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More insights from Abca4-/- mouse models of recessive Stargardt disease. Abca4-/-小鼠隐性Stargardt病模型的更多见解
IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2026-02-05 DOI: 10.1016/j.jbc.2026.111261
Jin Zhao, Diego Montenegro, Sihua Cheng, Hye Jin Kim, Janet R Sparrow

Mutations in the ATP-binding cassette transporter ABCA4 are responsible for recessive Stargardt disease (STGD1) a juvenile form of macular degeneration. In preclinical and clinical studies it has been shown that deficiency in ABCA4 leads to accelerated formation of the toxic bisretinoid fluorophores that form as the product of nonenzymatic reactions of retinaldehyde with phosphatidylethanolamine (PE) (2:1 ratio). Here, by comparing photoreceptor cell viability in albino versus agouti Abca4-/- mice and by dark-rearing albino Abca4-/- mice we show that photoreceptor cell degeneration in the Abca4-/- mouse is at least partially driven by light. Elevated vitamin A in chow and a high fat diet reduced photoreceptor cell viability. PE and N-retinylidiene-phosphatidylethanolamine were reduced as were steady state levels of retinoid in light-adapted eyes. As expected, bisretinoids, measured as short-wavelength fundus autofluorescence, were elevated in both pigmented and albino Abca4-/- mice. Hyperautofluorescent puncta in fundus autofluorescence images colocalized in spectral domain optical coherence tomography scans with aberrant hyperreflectivity that occupied photoreceptor-attributable bands and extended anteriorly to interrupt the ellipsoid zone and external limiting membrane. In epifluorescence images of Abca4-/- retina, retinal pigment epithelium was autofluorescent due to bisretinoid accumulation. Occasionally AF lesions extended anteriorly from the RPE to a horizontal band exhibiting less pronounced autofluorescence at the level of photoreceptor inner and outer segments. These lesions did not co-localize with IBA1-labeled microglia. The hyperautofluorescent foci that presented as hyperreflective lesions in SD-OCT form in photoreceptor inner segments and are reminiscent of fundus flecks in STGD1.

atp结合盒转运体ABCA4的突变是隐性Stargardt病(STGD1)的原因,STGD1是黄斑变性的一种幼年形式。临床前和临床研究表明,ABCA4的缺乏会导致毒性类双维甲酸荧光团的加速形成,这种荧光团是视黄醛与磷脂酰乙醇胺(PE)(2:1的比例)非酶反应的产物。在这里,通过比较白化Abca4-/-小鼠和黑饲养白化Abca4-/-小鼠的光感受器细胞活力,我们发现Abca4-/-小鼠的光感受器细胞退化至少部分是由光驱动的。食物中维生素A含量升高和高脂肪饮食降低了感光细胞的活力。PE和n -视黄二烯-磷脂酰乙醇胺在适应光的眼睛中的稳态水平降低。正如预期的那样,在色素沉着和白化Abca4-/-小鼠中,以短波眼底自身荧光测量的类双维甲酸均升高。眼底自体荧光图像中的超自体荧光点在光谱域光学相干断层扫描中共定位,具有异常的超反射率,占据光感受器可归因带并向前延伸以中断椭球区和外限制膜。在Abca4-/-视网膜的表观荧光图像中,由于类双维甲酸积累,视网膜色素上皮呈自体荧光。偶尔房颤病变从RPE向前延伸到水平带,在感光器内外节段水平表现出不太明显的自身荧光。这些病变没有与iba1标记的小胶质细胞共定位。在SD-OCT上表现为高反射病变的高自荧光灶形成于光感受器内节段,使人联想到STGD1的眼底斑点。
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引用次数: 0
GPCR-selective effects of endocytosis on cellular signaling through the cAMP / PKA cascade. 内吞作用通过cAMP / PKA级联对细胞信号传导的gpcr选择性影响。
IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2026-02-05 DOI: 10.1016/j.jbc.2026.111253
Emily E Blythe, Rita R Fagan, Mark von Zastrow

