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The two major splice variants of scavenger receptor BI differ by their interactions with lipoproteins and cellular localization in endothelial cells. 清道夫受体 BI 的两种主要剪接变体在与脂蛋白的相互作用以及在内皮细胞中的细胞定位方面存在差异。
IF 5 2区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-01 Epub Date: 2024-10-10 DOI: 10.1016/j.jlr.2024.100665
Anton Potapenko, Kathrin Frey, Eveline Schlumpf, Jérôme Robert, Bernd Wollscheid, Arnold von Eckardstein, Lucia Rohrer

The scavenger receptor BI (SR-BI) facilitates the transport of both HDL and LDL through endothelial cells. Its two splice variants, SR-BIvar1 and SR-BIvar2, differ in their carboxy terminal domains. Only SR-BIvar1 contains the putative binding sites for the adapter proteins PDZ domain containing protein 1 (PDZK1) and dedicator of cytokinesis 4 (DOCK4), which limit the cell surface abundance and internalization of the receptor. To investigate the cellular localization of the SR-BI variants and their interaction with lipoproteins in endothelial cells, EA.hy926 cells were stably transfected with vectors encoding untagged, GFP- or mCherry-tagged constructs of the two SR-BI variants. Additionally, the cells were transfected with shRNAs against PDZK1 or DOCK4. Microscopy investigation showed that SR-BIvar1 was predominantly localized on the cell surface together with clathrin whereas SR-BIvar2 was absent from the cell surface but retrieved in endosomes and lysosomes. Accordingly, only SR-BIvar1 increased lipoprotein binding to endothelial while HDL and LDL uptake were enhanced by both variants. Silencing of PDZK1 or DOCK4 only reduced HDL association in SR-BIvar2 overexpressing cells while LDL association was reduced both in WT and SR-BIvar2 overexpressing cells. In conclusion, either SR-BI variant facilitates the uptake of HDL and LDL into endothelial cells, however by different mechanisms and trafficking routes. This dual role may explain why the loss of DOCK4 or PDZK1 differently affects the uptake of HDL and LDL in different endothelial cells.

清道夫受体 SR-BI 可促进高密度脂蛋白和低密度脂蛋白通过内皮细胞的转运。它的两个剪接变体 SR-BIvar1 和 SR-BIvar2 在羧基末端结构域上有所不同。只有 SR-BIvar1 中的一个包含适配蛋白 PDZK1 和 DOCK4 的假定结合位点,这限制了受体在细胞表面的丰度和内化。为了研究SR-BI变体的细胞定位及其在内皮细胞中与脂蛋白的相互作用,用编码两种SR-BI变体的无标记、GFP或mCherry标记构建体的载体稳定转染EA.hy926细胞。此外,细胞还转染了针对 PDZK1 或 DOCK4 的 shRNA。显微镜调查显示,SR-BIvar1主要与凝集素一起定位于细胞表面,而SR-BIvar2则不在细胞表面,而是在内质体和溶酶体中。因此,只有 SR-BIvar1 能增加脂蛋白与内皮的结合,而两种变体都能增加高密度脂蛋白和低密度脂蛋白的摄取。在过表达 SR-BIvar2 的细胞中,PDZK1 或 DOCK3 的沉默只减少了高密度脂蛋白的结合,而在野生型和过表达 SR-BIvar2 的细胞中,低密度脂蛋白的结合都减少了。总之,SR-BI 的任一变体都能促进高密度脂蛋白和低密度脂蛋白被内皮细胞吸收,但机制和运输途径不同。这种双重作用可以解释为什么 DOCK4 或 PDZK1 的缺失会对不同内皮细胞摄取 HDL 和 LDL 产生不同的影响。
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引用次数: 0
Corrigendum to: Protein kinase C activation stabilizes LDL receptor mRNA via the JNK pathway in HepG2 cells [Journal of Lipid Research 50/3 (2008) 386-397]. 更正:蛋白激酶 C 激活通过 JNK 途径稳定 HepG2 细胞中的低密度脂蛋白受体 mRNA [《脂质研究杂志》50/3 (2008) 386-397]。
IF 5 2区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-01 Epub Date: 2024-11-16 DOI: 10.1016/j.jlr.2024.100661
Noelle B Vargas, Brandy Y Brewer, Terry B Rogers, Gerald M Wilson
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引用次数: 0
PCPE2: Expression of multifunctional extracellular glycoprotein associated with diverse cellular functions. PCPE2:多功能细胞外糖蛋白的表达与多种细胞功能有关。
IF 5 2区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-01 Epub Date: 2024-10-05 DOI: 10.1016/j.jlr.2024.100664
Michael J Thomas, Hao Xu, Angela Wang, Mirza Ahmar Beg, Mary G Sorci-Thomas

