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Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling. 评估小分子抑制剂马利司他对蛇毒毒素的靶向特异性:热蛋白质组图谱分析的新应用。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-06-01 Epub Date: 2024-04-27 DOI: 10.1016/j.mcpro.2024.100779
Cara F Smith, Cassandra M Modahl, David Ceja Galindo, Keira Y Larson, Sean P Maroney, Lilyrose Bahrabadi, Nicklaus P Brandehoff, Blair W Perry, Maxwell C McCabe, Daniel Petras, Bruno Lomonte, Juan J Calvete, Todd A Castoe, Stephen P Mackessy, Kirk C Hansen, Anthony J Saviola

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

规避传统抗蛇毒血清疗法缺陷的新疗法对于解决全球蛇咬伤问题至关重要。许多蛇毒毒素抑制剂已在体内和体外显示出对具有重要医疗意义的蛇毒毒素具有良好的跨物种中和作用。开发用于筛选此类抑制剂的高通量方法可加快抑制剂的鉴定、测试和应用,从而为改善全球蛇毒中毒的治疗和结果带来令人兴奋的潜力。基于能量学的蛋白质组学方法,包括热蛋白质组剖析(TPP)和蛋白质组整体溶解度改变(PISA)测定,代表了高通量、全蛋白质组鉴定药物靶点和配体的 "深度蛋白质组学 "方法。在下面的研究中,我们应用 TPP 和 PISA 方法描述了西部菱背响尾蛇(Crotalus atrox)的毒液毒素蛋白形式与蛇毒金属蛋白酶(SVMP)抑制剂马利司他之间的相互作用。我们研究了它对整个毒液蛋白体的影响,并描述了它与特定 SVMP 蛋白形式的相互作用,以及它对非 SVMP 毒液毒素家族的潜在靶向作用。我们还比较了使用两种浓度抑制剂的 PISA 热窗口和可溶性上清液与不溶性沉淀的性能,首次证明了基于灵敏的高通量 PISA 方法在评估蛇毒小分子抑制剂的直接靶标方面的实用性。
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引用次数: 0
Proteome, Lysine Acetylome, and Succinylome Identify Posttranslational Modification of STAT1 as a Novel Drug Target in Silicosis. 蛋白质组、赖氨酸乙酰基组和琥珀酰基组将 STAT1 的翻译后修饰确定为矽肺病的新型药物靶点。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-06-01 Epub Date: 2024-04-17 DOI: 10.1016/j.mcpro.2024.100770
Tiantian Zhang, Yiyang Wang, Youliang Sun, Meiyue Song, Junling Pang, Mingyao Wang, Zhe Zhang, Peiran Yang, Yiling Chen, Xianmei Qi, Huan Zhou, Zhenzong Han, Yanjiang Xing, Ying Liu, Baicun Li, Jiangfeng Liu, Juntao Yang, Jing Wang

Inhalation of crystalline silica dust induces incurable lung damage, silicosis, and pulmonary fibrosis. However, the mechanisms of the lung injury remain poorly understood, with limited therapeutic options aside from lung transplantation. Posttranslational modifications can regulate the function of proteins and play an important role in studying disease mechanisms. To investigate changes in posttranslational modifications of proteins in silicosis, combined quantitative proteome, acetylome, and succinylome analyses were performed with lung tissues from silica-injured and healthy mice using liquid chromatography-mass spectrometry. Combined analysis was applied to the three omics datasets to construct a protein landscape. The acetylation and succinylation of the key transcription factor STAT1 were found to play important roles in the silica-induced pathophysiological changes. Modulating the acetylation level of STAT1 with geranylgeranylacetone effectively inhibited the progression of silicosis. This report revealed a comprehensive landscape of posttranslational modifications in silica-injured mouse and presented a novel therapeutic strategy targeting the posttranslational level for silica-induced lung diseases.