Many GPCRs use endocytosis to promote gene transcription by prolonging signaling through the Gs-coupled cAMP / cAMP-dependent protein kinase (PKA) cascade. However, not all GPCRs efficiently internalize after agonist-induced activation and, among those that do, considerable differences have been observed in the ability of different GPCRs to stimulate endosomal cAMP production in different cell types. We asked if endocytosis distinguishes the signaling profiles of GPCRs that are naturally coexpressed in the same cells, focusing on three Gs-coupled GPCRs endogenous to human kidney-derived (HEK293) cells: the vasoactive intestinal peptide receptor-1 (VIPR1 / VPAC1) and β2-adrenergic receptor (β2AR / ADRB2) which both rapidly internalize after activation and the adenosine-2B receptor (A2BR / ADORA2B) which we show here does not. For VIPR1, endocytosis significantly prolongs both the global cAMP elevation and cytoplasmic PKA activity increase. For β2AR, endocytosis has little effect on the global cAMP elevation but, nonetheless, it significantly prolongs the cytoplasmic PKA activity increase. A2BR differs still further, with endocytosis having little effect on signal duration measured at either intermediate step. We then show that further downstream steps in the cascade, nuclear PKA activation and transcriptional induction, are significantly endocytosis-dependent when stimulated through VIPR1 and β2AR but endocytosis-independent through A2BR. We conclude that endocytosis indeed distinguishes the signaling profiles of endogenously coexpressed GPCRs. We propose that quantitative differences in GPCR internalization and activation in endosomes program in cells a GPCR-selective, spatiotemporal 'cAMP code' that is spatially 'decoded' by proximity to local cytoplasmic PKA stores and then temporally interpreted by the nucleus.

许多gpcr通过gs偶联cAMP / cAMP依赖性蛋白激酶(PKA)级联延长信号传导,利用内吞作用促进基因转录。然而,并不是所有的gpcr在激动剂诱导激活后都能有效内化,在那些内化的细胞中,在不同的细胞类型中,不同的gpcr刺激内体cAMP产生的能力存在相当大的差异。我们询问内吞作用是否能区分在同一细胞中自然共表达的GPCRs的信号谱,重点关注人肾源性(HEK293)细胞内源性的三种gs偶联GPCRs:血管活性肠肽受体-1 (VIPR1 / VPAC1)和β2-肾上腺素能受体(β2AR / ADRB2),它们在激活后都会迅速内化,而腺苷- 2b受体(A2BR / ADORA2B)则不会。对于VIPR1,内吞作用显著延长了cAMP的整体升高和细胞质PKA活性的增加。对于β2AR,内吞作用对cAMP的整体升高影响不大,但却显著延长了胞质PKA活性的升高。A2BR的差异更大,内吞作用对任何中间步骤测量的信号持续时间几乎没有影响。然后,我们发现级联的进一步下游步骤,核PKA激活和转录诱导,当通过VIPR1和β2AR刺激时,明显依赖于内吞,但通过A2BR不依赖于内吞。我们得出结论,内吞作用确实区分了内源性共表达gpcr的信号谱。我们提出,细胞内核体程序中GPCR内化和激活的数量差异是一个GPCR选择性的、时空的“cAMP代码”,该代码通过接近局部细胞质PKA储存而在空间上“解码”,然后由细胞核在时间上解释。
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引用次数: 0
An evolutionarily conserved salt bridge stabilizes the active site for GTP hydrolysis in Rho GTPases. 一个进化上保守的盐桥稳定了Rho GTP酶中GTP水解的活性位点。
IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2026-02-05 DOI: 10.1016/j.jbc.2026.111260
Kendra Marcus, Michael Schwabe, Ryan Knihtila, Carla Mattos

