils can establish a niche of chronic inflammation and promote degeneration. Studies investigated transcriptome of single-infiltrated neutrophils which could misinterpret molecular states of these post mitotic cells. However, neutrophil proteome characterization has been challenging due to low harvests from affected tissues. Here, we present a workflow to obtain proteome of 1000 murine and human tissue-infiltrated neutrophils. We generated spectral libraries containing ∼6200 mouse and ∼5300 human proteins from circulating neutrophils. 4800 mouse and 3400 human proteins were recovered from 1000 cells with 102-108 copies/cell. Neutrophils from stroke-affected mouse brains adapted to the glucose-deprived environment with increased mitochondrial activity and ROS-production, while cells invading inflamed human oral cavities increased phagocytosis and granule release. We provide an extensive protein repository for resting human and mouse neutrophils, identify proteins lost in low input samples, thus enabling the proteomic characterization of limited tissue-infiltrated neutrophils.

中性粒细胞是抵御病原体不可或缺的物质。损伤组织浸润的中性粒细胞可建立慢性炎症的生态位并促进退化。研究调查了单个浸润中性粒细胞的转录组,这可能会误解这些有丝分裂后细胞的分子状态。然而,由于从受影响组织中获取的蛋白质较少,嗜中性粒细胞蛋白质组的表征一直具有挑战性。在这里,我们介绍了一种获取 1000 个鼠和人组织浸润中性粒细胞蛋白质组的工作流程。我们从循环中性粒细胞中生成了包含 6,200 个小鼠蛋白质和 5,300 个人类蛋白质的谱库。从每细胞 102-108 个拷贝的 1000 个细胞中回收了 4,800 个小鼠蛋白质和 3,400 个人类蛋白质。受中风影响的小鼠大脑中的中性粒细胞适应了葡萄糖缺乏的环境,线粒体活性和 ROS 生成增加,而侵入发炎的人类口腔的细胞吞噬和颗粒释放增加。我们为静息的人和小鼠中性粒细胞提供了一个广泛的蛋白质库,识别了在低输入样本中丢失的蛋白质,从而实现了对有限组织浸润的中性粒细胞进行蛋白质组学表征。
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引用次数: 0
Retinal Proteome Profiling of Inherited Retinal Degeneration Across Three Different Mouse Models Suggests Common Drug Targets in Retinitis Pigmentosa. 通过对三种不同小鼠模型的遗传性视网膜变性进行视网膜蛋白质组分析,发现了视网膜色素变性的共同药物靶点。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-09 DOI: 10.1016/j.mcpro.2024.100855
Ahmed B Montaser, Fangyuan Gao, Danielle Peters, Katri Vainionpää, Ning Zhibin, Dorota Skowronska-Krawczyk, Daniel Figeys, Krzysztof Palczewski, Henri Leinonen

Inherited retinal degenerations (IRDs) are a leading cause of blindness among the population of young people in the developed world. Approximately half of IRDs initially manifest as gradual loss of night vision and visual fields, characteristic of retinitis pigmentosa (RP). Due to challenges in genetic testing, and the large heterogeneity of mutations underlying RP, targeted gene therapies are an impractical largescale solution in the foreseeable future. For this reason, identifying key pathophysiological pathways in IRDs that could be targets for mutation-agnostic and disease-modifying therapies (DMTs) is warranted. In this study, we investigated the retinal proteome of three distinct IRD mouse models, in comparison to sex- and age-matched wild-type mice. Specifically, we used the Pde6βRd10 (rd10) and RhoP23H/WT (P23H) mouse models of autosomal recessive and autosomal dominant RP, respectively, as well as the Rpe65-/- mouse model of Leber's congenital amaurosis type 2 (LCA2). The mice were housed at two distinct institutions and analyzed using LC-MS in three separate facilities/instruments following data-dependent and data-independent acquisition modes. This cross-institutional and multi-methodological approach signifies the reliability and reproducibility of the results. The large-scale profiling of the retinal proteome, coupled with in vivo electroretinography recordings, provided us with a reliable basis for comparing the disease phenotypes and severity. Despite evident inflammation, cellular stress, and downscaled phototransduction observed consistently across all three models, the underlying pathologies of RP and LCA2 displayed many differences, sharing only four general KEGG pathways. The opposite is true for the two RP models in which we identify remarkable convergence in proteomic phenotype even though the mechanism of primary rod death in rd10 and P23H mice is different. Our data highlights the cAMP and cGMP second-messenger signaling pathways as potential targets for therapeutic intervention. The proteomic data is curated and made publicly available, facilitating the discovery of universal therapeutic targets for RP.