吸入结晶二氧化硅粉尘会诱发不可治愈的肺损伤、矽肺和肺纤维化。然而,人们对肺损伤的机制仍然知之甚少,除了肺移植外,治疗方法也很有限。翻译后修饰可以调节蛋白质的功能,在研究疾病机理方面发挥着重要作用。为了研究矽肺病蛋白质翻译后修饰的变化,研究人员利用液相色谱-质谱联用技术对矽肺损伤小鼠和健康小鼠的肺组织进行了蛋白质组、乙酰组和琥珀酰组的定量分析。对这三个全息数据集进行了综合分析,以构建蛋白质图谱。研究发现,关键转录因子STAT1的乙酰化和琥珀酰化在二氧化硅诱导的病理生理变化中发挥了重要作用。用香叶基丙酮(GGA)调节STAT1的乙酰化水平可有效抑制矽肺的发展。该报告揭示了矽损伤小鼠体内翻译后修饰的全面情况,提出了针对矽诱导的肺部疾病的翻译后水平的新型治疗策略。
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引用次数: 0
Exploring the Early Molecular Pathogenesis of Osteoarthritis Using Differential Network Analysis of Human Synovial Fluid. 利用人体滑液的差异网络分析探索骨关节炎的早期分子发病机制。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-06-01 Epub Date: 2024-05-14 DOI: 10.1016/j.mcpro.2024.100785
Martin Rydén, Amanda Sjögren, Patrik Önnerfjord, Aleksandra Turkiewicz, Jon Tjörnstrand, Martin Englund, Neserin Ali

The molecular mechanisms that drive the onset and development of osteoarthritis (OA) remain largely unknown. In this exploratory study, we used a proteomic platform (SOMAscan assay) to measure the relative abundance of more than 6000 proteins in synovial fluid (SF) from knees of human donors with healthy or mildly degenerated tissues, and knees with late-stage OA from patients undergoing knee replacement surgery. Using a linear mixed effects model, we estimated the differential abundance of 6251 proteins between the three groups. We found 583 proteins upregulated in the late-stage OA, including MMP1, collagenase 3 and interleukin-6. Further, we selected 760 proteins (800 aptamers) based on absolute fold changes between the healthy and mild degeneration groups. To those, we applied Gaussian Graphical Models (GGMs) to analyze the conditional dependence of proteins and to identify key proteins and subnetworks involved in early OA pathogenesis. After regularization and stability selection, we identified 102 proteins involved in GGM networks. Notably, network complexity was lost in the protein graph for mild degeneration when compared to controls, suggesting a disruption in the regular protein interplay. Furthermore, among our main findings were several downregulated (in mild degeneration versus healthy) proteins with unique interactions in the healthy group, one of which, SLCO5A1, has not previously been associated with OA. Our results suggest that this protein is important for healthy joint function. Further, our data suggests that SF proteomics, combined with GGMs, can reveal novel insights into the molecular pathogenesis and identification of biomarker candidates for early-stage OA.

驱动骨关节炎(OA)发病和发展的分子机制在很大程度上仍不为人所知。在这项探索性研究中,我们使用蛋白质组学平台(SOMAscan 检测法)测量了健康或轻度退化组织的人体供体膝关节滑液(SF)和接受膝关节置换手术的晚期 OA 患者膝关节滑液(SF)中 6000 多种蛋白质的相对丰度。利用线性混合效应模型,我们估算了三组之间 6251 个蛋白质的丰度差异。我们发现有 583 个蛋白质在晚期 OA 中上调,包括 MMP1、MMP13 和 IL6。此外,我们还根据健康组和轻度退行性病变组之间的绝对折叠变化选取了 760 个蛋白质(800 个适配体)。对此,我们应用高斯图形模型(GGMs)分析了蛋白质的条件依赖性,并确定了参与早期OA发病机制的关键蛋白质和子网络。经过正则化和稳定性选择,我们确定了 102 个参与 GGM 网络的蛋白质。值得注意的是,与对照组相比,轻度退行性病变的蛋白质图失去了网络的复杂性,这表明有规律的蛋白质相互作用被破坏了。此外,我们的主要发现还包括几个在健康组中具有独特相互作用的下调蛋白(在轻度退化与健康组中),其中一个蛋白 SLCO5A1 以前从未与 OA 相关。我们的研究结果表明,这种蛋白质对健康关节功能非常重要。此外,我们的数据还表明,SF 蛋白组学结合 GGMs 可以揭示分子发病机制的新见解,并确定早期 OA 的候选生物标志物。
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引用次数: 0
Replisome Proximal Protein Associations and Dynamic Proteomic Changes at Stalled Replication Forks. 复制体近端蛋白关联和停滞复制叉的动态蛋白质组变化
IF 7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-04-13 DOI: 10.1016/j.mcpro.2024.100767
Carla-Marie Jurkovic, Jennifer Raisch, Stephanie Tran, Hoang Dong Nguyen, Dominique Lévesque, Michelle S Scott, Eric I Campos, François-Michel Boisvert