Procollagen C-endopeptidase enhancer 2, known as PCPE2 or PCOC2 (gene name, PCOLCE2) is a glycoprotein that resides in the extracellular matrix, and is similar in domain organization to PCPE1/PCPE, PCOC1 (PCOLCE1/PCOLCE). Due to the many similarities between the two related proteins, PCPE2 has been assumed to have biological functions similar to PCPE. PCPE is a well-established enhancer of procollagen processing activating the enzyme, BMP-1. However, reports show that PCPE2 has a strikingly different tissue expression profile compared to PCPE. With that in mind and given the paucity of published studies on PCPE2, this review examines the current literature citing PCPE2 and its association with specific cell types and signaling pathways. Additionally, this review will present a brief history of PCPE2's discovery, highlighting structural and functional similarities and differences compared to PCPE. Considering the widespread use of RNA sequencing techniques to examine associations between cell-specific gene expression and disease states, we will show that PCPE2 is repeatedly found as a differentially regulated gene (DEG) significantly associated with a number of cellular processes, well beyond the scope of procollagen fibril processing.

Procollagen C-endopeptidase enhancer 2(又称 PCPE2 或 PCOC2,基因名称为 PCOLCE2)是一种存在于细胞外基质中的糖蛋白,其结构域与 PCPE1 / PCPE、PCOC1(PCOLCE1 /PCOLCE)相似。由于这两种相关蛋白之间有许多相似之处,人们认为 PCPE2 具有与 PCPE 相似的生物学功能。PCPE 是一种公认的促进胶原蛋白加工的酶,可激活 BMP-1 酶。然而,有报告显示,与 PCPE 相比,PCPE2 的组织表达谱有显著不同。有鉴于此,并考虑到已发表的有关 PCPE2 的研究很少,本综述将对引用 PCPE2 及其与特定细胞类型和信号通路的关联的现有文献进行研究。此外,本综述还将简要介绍 PCPE2 的发现历史,并着重说明其与 PCPE 在结构和功能上的异同。考虑到 RNA 测序技术已被广泛用于研究细胞特异性基因表达与疾病状态之间的关联,我们将展示 PCPE2 作为一种差异调控基因(DEG)多次被发现与许多细胞过程有显著关联,远远超出了胶原纤维加工的范围。
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引用次数: 0
Apolipoprotein B gene expression and regulation in relation to Alzheimer's disease pathophysiology. 载脂蛋白 B 基因表达和调控与阿尔茨海默病病理生理学的关系。
IF 5 2区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-01 Epub Date: 2024-10-10 DOI: 10.1016/j.jlr.2024.100667
Gabriel Aumont-Rodrigue, Cynthia Picard, Anne Labonté, Judes Poirier

Apolipoprotein B (APOB), a receptor-binding protein present in cholesterol-rich lipoproteins, has been implicated in Alzheimer's disease (AD). High levels of APOB-containing low-density lipoproteins (LDL) are linked to the pathogenesis of both early-onset familial and late-onset sporadic AD. Rare coding mutations in the APOB gene are associated with familial AD, suggesting a role for APOB-bound lipoproteins in the central nervous system. This research explores APOB gene regulation across the AD spectrum using four cohorts: BRAINEAC (elderly control brains), DBCBB (controls, AD brains), ROSMAP (controls, MCI, AD brains), and ADNI (control, MCI, AD clinical subjects). APOB protein levels, measured via mass spectrometry and ELISA, positively correlated with AD pathology indices and cognition, while APOB mRNA levels showed negative correlations. Brain APOB protein levels are also correlated with cortical Aβ levels. A common coding variant in the APOB gene locus affected its expression but didn't impact AD risk or brain cholesterol concentrations, except for 24-S-hydroxycholesterol. Polymorphisms in the CYP27A1 gene, notably rs4674344, were associated with APOB protein levels. A negative correlation was observed between brain APOB gene expression and AD biomarker levels. CSF APOB correlated with Tau pathology in presymptomatic subjects, while cortical APOB was strongly associated with cortical Aβ deposition in late-stage AD. The study discusses the potential link between blood-brain barrier dysfunction and AD symptoms in relation to APOB neurobiology. Overall, APOB's involvement in lipoprotein metabolism appears to influence AD pathology across different stages of the disease.