遗传性视网膜变性(IRDs)是发达国家年轻人失明的主要原因。大约一半的遗传性视网膜变性最初表现为夜视和视野逐渐丧失,这是视网膜色素变性(RP)的特征。由于基因检测方面的挑战以及 RP 基因突变的巨大异质性,在可预见的未来,大规模的靶向基因疗法是不切实际的。因此,有必要确定IRD的关键病理生理通路,以作为突变诊断和疾病改变疗法(DMT)的靶点。在本研究中,我们研究了三种不同 IRD 小鼠模型的视网膜蛋白质组,并与性别和年龄匹配的野生型小鼠进行了比较。具体来说,我们使用了 Pde6βRd10 (rd10) 和 RhoP23H/WT (P23H) 小鼠模型(分别为常染色体隐性和常染色体显性 RP),以及 Rpe65-/- 小鼠模型(Leber´s 先天性羊角疯 2 型 (LCA2))。小鼠分别饲养在两个不同的机构,并在三个不同的设施/仪器中使用 LC-MS 进行分析,采用数据依赖型和数据非依赖型采集模式。这种跨机构、多方法的研究方法标志着研究结果的可靠性和可重复性。大规模视网膜蛋白质组分析与体内视网膜电图记录相结合,为我们比较疾病表型和严重程度提供了可靠的依据。尽管在所有三个模型中都观察到了明显的炎症、细胞应激和光传导下调,但 RP 和 LCA2 的基本病理却显示出许多差异,只有四个通用 KEGG 通路是相同的。两种 RP 模型的情况恰恰相反,尽管 rd10 和 P23H 小鼠的初级杆状病毒死亡机制不同,但我们发现它们的蛋白质组表型有显著的趋同性。我们的数据强调了 cAMP 和 cGMP 第二信使信号通路是治疗干预的潜在靶点。我们对蛋白质组数据进行了整理并将其公开,这有助于发现RP的通用治疗靶点。
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引用次数: 0
Hydrogen/Deuterium Exchange Mass Spectrometry: Fundamentals, Limitations, and Opportunities. 氢/氘交换质谱法:基本原理、局限性和机遇。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-09 DOI: 10.1016/j.mcpro.2024.100853
Lars Konermann, Pablo M Scrosati

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) probes dynamic motions of proteins by monitoring the kinetics of backbone amide deuteration. Dynamic regions exhibit rapid HDX, while rigid segments are more protected. Current data readouts focus on qualitative comparative observations (such as "residues X to Y become more protected after protein exposure to ligand Z"). At present, it is not possible to decode HDX protection patterns in an atomistic fashion. In other words, the exact range of protein motions under a given set of conditions cannot be uncovered, leaving space for speculative interpretations. Amide back exchange is an under-appreciated problem, as the widely used (m-m0)/(m100-m0) correction method can distort HDX kinetic profiles. Future data analysis strategies require a better fundamental understanding of HDX events, going beyond the classical Linderstrøm-Lang model. Combined with experiments that offer enhanced spatial resolution and suppressed back exchange, it should become possible to uncover the exact range of motions exhibited by a protein under a given set of conditions. Such advances would provide a greatly improved understanding of protein behavior in health and disease.