DNA replication is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place in S phase and requires a dynamic and highly coordinated recruitment of multiple proteins at replication forks. Various genotoxic stressors lead to fork instability and collapse, hence the need for DNA repair pathways. By identifying the multitude of protein interactions implicated in those events, we can better grasp the complex and dynamic molecular mechanisms that facilitate DNA replication and repair. Proximity-dependent biotin identification was used to identify associations with 17 proteins within four core replication components, namely the CDC45/MCM2-7/GINS helicase that unwinds DNA, the DNA polymerases, replication protein A subunits, and histone chaperones needed to disassemble and reassemble chromatin. We further investigated the impact of genotoxic stress on these interactions. This analysis revealed a vast proximity association network with 108 nuclear proteins further modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, hydroxyurea treatment also caused a redistribution of associations with 11 interactors, meaning that the replisome is dynamically reorganized when stressed. The analysis identified several poorly characterized proteins, thereby uncovering new putative players in the cellular response to DNA replication arrest. It also provides a new comprehensive proteomic framework to understand how cells respond to obstacles during DNA replication.

DNA 复制是细胞分裂过程中确保遗传信息传递的基本细胞过程。基因组复制发生在 S 期,需要在复制叉上动态、高度协调地招募多种蛋白质。各种基因毒性应激源会导致分叉不稳定和崩溃,因此需要 DNA 修复途径。通过识别这些事件中涉及的多种蛋白质相互作用,我们可以更好地掌握促进 DNA 复制和修复的复杂而动态的分子机制。我们利用亲近依赖性生物素鉴定(BioID)技术鉴定了四种核心复制元件中 17 种蛋白质的关联,这四种核心复制元件分别是拆分 DNA 的 CDC45/MCM2-7/GINS (CMG) 螺旋酶、DNA 聚合酶、复制蛋白 A 亚基以及拆分和重新组装染色质所需的组蛋白伴侣。我们进一步研究了基因毒性应激对这些相互作用的影响。这项分析表明,在羟基脲存在的情况下,有108个核蛋白进一步调节了庞大的邻近关联网络;其中45个核蛋白富集,63个核蛋白减少。有趣的是,羟基脲处理还导致与 11 个相互作用者的关联重新分布,这意味着复制体在受压时会发生动态重组。这项分析发现了几种特征不清的蛋白质,从而揭示了细胞对 DNA 复制停滞反应的新的假定参与者。它还提供了一个新的综合蛋白质组框架,以了解细胞如何应对 DNA 复制过程中的障碍。
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引用次数: 0
TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics. TIMAHAC:简化串联 IMAC-HILIC 工作流程,实现同步高通量植物磷蛋白组学和 N-糖蛋白组学。
IF 7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-04-11 DOI: 10.1016/j.mcpro.2024.100762
Chin-Wen Chen, Pei-Yi Lin, Ying-Mi Lai, Miao-Hsia Lin, Shu-Yu Lin, Chuan-Chih Hsu

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.

蛋白质翻译后修饰(PTM)在植物细胞过程中至关重要,尤其是在蛋白质折叠和信号转导过程中。N-糖基化和磷酸化是特别重要的 PTM,在调节植物对环境刺激的反应中起着至关重要的作用。然而,目前同时分析磷酸化蛋白质组和 N-糖蛋白组的序列富集方法耗费大量人力和时间,限制了其通量。为了应对这一挑战,本研究引入了一种新型串联 S-Trap-IMAC-HILIC(S-Trap:悬浮捕集;IMAC:固定金属离子亲和层析;HILIC:亲水作用层析)策略,称为 TIMAHAC,用于同时分析植物磷酸蛋白组和 N-糖蛋白组。这种方法将 IMAC 和 HILIC 集成到串联尖端格式中,简化了磷酸肽和 N-糖肽的富集过程。其创新之处在于使用了统一的缓冲液系统和优化的富集序列,从而提高了效率和可重复性。通过分析拟南芥磷酸蛋白质组和N-糖蛋白组对脱落酸(ABA)处理的反应,证明了TIMAHAC的适用性。从拟南芥中鉴定出了多达 1,954 个 N-糖肽和 11,255 个磷酸肽,这表明该方法适用于植物组织。值得注意的是,在磷酸蛋白组和 N-糖蛋白组中观察到了不同的扰动模式,这表明它们对 ABA 反应有独特的贡献。我们的研究结果表明,TIMAHAC 为研究植物生物学中复杂的调控机制和 PTM 相互作用提供了一种全面的方法,为深入研究植物信号网络铺平了道路。
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引用次数: 0
Assessment of Kinome-Wide Activity Remodeling upon Picornavirus Infection. 评估皮卡病毒感染时整个基因组的活动重塑
IF 7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-03-30 DOI: 10.1016/j.mcpro.2024.100757
Tim S Veth, Lonneke V Nouwen, Marleen Zwaagstra, Heyrhyoung Lyoo, Kathryn A Wierenga, Bart Westendorp, Maarten A F M Altelaar, Celia Berkers, Frank J M van Kuppeveld, Albert J R Heck