载脂蛋白 B(APOB)是一种存在于富含胆固醇的脂蛋白中的受体结合蛋白,与阿尔茨海默病(AD)有关。含有高水平 APOB 的低密度脂蛋白(LDL)与早发家族性和晚发散发性阿尔茨海默病的发病机制有关。APOB 基因的罕见编码突变与家族性注意力缺失症有关,这表明与 APOB 结合的脂蛋白在中枢神经系统中发挥作用。这项研究利用四个队列探索了AD谱系中的APOB基因调控:BRAINEAC(老年对照组大脑)、DBCBB(对照组、AD 大脑)、ROSMAP(对照组、MCI、AD 大脑)和 ADNI(对照组、MCI、AD 临床受试者)。通过质谱法和酶联免疫吸附法测量的APOB蛋白水平与AD病理指数和认知能力呈正相关,而APOB mRNA水平则呈负相关。大脑 APOB 蛋白水平也与皮质 Aβ 水平相关。APOB基因位点的一个常见编码变异会影响其表达,但不会影响AD风险或脑胆固醇浓度,24-S-羟基胆固醇除外。CYP27A1基因的多态性,特别是rs4674344,与APOB蛋白水平有关。大脑 APOB 基因表达与 AD 生物标志物水平之间呈负相关。CSF APOB 与无症状受试者的 Tau 病理学相关,而皮质 APOB 与 AD 晚期的皮质 Aβ 沉积密切相关。该研究讨论了血脑屏障功能障碍与AD症状之间的潜在联系,以及与APOB神经生物学的关系。总之,APOB参与脂蛋白代谢似乎会影响AD不同阶段的病理变化。
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引用次数: 0
Triglyceride-rich lipoproteins cholesterol, 10-years atherosclerotic cardiovascular disease risk, and risk of myocardial infarction and ischemic stroke. 富含甘油三酯的脂蛋白胆固醇、10 年动脉粥样硬化性心血管疾病风险以及心肌梗死和缺血性中风风险。
IF 5 2区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-01 Epub Date: 2024-09-20 DOI: 10.1016/j.jlr.2024.100653
Yi-Ping Jia, Jia-Min Wang, Jie-Qiong Lyu, Huan-Huan Yang, Meng-Yuan Miao, Xiaowen Wang, Zhong-Xiao Wan, Yan Zheng, Li-Qiang Qin, Fu-Rong Li, Guo-Chong Chen

Triglyceride-rich lipoproteins cholesterol (TRLs-C) has been associated with atherosclerotic cardiovascular disease (ASCVD), even among individuals with low-density lipoprotein cholesterol in the targeted range. We assessed the associations of TRLs-C with myocardial infarction (MI) and ischemic stroke (IS) and compared the associations with those for other traditional lipids (i.e., triglycerides and non-high-density lipoprotein cholesterol [non-HDL-C]). Included were 327,899 participants from the UK Biobank who were free of MI or IS and did not receive lipid-lowering treatment at baseline. Ten-year risk for ASCVD was estimated by the Pooled Cohort Equations and was grouped as low (<7.5%), intermediate (7.5% to <20%), and high risk (≥20%). Multivariable Cox regression models were used to examine the associations of TRLs-C, triglycerides, and non-HDL-C with risk of MI and IS, overall and by the 10-years risk categories. During a median of 12.3 years of follow-up, 8,358 incident MI and 4,400 incident IS cases were identified. After multivariable adjustment, higher TRLs-C was associated with a higher risk of MI (p-trend <0.0001) but not IS (p-trend = 0.074), with similar associations for triglycerides and non-HDL-C. There were interactions between TRLs-C and 10-years ASCVD risk on risk of MI (p-interaction <0.0001) and IS (p-interaction = 0.0003). Hazard ratios (95% CIs) of MI comparing the highest with the lowest quartiles of TRLs-C were 2.10 (1.23-1.30) in the low-risk group, 1.52 (1.38-1.69) in the intermediate-risk group, and 1.22 (1.03-1.45) in the high-risk group. The corresponding estimates for IS were 1.24 (1.05-1.45), 0.94 (0.83-1.07), and 0.83 (0.67-1.04), respectively. Similar interactions with the 10-years ASCVD risk were observed for triglycerides and non-HDL-C on risk of MI and for triglycerides on risk of IS. Elevated levels of TRLs-C (or triglycerides or non-HDL-C) are associated with a higher risk of developing MI and IS (except non-HDL-C) predominantly among individuals who are typically classified as being low-risk. These findings may have implications for more detailed risk stratification and early intervention.