氢/氘交换质谱(HDX-MS)通过监测骨架酰胺脱氘的动力学来探测蛋白质的动态运动。动态区域表现出快速的 HDX,而刚性部分则受到更多保护。目前的数据读取侧重于定性比较观察(如 "蛋白质暴露于配体 Z 后,X 至 Y 残基受到更多保护")。目前,还无法以原子论的方式解码 HDX 保护模式。换句话说,无法揭示特定条件下蛋白质运动的确切范围,这就为推测解释留下了空间。酰胺反向交换是一个未得到充分重视的问题,因为广泛使用的(m-m0)/(m100-m0)校正方法会扭曲 HDX 动力曲线。未来的数据分析策略需要从根本上更好地理解 HDX 事件,超越经典的林德斯特伦-朗(Linderstrøm-Lang)模型。结合提供更高的空间分辨率和抑制反向交换的实验,应该有可能发现蛋白质在特定条件下表现出的确切运动范围。这些进展将大大提高人们对蛋白质在健康和疾病中行为的理解。
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引用次数: 0
Proteomic characterization of ubiquitin carboxyl-terminal hydrolase 19 deficient cells reveals a role for USP19 in secretion of lysosomal proteins. 泛素羧基末端水解酶 19 缺陷细胞的蛋白质组学特征揭示了 USP19 在溶酶体蛋白分泌中的作用。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-08 DOI: 10.1016/j.mcpro.2024.100854
Simone Bonelli, Margot Lo Pinto, Yihong Ye, Stephan A Mueller, Stefan F Lichtenthaler, Simone D Scilabra

Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase (DUB), characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain that anchors them to the endoplasmic reticulum (ER). Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from ER-associated protein degradation (ERAD) in a catalytic-independent manner, promote autophagy and address proteins to lysosomal degradation via endosomal microautophagy. USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson's disease-associated protein α-synuclein. Despite mounting evidence that USP19 plays crucial roles in several biological processes, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by loss of USP19 to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired the release of several lysosomal proteins, including legumain (LGMN) and several cathepsins. In order to understand the underlaying mechanism, we dissected the USP19-regulated secretion of LGMN in several cell types. We found that LGMN was not a DUB substrate of USP19 and that its USP19-dependent release did not require their direct interaction. LGMN secretion occurred by a mechanism that involved the Golgi apparatus, autophagosome formation and lysosome function. This mechanism resembled the recently described "lysosomal exocytosis", by which lysosomal hydrolases are secreted, when ubiquitination of p62 is increased in cells lacking deubiquitinases such as USP15 and USP17. In conclusion, our proteomic characterization of USP19 has identified a collection of proteins in the secretome and within the cell that are regulated by USP19, which link USP19 to secretion of lysosomal proteins, including LGMN.

泛素羧基末端水解酶 19(USP19)是一种独特的去泛素化酶(DUB),其特点是通过替代剪接产生多个变体。有几个变体带有一个 C 端跨膜结构域,可将它们锚定在内质网(ER)上。除了通过防止蛋白酶体降解来调节蛋白质的稳定性外,USP19 还能以催化无关的方式将底物从 ER 相关蛋白质降解(ERAD)中解救出来,促进自噬,并通过内体微自噬将蛋白质送到溶酶体降解。USP19 是最近出现的一种蛋白质,它负责错误折叠蛋白(包括帕金森病相关蛋白 α-突触核蛋白)的非常规分泌。尽管越来越多的证据表明 USP19 在多个生物过程中发挥着关键作用,但由于缺乏有关 USP19 生理底物的信息,其基本机制尚不清楚。在这里,我们利用高分辨率定量蛋白质组学分析了USP19缺失诱导的分泌组和细胞蛋白质组的变化,以确定其分泌或周转受USP19调控的蛋白质。我们发现,消减 USP19 会引起细胞内外蛋白质组的显著变化。USP19 的缺失损害了几种溶酶体蛋白的释放,包括豆豆蛋白酶(LGMN)和几种酪蛋白。为了了解其基本机制,我们剖析了几种细胞类型中受 USP19 调节的 LGMN 分泌。我们发现 LGMN 并非 USP19 的 DUB 底物,其释放不需要 USP19 的直接作用。LGMN 的分泌机制涉及高尔基体、自噬体的形成和溶酶体的功能。这种机制类似于最近描述的 "溶酶体外泌",当缺乏 USP15 和 USP17 等去泛素化酶的细胞中 p62 泛素化增加时,溶酶体水解酶就会分泌。总之,我们对 USP19 进行的蛋白质组学表征确定了分泌组和细胞内一系列受 USP19 调节的蛋白质,这些蛋白质将 USP19 与溶酶体蛋白质(包括 LGMN)的分泌联系起来。
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引用次数: 0
Profiling Cullin4-E3 ligases interactomes and their rewiring in influenza A virus infection. 剖析Cullin4-E3连接酶相互作用组及其在甲型流感病毒感染中的重新布线。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-07 DOI: 10.1016/j.mcpro.2024.100856
Guillaume Dugied, Thibaut Douche, Melanie Dos Santos, Quentin Giai Gianetto Q, Camille Cassonnet, Françoise Vuillier, Patricia Cassonnet, Yves Jacob, Sylvie van der Werf, Anastassia Komarova, Mariette Matondo, Marwah Karim, Caroline Demeret