Rho GTPases are members of the Ras superfamily of small GTPases that regulate cell morphology, motility, polarization and cell cycling. Like members of the Ras subfamily, Rho subfamily GTPases dysregulation is implicated in a range of tumors and can serve as a valid drug target. In this work, we investigate the evolutionary trajectory of Rho GTPases within a region of the protein that has been exploited for cancer drug discovery within the Ras subfamily branch - the "switch II pocket". Our previous work has illustrated the role of allostery in this region of H-Ras in modulation of intrinsic hydrolysis and effector-binding capacity. Here, we report that a highly conserved salt bridge within the Rho subfamily stabilizes the RhoA GTPase active site in a catalytically favorable conformation. We probed the roles of the Rho salt bridge via X-ray crystallography, accelerated molecular dynamics simulations (aMD), and enzymatic studies. We showed that the removal of a residue within switch II of RhoA, the salt bridge residue R70, can impart catastrophic effects on active site organization and GTP hydrolysis. As expected, removal of the analogous R68 in H-Ras, which is not involved in a salt bridge interaction, results in a structure with changes in the active site and a decrease in GTP hydrolysis rate constant that are more moderate than observed for RhoA. The anionic partner of R70, E102, also modulates active site conformation and, upon removal, decreases intrinsic hydrolysis. Based on aMD simulations, we uncovered evidence of epistatic relationships between the Rho salt bridge, the distal residue K98 and P-loop residue D13 which coordinate allosteric communication from the switch regions directly to the active site. Finally, we describe the functional landscape of switch II pocket in the context of both Rho subfamily evolution and potential for drug discovery.

Rho gtpase是Ras超家族的成员,它是调节细胞形态、运动、极化和细胞周期的小gtpase。与Ras亚家族成员一样,Rho亚家族GTPases失调与一系列肿瘤有关,可以作为有效的药物靶点。在这项工作中,我们研究了Rho gtpase在Ras亚家族分支中用于癌症药物发现的蛋白质区域内的进化轨迹-“开关II口袋”。我们之前的工作已经说明了H-Ras区域的变构在调节内在水解和效应物结合能力中的作用。在这里,我们报道了Rho亚家族中的一个高度保守的盐桥,使RhoA GTPase活性位点稳定在催化有利的构象中。我们通过x射线晶体学、加速分子动力学模拟(aMD)和酶学研究探讨了Rho盐桥的作用。我们发现去除RhoA开关II中的残基,即盐桥残基R70,会对活性位点的组织和GTP水解产生灾难性的影响。正如预期的那样,去除H-Ras中类似的R68,它不参与盐桥相互作用,导致活性位点的结构变化和GTP水解速率常数的降低,比RhoA观察到的更温和。R70的阴离子伴侣E102也能调节活性位点的构象,并在去除后减少内在水解。基于aMD模拟,我们发现了Rho盐桥、远端残基K98和p环残基D13之间的上位性关系,它们协调从开关区直接到活性位点的变构通信。最后,我们描述了开关II口袋在Rho亚家族进化和药物发现潜力的背景下的功能景观。
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引用次数: 0
Alix Mediated Selective Packaging of β-Catenin into Extracellular Vesicles Enhances Their Pro-Angiogenic Function. Alix介导的β-连环蛋白选择性包装细胞外囊泡增强其促血管生成功能。
IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2026-02-05 DOI: 10.1016/j.jbc.2026.111254
Rui Li, Kai Pan, Qiaonan Zhang, Yu Guo, Nijing Jung, Zhibo Han, Zhong-Chao Han, Jun Zhang, Zongjin Li

The function of extracellular vesicles (EVs) is determined by the molecular cargo they carry from their parent cells. Although Apoptosis-linked gene 2-interacting protein X (Alix) is known to regulate EV cargo loading and functional properties, the specific mechanisms underlying its role in mediating β-catenin sorting and function remain unclear. In this study, we first observed the co-localization of Alix and β-catenin through immunofluorescence staining. To assess whether the interaction between Alix and β-catenin affects the function of mesenchymal stem cell (MSC)-derived EVs, we generated Alix-knockdown (KD) and Alix-overexpressing (OE) MSCs via viral transduction. Analysis of secreted EVs revealed that those derived from Alix-OE-MSCs promoted angiogenesis both in vitro and in a mouse model of hindlimb ischemia, whereas EVs from Alix-KD-MSCs suppressed angiogenesis. Mechanistically, we confirmed that the Alix-β-catenin interaction selectively enhances β-catenin enrichment within EVs. In conclusion, our findings demonstrate that Alix plays a critical role in selectively packaging β-catenin into EVs, thereby enhancing their pro-angiogenic potency. Modified.