背景:富含甘油三酯的脂蛋白胆固醇(TRLs-C富含甘油三酯的脂蛋白胆固醇(TRLs-C)与动脉粥样硬化性心血管疾病(ASCVD)有关,即使低密度脂蛋白胆固醇在目标范围内的人也是如此。我们评估了 TRLs-C 与心肌梗死(MI)和缺血性中风(IS)的关系是否因传统心血管风险因素的负担而异,这反映在预测的 10 年 ASCVD 风险上:研究对象包括英国生物库中的 327,899 名参与者,他们没有发生过心肌梗死或缺血性中风,基线时也没有接受降脂治疗。根据集合队列方程(Pooled Cohort Equations)估算了ASCVD的十年风险,并将其归为低风险组(结果:在中位 12.3 年的随访期间,共发现 8,358 例心肌梗死和 4,400 例心肌梗死病例。总体而言,TRLs-C越高,发生心肌梗死的风险越高(p-趋势 结论:TRLs-C与心肌梗死的关系是一个非常重要的因素:在低风险组中,TRLs-C与心肌梗死和IS的相关性显著。在确定风险方面,甘油三酯和非高密度脂蛋白胆固醇与 TRLs-C 大致相当。这些发现可能会对更详细的风险分层和早期干预产生影响。
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引用次数: 0
Development of LC-FAIMS-MS and its application to lipidomics study of Acinetobacter baumannii infection. 开发 LC-FAIMS-MS 并将其应用于鲍曼不动杆菌感染的脂质组学研究。
IF 5 2区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-01 Epub Date: 2024-10-10 DOI: 10.1016/j.jlr.2024.100668
Jianjun Li, Jacek Stupak, Arsalan S Haqqani, Greg Harris, Hongyan Zhou, Sam Williamson, Rui Chen, H Howard Xu, Wangxue Chen

The recent advances in mass spectrometry (MS) technologies have enabled comprehensive lipid profiling in biological samples. However, the robustness and efficiency of MS-based lipidomics is compromised by the complexity of biological samples. High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a technology that can continuously transmit one type of ion, independent of the mass-to-charge ratio. Here we present the development and application of LC-FAIMS-MS/MS-based platform for untargeted lipidomics. We used 3 optimally balanced compensation voltages, i.e., 29 V, 34 V and 39 V, to analyze all subclasses of glycerophospholipids. The reproducibility of the method was evaluated using reference standards. The reproducibility of retention times ranged from 0.9% to 1.5% RSD; whereas RSD values of 5%-10% were observed for peak areas. More importantly, the coupling of a FAIMS device can significantly improve the robustness and efficiency. We exploited this NPLC-FAIMS-HRMS to analyze the serum lipid profiles in mice infected intranasally with Acinetobacter baumannii. The temporal profiles of serum lipids after A. baumannii inoculation were obtained for 4 h, 8 h, and 24 h. We found that nearly all ether PC and ether PE lipids were significantly decreased 8 h after inoculation. The resultant volcano plot illustrated the distribution of 28 increased and 28 decreased lipid species in mouse sera 24 h after inoculation. We also found that a single ether PE composition can comprise multiple isomeric structures, and the relative abundance of each isomer could be quantified using the newly developed NPLC-FAIMS-PRM method. We have demonstrated that the proposed LC-FAIMS-MS is a valuable platform for lipidomics.