Picornaviridae represent a large family of single-stranded positive RNA viruses of which different members can infect both humans and animals. These include the enteroviruses (e.g., poliovirus, coxsackievirus, and rhinoviruses) as well as the cardioviruses (e.g., encephalomyocarditis virus). Picornaviruses have evolved to interact with, use, and/or evade cellular host systems to create the optimal environment for replication and spreading. It is known that viruses modify kinase activity during infection, but a proteome-wide overview of the (de)regulation of cellular kinases during picornavirus infection is lacking. To study the kinase activity landscape during picornavirus infection, we here applied dedicated targeted mass spectrometry-based assays covering ∼40% of the human kinome. Our data show that upon infection, kinases of the MAPK pathways become activated (e.g., ERK1/2, RSK1/2, JNK1/2/3, and p38), while kinases involved in regulating the cell cycle (e.g., CDK1/2, GWL, and DYRK3) become inactivated. Additionally, we observed the activation of CHK2, an important kinase involved in the DNA damage response. Using pharmacological kinase inhibitors, we demonstrate that several of these activated kinases are essential for the replication of encephalomyocarditis virus. Altogether, the data provide a quantitative understanding of the regulation of kinome activity induced by picornavirus infection, providing a resource important for developing novel antiviral therapeutic interventions.

脊髓灰质炎病毒科(Picornaviridae)是一个庞大的单链正RNA病毒家族,其不同成员可感染人类和动物。这些病毒包括肠道病毒(如脊髓灰质炎病毒、柯萨奇病毒和鼻病毒)以及心道病毒(如脑心肌炎病毒 (EMCV))。皮卡病毒在进化过程中与细胞宿主系统相互作用,利用和/或逃避细胞宿主系统,为复制和传播创造最佳环境。众所周知,病毒在感染过程中会改变激酶的活性,但目前还缺乏关于皮卡病毒感染过程中细胞激酶(去)调控的蛋白质组概览。为了研究皮卡病毒感染期间的激酶活性状况,我们在此采用了基于质谱的专用靶向检测方法,覆盖了人类激酶组的 40%。我们的数据显示,感染后,MAPK 通路的激酶会被激活(如 ERK1/2、RSK1/2、JNK1/2/3、p38),而参与调节细胞周期的激酶(如 CDK1/2、GWL、DYRK3)则会失活。此外,我们还观察到参与 DNA 损伤反应的重要激酶 CHK2 被激活。通过使用药理激酶抑制剂,我们证明了这些被激活的激酶中,有几个对 EMCV 的复制至关重要。总之,这些数据提供了对皮卡病毒感染诱导的激酶组活性调控的定量理解,为开发新型抗病毒治疗干预措施提供了重要资源。
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引用次数: 0
Dynamic Proteomic Changes in Tumor and Immune Organs Reveal Systemic Immune Response to Tumor Development. 肿瘤和免疫器官的动态蛋白质组变化揭示了肿瘤发生的全身免疫反应。
IF 7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-03-28 DOI: 10.1016/j.mcpro.2024.100756
Zhike Li, Shuwen Liu, Zhouyong Gao, Linlin Ji, Jiaqi Jiao, Nairen Zheng, Xianju Li, Guangshun Wang, Jun Qin, Yi Wang