细胞外囊泡(EVs)的功能取决于它们从亲本细胞携带的分子货物。虽然已知凋亡相关基因2-相互作用蛋白X (Alix)调节EV载货量和功能特性,但其介导β-连环蛋白分选和功能的具体机制尚不清楚。在本研究中,我们首先通过免疫荧光染色观察到Alix和β-catenin的共定位。为了评估Alix和β-catenin之间的相互作用是否影响间充质干细胞(MSC)衍生的ev的功能,我们通过病毒转导生成了Alix敲低(KD)和Alix过表达(OE)的MSCs。体外和小鼠后肢缺血模型中,Alix-OE-MSCs衍生的EVs均能促进血管生成,而Alix-KD-MSCs衍生的EVs则抑制血管生成。在机制上,我们证实了Alix-β-catenin相互作用选择性地增强了ev内β-catenin的富集。总之,我们的研究结果表明,Alix在选择性地将β-catenin包装到ev中,从而增强其促血管生成能力方面发挥了关键作用。修改。
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引用次数: 0
Writers and Readers of Sialylation in Immunoregulation in Cancer. 唾液酰化在癌症免疫调节中的作用。
IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2026-02-04 DOI: 10.1016/j.jbc.2026.111249
Mathieu Decloquement, Matthew S Macauley

Sialic acids are the terminal monosaccharides of the glycocalyx that critically shape cell-cell interactions, and are strongly implicated in regulating immune recognition and tissue homeostasis. In cancer, aberrant sialylation rewires the tumor microenvironment by enhancing ligands of the inhibitory Siglecs, suppressing immune effector functions, and facilitating metastatic dissemination. This review provides a comprehensive synthesis of the dual role of sialyltransferases (the "writers") and Siglecs/Selectins (the "readers") in cancer progression. We examine the structural and functional diversity of these molecules, their dysregulation in malignancy, and their impact on tumor-immune dynamics. Finally, we highlight emerging therapeutic strategies, including sialyltransferase inhibitors, sialidase conjugates, and Siglec-targeted immunotherapies, which collectively position the sialome as a tractable frontier in cancer treatment.

唾液酸是糖萼的末端单糖,它对细胞与细胞的相互作用起关键作用,并与调节免疫识别和组织稳态密切相关。在癌症中,异常唾液酰化通过增强抑制Siglecs的配体、抑制免疫效应功能和促进转移性传播来重新连接肿瘤微环境。本文综述了唾液转移酶(“书写者”)和Siglecs/选择蛋白(“阅读者”)在癌症进展中的双重作用。我们研究了这些分子的结构和功能多样性,它们在恶性肿瘤中的失调,以及它们对肿瘤免疫动力学的影响。最后,我们重点介绍了新兴的治疗策略,包括唾液酸转移酶抑制剂、唾液酸转移酶偶联物和siglecl靶向免疫疗法,它们共同将唾液酸转移酶定位为癌症治疗的一个可处理的前沿。
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引用次数: 0
Development of an inhibitory monoclonal nanobody targeting Streptococcus pyogenes siderophore binding protein FtsB. 一种针对化脓性链球菌铁载体结合蛋白FtsB的抑制单克隆纳米体的建立。
IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2026-02-04 DOI: 10.1016/j.jbc.2026.111224
Jorge Fernandez-Perez, Susana de Vega, Jose M M Caaveiro, Makoto Nakakido, Satoru Nagatoishi, Akinobu Senoo, Keitaro Tanoi, Takashi Nozawa, Ichiro Nakagawa, Kouhei Tsumoto