质谱(MS)技术的最新进展实现了对生物样本进行全面的脂质分析。然而,由于生物样本的复杂性,基于质谱的脂质组学的稳健性和效率受到了影响。高场非对称波形离子迁移谱(FAIMS)是一种可以连续传输一种离子的技术,与质荷比无关。在此,我们介绍了基于 LC-FAIMS-MS/MS 平台的非靶向脂质组学的开发与应用。我们使用了 3 个最佳平衡补偿电压,即 29 V、34 V 和 39 V,来分析所有亚类的甘油磷脂。使用参考标准对该方法的重现性进行了评估。保留时间的重现性在 0.9 到 1.5 % RSD 之间,而峰面积的 RSD 值为 5-10%。更重要的是,FAIMS 装置的耦合可以显著提高稳健性和效率。我们利用这种 NPLC-FAIMS-HRMS 分析了小鼠经鼻感染鲍曼不动杆菌后的血清脂质分布。我们发现几乎所有的醚PC和醚PE脂质在接种8小时后都显著下降。由此绘制的火山图显示了接种 24 小时后小鼠血清中 28 种增加的脂质和 28 种减少的脂质的分布情况。我们还发现,单一的醚聚乙烯成分可包括多种异构体结构,而每种异构体的相对丰度可通过新开发的 NPLC-FAIMS-PRM 方法进行量化。
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引用次数: 0
Potential causal and temporal relationship between plasma triglyceride levels and circulating leukocyte. 血浆甘油三酯水平与循环白细胞之间的潜在因果关系和时间关系。
IF 5 2区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-01 Epub Date: 2024-10-05 DOI: 10.1016/j.jlr.2024.100662
Jing Xian Fang, Hui Min Zou, Jian Meng, Yu Han, Xue Hu, Qing Gu, Sui Jun Wang, Xing Zhen Liu

Circulating triglyceride (TG) and leukocytes, the main components of the vascular system, may impact each other and co-fuel atherosclerosis. While the causal relationship between plasma TG levels and leukocyte counts remains unclear. Bidirectional Mendelian randomization (MR) analysis was conducted to investigate the potential causal relationship between TG levels and the counts of leukocytes and their subtypes. A cross-lagged panel model (CLPM) using longitudinal healthy screening data (13,389 adults with a follow-up of 4 years) was fitted to examine the temporal relationship between them. Genetically predicted plasma TG levels were positively associated with total leukocyte counts (TLC) [β(se) = 0.195(0.01)], lymphocyte counts (LC) [β(se) = 0.196(0.019)], and neutrophil counts (NC) [β(se) = 0.086(0.01)], which remained significant after adjusting for several confounders. Inversely, the genetically predicted TLC [β(se) = 0.033(0.008)], LC [β(se) = 0.053(0.008)], and NC [β(se) = 0.034(0.008)] were positively associated with plasma TG levels. However, when all three of them were put into the MR model adjusted for each other, only LC was significantly associated with TG levels. There was no association between genetically predicted TG levels and monocyte counts (MC), basophil counts, and eosinophil counts. The results of CLPM showed that the temporal effect of elevated TLC, MC, LC, and NC on plasma TG levels was stronger than the inverse effect. Our findings suggest causal associations of plasma TG levels with TLC, LC, and NC. In turn, LC was positively associated with plasma TG levels. Additionally, elevated circulating LC may precede high plasma TG levels.