Understanding the integrated regulation of cellular processes during viral infection is crucial for developing host-targeted approaches. We have previously reported that an optimal in vitro infection by influenza A (IAV) requires three components of Cullin 4-RING E3 ubiquitin ligases (CRL4) complexes, namely the DDB1 adaptor and two Substrate Recognition Factors (SRF), DCAF11 and DCAF12L1, which mediate non-degradative poly-ubiquitination of the PB2 subunit of the viral polymerase. However, the impact of IAV infection on the CRL4 interactome remains elusive. Here, using Affinity Purification coupled with Mass Spectrometry (AP-MS) approaches, we identified cellular proteins interacting with these CRL4 components in IAV-infected and non-infected contexts. IAV infection induces significant modulations in protein interactions, resulting in a global loss of DDB1 and DCAF11 interactions, and an increase in DCAF12L1-associated proteins. The distinct rewiring of CRL4's associations upon infection impacted cellular proteins involved in protein folding, ubiquitination, translation, splicing, and stress responses. Using a split-nanoluciferase-based assay, we identified direct partners of CRL4 components and via siRNA-mediated silencing validated their role in IAV infection, representing potential substrates or regulators of CRL4 complexes. Our findings unravel the dynamic remodeling of the proteomic landscape of CRL4's E3 ubiquitin ligases during IAV infection, likely involved in shaping a cellular environment conducive to viral replication and offer potential for the exploration of future host-targeted antiviral therapeutic strategies.

了解病毒感染过程中细胞过程的综合调控对于开发针对宿主的方法至关重要。我们以前曾报道,甲型流感(IAV)的最佳体外感染需要Cullin 4-RING E3泛素连接酶(CRL4)复合物的三个组分,即DDB1适配体和两个底物识别因子(SRF)DCAF11和DCAF12L1,它们介导病毒聚合酶PB2亚基的非降解性多泛素化。然而,IAV感染对CRL4相互作用组的影响仍然难以捉摸。在这里,我们利用亲和纯化与质谱联用(AP-MS)方法,鉴定了在感染 IAV 和未感染 IAV 的情况下与这些 CRL4 成分相互作用的细胞蛋白。IAV 感染会引起蛋白质相互作用的显著改变,导致 DDB1 和 DCAF11 相互作用的全面丧失,以及 DCAF12L1 相关蛋白质的增加。感染后,CRL4关联的独特重构影响了参与蛋白质折叠、泛素化、翻译、剪接和应激反应的细胞蛋白质。利用基于分体荧光素酶的检测方法,我们确定了 CRL4 成分的直接伙伴,并通过 siRNA 介导的沉默验证了它们在 IAV 感染中的作用,它们代表了 CRL4 复合物的潜在底物或调节因子。我们的研究结果揭示了在 IAV 感染过程中 CRL4 的 E3 泛素连接酶蛋白质组格局的动态重塑,这可能参与了有利于病毒复制的细胞环境的形成,并为探索未来宿主靶向抗病毒治疗策略提供了潜力。
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Molecular & Cellular Proteomics
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