Due to the limited availability of metals inside the human body, pathogenic bacteria must produce multiple highly specialized metal transporters to cause infection. These transporters constitute attractive targets for developing novel antibacterial strategies. Streptococcus pyogenes possesses three iron transporters, of which the FtsABCD system is specialized in the uptake of ferric hydroxamates. The role of this transporter in infection remains unclear. In this study, we developed a monoclonal alpaca VHH, or nanobody, Nb1, targeting FtsB. Nb1 binds to FtsB with sub-nM affinity, in an enthalpy-driven manner, and with a characteristically slow dissociation rate. Solvent accessibility analysis by HDX-MS, mutational analyses, and X-ray crystallography revealed that the epitope of Nb1 is in the binding pocket of FtsB. The nanobody competitively inhibited the binding of multiple hydroxamate siderophores and partially inhibited the uptake of siderophores in Streptococcus pyogenes cells. The inhibitory activity of Nb1 on siderophore transport represents a new tool to study the role of the FtsABCD transporter and can be used as a potential inhibitor of Streptococcus pyogenes growth under iron-limited conditions.

由于人体内金属的可用性有限,致病菌必须产生多种高度专门化的金属转运体才能引起感染。这些转运体构成了开发新型抗菌策略的有吸引力的靶点。化脓性链球菌具有三种铁转运体,其中FtsABCD系统专门用于铁羟基酸盐的摄取。这种转运体在感染中的作用尚不清楚。在这项研究中,我们开发了一种针对FtsB的单克隆羊驼VHH或纳米体Nb1。Nb1以低于nm的亲和力与FtsB结合,以焓驱动的方式,并具有典型的缓慢解离速率。通过HDX-MS、突变分析和x射线晶体学分析发现,Nb1的表位位于FtsB的结合口袋中。该纳米体竞争性地抑制了多个羟酸盐铁载体的结合,并部分抑制了化脓性链球菌细胞对铁载体的摄取。Nb1对铁载体运输的抑制活性为研究FtsABCD转运体的作用提供了新的工具,可以作为一种潜在的化脓性链球菌在铁限制条件下生长的抑制剂。
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引用次数: 0
Modernizing Biomolecular NMR: the POKY Suite. 现代化生物分子核磁共振:POKY套件。
IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2026-02-04 DOI: 10.1016/j.jbc.2026.111246
Abigail Chiu, Woonghee Lee

Biomolecular NMR spectroscopy has been a keystone in structural biology for decades. It can provide unique, atomic-level insights into protein dynamics, interactions, and conformational ensembles. However, its complex workflows and fragmented data analysis pipelines are often perceived as significant barriers to entry. This review highlights the POKY suite as a comprehensive solution that modernizes and streamlines the entire biomolecular NMR process. From spectral processing to structure calculation, POKY creates a single user-friendly cyberinfrastructure for a seamless and efficient NMR data analysis environment. A key aspect of its design is the integration of various artificial intelligence (AI) components to streamline complex tasks and reduce user burden, such as automation, unsupervised learning, and more. While recent advances within in silico AI prediction models have raised questions about the role of experimental data, POKY provides a clear answer. This ecosystem can create a powerful synergy between the experimental data with structure prediction. modernizing the experimental workflow, POKY makes NMR more accessible and powerful, reinforcing its vital role instructural biology.

几十年来,生物分子核磁共振波谱一直是结构生物学的基石。它可以为蛋白质动力学,相互作用和构象集成提供独特的,原子水平的见解。然而,其复杂的工作流程和碎片化的数据分析管道通常被视为进入的重大障碍。这篇综述强调了POKY套件作为一个全面的解决方案,现代化和简化了整个生物分子核磁共振过程。从光谱处理到结构计算,POKY为无缝和高效的核磁共振数据分析环境创建了一个单一的用户友好的网络基础设施。其设计的一个关键方面是集成各种人工智能(AI)组件,以简化复杂的任务并减轻用户负担,例如自动化,无监督学习等。虽然最近在计算机人工智能预测模型中的进展引发了对实验数据作用的质疑,但POKY提供了一个明确的答案。这个生态系统可以在实验数据和结构预测之间产生强大的协同作用。现代化的实验工作流程,POKY使核磁共振更容易获得和强大,加强其重要作用,指导生物学。
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引用次数: 0
Unusual and atypical cyclooxygenase reactions. 异常和非典型环加氧酶反应。
IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2026-02-04 DOI: 10.1016/j.jbc.2026.111247
Claus Schneider, Alan R Brash