背景:循环中的甘油三酯(TG)和白细胞是血管系统的主要组成部分,两者可能相互影响并共同引发动脉粥样硬化。但血浆甘油三酯水平与白细胞数量之间的因果关系仍不清楚:方法:采用双向泯灭随机化(MR)分析方法,研究血浆胆固醇水平与白细胞数量及其亚型之间的潜在因果关系。利用纵向健康筛查数据(13389 名成年人,随访 4 年)拟合了一个交叉滞后面板模型(CLPM),以研究它们之间的时间关系:结果:基因预测的血浆 TG 水平与白细胞总数 (TLC) [β(se)=0.195(0.01)]、淋巴细胞计数 (LC) [β(se)=0.196(0.019)]和中性粒细胞计数 (NC) [β(se)=0.086(0.01)]呈正相关,在调整了几种混杂因素后仍有显著性。相反,基因预测的 TLC [β(se)=0.033(0.008)] 、LC [β(se)=0.053(0.008)] 和 NC [β(se)=0.034(0.008)] 与血浆 TG 水平呈正相关。然而,当将这三者放入相互调整的 MR 模型中时,只有 LC 与血浆 TG 水平显著相关。基因预测的 TG 水平与单核细胞计数(MC)、嗜碱性粒细胞计数和嗜酸性粒细胞计数之间没有关联。CLPM结果显示,TLC、MC、LC和NC升高对血浆TG水平的时间效应强于反向效应:我们的研究结果表明,血浆 TG 水平与 TLC、LC 和 NC 存在因果关系。反过来,LC 与血浆 TG 水平呈正相关。此外,循环中低密度脂蛋白的升高可能先于高血浆 TG 水平。
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引用次数: 0
A fatty acid-ordered plasma membrane environment is critical for Ebola virus matrix protein assembly and budding. 脂肪酸有序的质膜环境对埃博拉病毒基质蛋白的组装和出芽至关重要。
IF 5 2区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-01 Epub Date: 2024-10-05 DOI: 10.1016/j.jlr.2024.100663
Souad Amiar, Kristen A Johnson, Monica L Husby, Andrea Marzi, Robert V Stahelin

Plasma membrane (PM) domains and order phases have been shown to play a key role in the assembly, release, and entry of several lipid-enveloped viruses. In the present study, we provide a mechanistic understanding of the Ebola virus (EBOV) matrix protein VP40 interaction with PM lipids and their effect on VP40 oligomerization, a crucial step for viral assembly and budding. VP40 matrix formation is sufficient to induce changes in the PM fluidity. We demonstrate that the distance between the lipid headgroups, the fatty acid tail saturation, and the PM order are important factors for the stability of VP40 binding and oligomerization at the PM. The use of FDA-approved drugs to fluidize the PM destabilizes the viral matrix assembly leading to a reduction in budding efficiency. Overall, these findings support an EBOV assembly mechanism that reaches beyond lipid headgroup specificity by using ordered PM lipid regions independent of cholesterol.

研究表明,质膜(PM)结构域和有序相在几种脂质包膜病毒的组装、释放和进入过程中发挥着关键作用。在本研究中,我们从机理上理解了埃博拉病毒(EBOV)基质蛋白VP40与PM脂质的相互作用及其对VP40寡聚化的影响,这是病毒组装和出芽的关键步骤。VP40 基质的形成足以引起 PM 流动性的变化。我们证明,脂质头基之间的距离、脂肪酸尾饱和度和 PM 顺序是 VP40 在 PM 上结合和低聚作用稳定性的重要因素。使用 FDA 批准的药物使 PM 流体化,会破坏病毒基质组装的稳定性,导致出芽效率降低。总之,这些发现支持了EBOV的组装机制,该机制利用独立于胆固醇的有序PM脂质区域,超越了脂质头基的特异性。
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引用次数: 0
Highly reliable LC-MS lipidomics database for efficient human plasma profiling based on NIST SRM 1950. 基于 NIST SRM 1950 的高度可靠的 LC-MS 脂质组学数据库,可用于高效的人体血浆分析。
IF 5 2区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-01 Epub Date: 2024-10-10 DOI: 10.1016/j.jlr.2024.100671
Sara Martínez, Miguel Fernández-García, Sara Londoño-Osorio, Coral Barbas, Ana Gradillas

Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS)-based methods have become the gold standard methodology for the comprehensive profiling of the human plasma lipidome. However, both the complexity of lipid chemistry and LC-HRMS-associated data pose challenges to the characterization of this biological matrix. In accordance with the current consensus of quality requirements for LC-HRMS lipidomics data, we aimed to characterize the NIST® Standard Reference Material for Human Plasma (SRM 1950) using an LC-ESI(+/-)-MS method compatible with high-throughput lipidome profiling. We generated a highly curated lipid database with increased coverage, quality, and consistency, including additional quality assurance procedures involving adduct formation, within-method m/z evaluation, retention behavior of species within lipid chain isomers, and expert-driven resolution of isomeric and isobaric interferences. As a proof-of-concept, we showed the utility of our in-house LC-MS lipidomic database -consisting of 592 lipid entries- for the fast, comprehensive, and reliable lipidomic profiling of the human plasma from healthy human volunteers. We are confident that the implementation of this robust resource and methodology will have a significant impact by reducing data redundancy and the current delays and bottlenecks in untargeted plasma lipidomic studies.