In orthotopic mouse tumor models, tumor progression is a complex process, involving interactions among tumor cells, host cell-derived stromal cells, and immune cells. Much attention has been focused on the tumor and its tumor microenvironment, while the host's macroenvironment including immune organs in response to tumorigenesis is poorly understood. Here, we report a temporal proteomic analysis on a subcutaneous tumor and three immune organs (LN, MLN, and spleen) collected on Days 0, 3, 7, 10, 14, and 21 after inoculation of mouse forestomach cancer cells in a syngeneic mouse model. Bioinformatics analysis identified key biological processes during distinct tumor development phases, including an initial acute immune response, the attack by the host immune system, followed by the adaptive immune activation, and the build-up of extracellular matrix. Proteomic changes in LN and spleen largely recapitulated the dynamics of the immune response in the tumor, consistent with an acute defense response on D3, adaptive immune response on D10, and immune evasion by D21. In contrast, the immune response in MLN showed a gradual and sustained activation, suggesting a delayed response from a distal immune organ. Combined analyses of tumors and host immune organs allowed the identification of potential therapeutic targets. A proof-of-concept experiment demonstrated that significant growth reduction can be achieved by dual inhibition of MEK and DDR2. Together, our temporal proteomic dataset of tumors and immune organs provides a useful resource for understanding the interaction between tumors and the immune system and has the potential for identifying new therapeutic targets for cancer treatment.

在正位小鼠肿瘤模型中,肿瘤进展是一个复杂的过程,涉及肿瘤细胞、宿主细胞衍生的基质细胞和免疫细胞之间的相互作用。人们的注意力主要集中在肿瘤及其肿瘤微环境上,而对宿主的大环境(包括免疫器官)在肿瘤发生过程中的反应却知之甚少。在这里,我们报告了在共生小鼠模型中接种小鼠森林胃癌(MFC)细胞后的第 0、3、7、10、14 和 21 天收集的皮下肿瘤和三个免疫器官(LN、MLN 和脾脏)的时间蛋白质组分析。生物信息学分析确定了不同肿瘤发生阶段的关键生物过程,包括最初的急性免疫反应、宿主免疫系统的攻击、随后的适应性免疫激活和细胞外基质的形成。LN和脾脏的蛋白质组变化在很大程度上再现了肿瘤免疫反应的动态变化,与D3的急性防御反应、D10的适应性免疫反应和D21的免疫逃避相一致。与此相反,MLN 中的免疫反应表现出逐渐和持续的激活,这表明来自远端免疫器官的反应是延迟的。通过对肿瘤和宿主免疫器官的综合分析,可以确定潜在的治疗靶点。一项概念验证实验表明,通过对 MEK 和 DDR2 的双重抑制,可以显著减少肿瘤的生长。我们的肿瘤和免疫器官时空蛋白质组数据集为了解肿瘤和免疫系统之间的相互作用提供了有用的资源,并有可能为癌症治疗确定新的治疗靶点。
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引用次数: 0
Proteomics Impact on Cell Biology to Resolve Cell Structure and Function. 蛋白质组学对细胞生物学的影响,以解析细胞结构和功能。
IF 7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-04-02 DOI: 10.1016/j.mcpro.2024.100758
John J M Bergeron

The acceleration of advances in proteomics has enabled integration with imaging at the EM and light microscopy levels, cryo-EM of protein structures, and artificial intelligence with proteins comprehensively and accurately resolved for cell structures at nanometer to subnanometer resolution. Proteomics continues to outpace experimentally based structural imaging, but their ultimate integration is a path toward the goal of a compendium of all proteins to understand mechanistically cell structure and function.

蛋白质组学的加速发展使其能够与电磁显微镜和光学显微镜成像、蛋白质结构的冷冻电镜以及人工智能相结合,以纳米到亚纳米分辨率全面准确地解析细胞结构中的蛋白质。蛋白质组学的发展速度继续超过以实验为基础的结构成像,但它们的最终整合是实现所有蛋白质汇编目标的必经之路,从而从机理上了解细胞的结构和功能。
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引用次数: 0
Galectin-4 Antimicrobial Activity Primarily Occurs Through its C-Terminal Domain. Galectin-4 的抗菌活性主要通过其 C 端结构域产生。
IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-03-13 DOI: 10.1016/j.mcpro.2024.100747
Hau-Ming Jan, Shang-Chuen Wu, Carter J Stowell, Mary L Vallecillo-Zúniga, Anu Paul, Kashyap R Patel, Sasikala Muthusamy, Hsien-Ya Lin, Diyoly Ayona, Ryan Philip Jajosky, Samata P Varadkar, Hirotomo Nakahara, Rita Chan, Devika Bhave, William J Lane, Melissa Y Yeung, Marie A Hollenhorst, Seth Rakoff-Nahoum, Richard D Cummings, Connie M Arthur, Sean R Stowell