Mechanistic studies have yielded novel prostaglandin analogs and acyclic products initially of interest in understanding cyclooxygenase structure-function, later found in vivo and of interest due to unique biological activities. Beyond arachidonic acid, fatty acid substrates span from 18 to 22 carbons and may contain ester/amide modification or epoxide/hydroxy moieties at the first double bond. Stereo control with the unconventional substrates remains largely intact although cyclization may be diverted or halted altogether, and catalysis proceeds with insertion of one, two, or three molecules of oxygen into substrates. A switch in stereochemistry at the 15-carbon occurs in a natural cyclooxygenase from coral and has received attention upon aspirin treatment of COX-2. The latter produces 15R-HETE and analogs from other fatty acids that may be further oxygenated by lipoxygenases. Functional plasticity in cyclooxygenase catalysis has enabled the formation and discovery of a host of novel eicosanoids and provided mechanistic insight into the COX reaction mechanisms.

机制研究产生了新的前列腺素类似物和无环产物,最初对理解环加氧酶的结构和功能感兴趣,后来在体内发现,由于独特的生物活性而感兴趣。在花生四烯酸之外,脂肪酸底物从18到22个碳,在第一个双键上可能含有酯/酰胺修饰或环氧化物/羟基部分。非常规底物的立体控制在很大程度上是完整的,尽管环化可能被转移或完全停止,催化过程通过在底物中插入一、二或三分子氧来进行。在珊瑚中的天然环加氧酶中出现了立体化学中的15碳开关,并且在阿司匹林治疗COX-2时引起了人们的注意。后者产生15R-HETE和其他脂肪酸的类似物,这些脂肪酸可能被脂肪加氧酶进一步氧化。环加氧酶催化的功能可塑性使许多新型类二十烷化合物的形成和发现成为可能,并为COX反应机制提供了机制见解。
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引用次数: 0
Emerging Biologic Modalities for Targeted Protein Degradation. 靶向蛋白质降解的新兴生物模式。
IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2026-02-04 DOI: 10.1016/j.jbc.2026.111248
Alana G Caldwell, Harshil Parmar, Xiaoyu Zhang

Targeted protein degradation (TPD) has emerged as a powerful approach for eliminating disease-associated proteins by harnessing the ubiquitin-proteasome system. Biologic degraders are modular protein chimeras that recruit ubiquitin machinery to target proteins. They offer high specificity, modular design, and the ability to access targets traditionally considered challenging for small molecule ligands. This review surveys the expanding landscape of biologic TPD modalities, highlighting E3 ligase- and E2 enzyme-based degraders, TRIM-Away and TRIMbody-Away systems, and diverse biologics-based ligands that serve as target-binding components. We also discuss emerging peptide-based strategies, which bridge biologic and synthetic approaches. Finally, we highlight future opportunities to improve biologic degraders and their potential to expand the scope of targeted protein degradation.