基于液相色谱-高分辨质谱(LC-HRMS)的方法已成为全面分析人体血浆脂质体的黄金标准方法。然而,脂质化学的复杂性和 LC-HRMS 相关数据给这一生物基质的表征带来了挑战。根据目前对 LC-HRMS 脂质组学数据质量要求的共识,我们旨在使用与高通量脂质组分析兼容的 LC-ESI(+/-)-MS 方法表征 NIST® 人血浆标准参考物质(SRM 1950)。我们生成了一个覆盖面更广、质量更高和一致性更强的脂质数据库,其中包括额外的质量保证程序,涉及加合物形成、方法内 m/z 评估、脂质链异构体内物种的保留行为,以及专家驱动的异构体和同位干扰解析。作为概念验证,我们展示了由 592 个脂质条目组成的内部 LC-MS 脂质组数据库在快速、全面、可靠地分析健康人体志愿者血浆脂质组方面的实用性。我们相信,这一强大的资源和方法的实施将产生重大影响,减少数据冗余以及目前非靶向血浆脂质体研究中的延迟和瓶颈。
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引用次数: 0
Development of a monoclonal antibody to study MARCH6, an E3 ligase that regulates proteins that control lipid homeostasis. 开发一种单克隆抗体,用于研究 MARCH6(一种调节控制脂质稳态的蛋白质的 E3 连接酶)。
IF 5 2区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-01 Epub Date: 2024-09-19 DOI: 10.1016/j.jlr.2024.100650
Shimeng Xu, Linda Donnelly, Daniel L Kober, Myra Mak, Arun Radhakrishnan

Membrane-associated ring-CH-type finger 6 (MARCH6), also designated as TEB4 or RNF176, is an E3 ligase that is embedded in membranes of the endoplasmic reticulum where it ubiquitinates many substrate proteins to consign them to proteasome-mediated degradation. In recent years, MARCH6 has been identified as a key regulator of several metabolic pathways, including cholesterol and lipid droplet homeostasis, protein quality control, ferroptosis, and tumorigenesis. Despite its importance, there are currently no specific antibodies to detect and monitor MARCH6 levels in cultured cells and animals. Here, we address this deficiency by generating a monoclonal antibody that specifically detects MARCH6 in cultured cells of insect, mouse, hamster, and human origin, as well as in mouse tissues, with minimal cross-reactivity against other proteins. We then used this antibody to assess two properties of MARCH6. First, analysis of mouse tissues with this antibody revealed that the liver contained the highest levels of March6. Second, analysis of five different cell lines with this antibody showed that endogenous levels of MARCH6 are unchanged as the cellular content of cholesterol is varied. This reagent promises to be a useful tool in interrogating additional signaling roles of MARCH6.

MARCH6也被称为TEB4或RNF176,是一种E3连接酶,它嵌入内质网(ER)的膜中,在那里泛素化许多底物蛋白,使其进入蛋白酶体介导的降解过程。近年来,MARCH6 已被确定为多种代谢途径的关键调节因子,包括胆固醇和脂滴稳态、蛋白质质量控制、铁变态反应和肿瘤发生。尽管MARCH6非常重要,但目前还没有特异性抗体来检测和监控培养细胞和动物中的MARCH6水平。在这里,我们生成了一种单克隆抗体来解决这一不足,这种抗体能特异性地检测昆虫、小鼠、仓鼠和人类培养细胞以及小鼠组织中的 MARCH6,而且与其他蛋白质的交叉反应极少。然后,我们用这种抗体评估了 MARCH6 的两个特性。首先,用该抗体分析小鼠组织发现,肝脏中的 MARCH6 含量最高。其次,用该抗体对五种不同细胞系进行的分析表明,随着细胞中胆固醇含量的变化,MARCH6 的内源性水平也不会改变。这种试剂有望成为研究 MARCH6 其他信号作用的有用工具。
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引用次数: 0
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Journal of Lipid Research
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