Although immune tolerance evolved to reduce reactivity with self, it creates a gap in the adaptive immune response against microbes that decorate themselves in self-like antigens. This is particularly apparent with carbohydrate-based blood group antigens, wherein microbes can envelope themselves in blood group structures similar to human cells. In this study, we demonstrate that the innate immune lectin, galectin-4 (Gal-4), exhibits strain-specific binding and killing behavior towards microbes that display blood group-like antigens. Examination of binding preferences using a combination of microarrays populated with ABO(H) glycans and a variety of microbial strains, including those that express blood group-like antigens, demonstrated that Gal-4 binds mammalian and microbial antigens that have features of blood group and mammalian-like structures. Although Gal-4 was thought to exist as a monomer that achieves functional bivalency through its two linked carbohydrate recognition domains, our data demonstrate that Gal-4 forms dimers and that differences in the intrinsic ability of each domain to dimerize likely influences binding affinity. While each Gal-4 domain exhibited blood group-binding activity, the C-terminal domain (Gal-4C) exhibited dimeric properties, while the N-terminal domain (Gal-4N) failed to similarly display dimeric activity. Gal-4C not only exhibited the ability to dimerize but also possessed higher affinity toward ABO(H) blood group antigens and microbes expressing glycans with blood group-like features. Furthermore, when compared to Gal-4N, Gal-4C exhibited more potent antimicrobial activity. Even in the context of the full-length protein, where Gal-4N is functionally bivalent by virtue of Gal-4C dimerization, Gal-4C continued to display higher antimicrobial activity. These results demonstrate that Gal-4 exists as a dimer and exhibits its antimicrobial activity primarily through its C-terminal domain. In doing so, these data provide important insight into key features of Gal-4 responsible for its innate immune activity against molecular mimicry.

虽然免疫耐受的进化是为了降低与自身的反应性,但它却造成了适应性免疫反应的缺失,无法抵御用类似自身的抗原装饰自身的微生物。这一点在以碳水化合物为基础的血型抗原上表现得尤为明显,微生物可将自身包裹在与人体细胞相似的血型结构中。在这项研究中,我们证明了先天性免疫凝集素--galectin-4(Gal-4)对显示血型类抗原的微生物具有菌株特异性结合和杀伤行为。通过结合使用 ABO(H)聚糖的微阵列和多种微生物菌株(包括表达血型样抗原的微生物菌株)对其结合偏好的研究表明,Gal-4 能结合具有血型和哺乳动物样结构特征的哺乳动物和微生物抗原。尽管人们认为 Gal-4 是作为单体存在的,通过其两个相连的碳水化合物识别结构域(CRDs)实现功能上的双价性,但我们的数据表明 Gal-4 形成了二聚体,而且每个结构域二聚化的内在能力差异可能会影响结合亲和力。虽然每个 Gal-4 结构域都表现出血型结合活性,但 C 端结构域(Gal-4C)表现出二聚体特性,而 N 端结构域(Gal-4N)未能表现出类似的二聚体活性。Gal-4C 不仅具有二聚化能力,而且对 ABO(H)血型抗原和表达具有血型相似特征的聚糖的微生物具有更高的亲和力。此外,与 Gal-4N 相比,Gal-4C 表现出更强的抗菌活性。即使在全长蛋白质中,Gal-4N 通过 Gal-4C 二聚化而具有二价功能,Gal-4C 仍然显示出更高的抗菌活性。这些结果表明,Gal-4 是以二聚体形式存在的,主要通过其 C 端结构域表现出抗菌活性。因此,这些数据为我们深入了解 Gal-4 对抗分子拟态的先天免疫活性的关键特征提供了重要依据。
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引用次数: 0
Corrigendum to "Red Blood Cells Protein Profile Is Modified in Breast Cancer Patients". 乳腺癌患者的红细胞蛋白谱发生改变 "的更正。
IF 7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-05-11 DOI: 10.1016/j.mcpro.2024.100774
Thais Pereira-Veiga, Susana Bravo, Antonio Gómez-Tato, Celso Yáñez-Gómez, Carmen Abuín, Vanesa Varela, Juan Cueva, Patricia Palacios, Ana B Dávila-Ibáñez, Roberto Piñeiro, Ana Vilar, María Del Pilar Chantada-Vázquez, Rafael López-López, Clotilde Costa
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引用次数: 0
期刊
Molecular & Cellular Proteomics
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