靶向蛋白降解(TPD)是一种利用泛素-蛋白酶体系统消除疾病相关蛋白的有效方法。生物降解物是模块化的蛋白质嵌合体,它招募泛素机制来靶向蛋白质。它们具有高特异性、模块化设计和接近传统上被认为对小分子配体具有挑战性的目标的能力。本文综述了生物TPD模式的发展前景,重点介绍了基于E3连接酶和E2酶的降解剂,TRIM-Away和TRIMbody-Away系统,以及作为靶标结合成分的多种基于生物的配体。我们还讨论了新兴的基于肽的策略,它连接了生物和合成方法。最后,我们强调了未来改进生物降解剂的机会及其扩大靶向蛋白质降解范围的潜力。
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引用次数: 0
Profiling the Pancreatic Cancer Secretome with Metabolic Glycoengineering. 用代谢糖工程分析胰腺癌分泌组。
IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2026-02-04 DOI: 10.1016/j.jbc.2026.111243
Kris Dammen-Brower, Stanley Zhu, Christian Agatemor, Safiya Aafreen, Vrinda Dharharma, Christopher T Saeui, Hui Li, Jian Song, Matthew J Buettner, Keith R Kwagala, Hui Zhang, Howard E Katz, Guanshu Liu, Kevin J Yarema

Profiling the secretome for biomarkers offers an attractive, minimally invasive strategy to detect and monitor cancer. Several challenges, however, must be overcome including the broad dynamic range of biomolecules in the secretome and the requirement for selective detection of tumor-associated markers. Here, we employed a metabolic glycoengineering (MGE) strategy, using 1,3,4-O-Bu3ManNAz, an azido-tagged, bioorthogonal metabolic precursor of sialic acid, to label the glycome of pancreatic near-normal and cancer cells to improve conventional LC-MS/MS proteomics-based biomarker discovery. By using this "MGE-LC-MS/MS" approach that incorporates MGE-enrichment into conventional LC-MS/MS proteomics, we identified several unique proteins from the secretomes of cancer cells evaluated in vitro. In addition to proteins known to be secreted, we identified several putatively intracellular, non-N-glycosylated proteins such β-glucocerebrosidase and paladin linked to pancreatic cancer (PC) as well as proteins associated with extracellular vesicles (EV) in PC such as DCTPP1. The identification of EV-associated proteins was consistent with our discovery that ManNAc analogs used in the MGE-LC-MS/MS workflow enhance EV production, creating a more complete secretome profile of PC cells. Pointing towards clinical relevance, we used MGE-LC-MS/MS to enrich PC-derived glycoproteins from plasma harvested from mice bearing xenografted human pancreatic tumors, unambiguously demonstrating that this approach can interrogate the secretomes of cancer cells for biomarker discovery. Finally, we discovered that MGE dramatically improved the production of EVs, which both aids in biomarker discovery (this study) and holds potential to facilitate biomanufacturing of these nascent drugs.

分析分泌组的生物标志物提供了一个有吸引力的,微创的策略来检测和监测癌症。然而,必须克服几个挑战,包括分泌组中生物分子的广泛动态范围和选择性检测肿瘤相关标记物的要求。在这里,我们采用代谢糖工程(MGE)策略,使用1,3,4- o- bu3mannaz(一种叠氮标记的唾液酸生物正交代谢前体)标记胰腺近正常细胞和癌细胞的糖,以改进传统的LC-MS/MS基于蛋白质组学的生物标志物发现。通过将MGE-LC-MS/MS富集纳入常规LC-MS/MS蛋白质组学的“MGE-LC-MS/MS”方法,我们从体外评估的癌细胞分泌组中鉴定了几种独特的蛋白质。除了已知的分泌蛋白外,我们还鉴定了几种假定的细胞内非n-糖基化蛋白,如β-葡萄糖脑苷酶和与胰腺癌(PC)相关的paladin,以及与PC中细胞外囊泡(EV)相关的蛋白,如DCTPP1。EV相关蛋白的鉴定与我们的发现一致,MGE-LC-MS/MS工作流程中使用的ManNAc类似物提高了EV的产量,创建了更完整的PC细胞分泌组谱。针对临床相关性,我们使用MGE-LC-MS/MS从携带异种移植人类胰腺肿瘤的小鼠的血浆中富集pc衍生的糖蛋白,明确表明该方法可以询问癌细胞的分泌组以发现生物标志物。最后,我们发现MGE显著提高了电动汽车的生产,这既有助于生物标志物的发现(本研究),也有可能促进这些新兴药物的生物制造。
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引用次数: 0
期刊
Journal of Biological Chemistry
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