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Extracellular Vesicles: Translational Agenda Questions for Three Protozoan Parasites 细胞外囊泡:三种原生动物寄生虫的转化议程问题
IF 4.5 3区 生物学 Q3 CELL BIOLOGY Pub Date : 2024-04-17 DOI: 10.1111/tra.12935
Kwesi Z. Tandoh, Ana Victoria Ibarra‐Meneses, David Langlais, Martin Olivier, Ana Claudia Torrecilhas, Christopher Fernandez‐Prada, Neta Regev‐Rudzki, Nancy O. Duah‐Quashie
The protozoan parasites Plasmodium falciparum, Leishmania spp. and Trypanosoma cruzi continue to exert a significant toll on the disease landscape of the human population in sub‐Saharan Africa and Latin America. Control measures have helped reduce the burden of their respective diseases—malaria, leishmaniasis and Chagas disease—in endemic regions. However, the need for new drugs, innovative vaccination strategies and molecular markers of disease severity and outcomes has emerged because of developing antimicrobial drug resistance, comparatively inadequate or absent vaccines, and a lack of trustworthy markers of morbid outcomes. Extracellular vesicles (EVs) have been widely reported to play a role in the biology and pathogenicity of P. falciparum, Leishmania spp. and T. cruzi ever since they were discovered. EVs are secreted by a yet to be fully understood mechanism in protozoans into the extracellular milieu and carry a cargo of diverse molecules that reflect the originator cell's metabolic state. Although our understanding of the biogenesis and function of EVs continues to deepen, the question of how EVs in P. falciparum, Leishmania spp. and T. cruzi can serve as targets for a translational agenda into clinical and public health interventions is yet to be fully explored. Here, as a consortium of protozoan researchers, we outline a plan for future researchers and pose three questions to direct an EV's translational agenda in P. falciparum, Leishmania spp. and T. cruzi. We opine that in the long term, executing this blueprint will help bridge the current unmet needs of these medically important protozoan diseases in sub‐Saharan Africa and Latin America.
原生动物寄生虫恶性疟原虫、利什曼原虫和南美锥虫继续对撒哈拉以南非洲和拉丁美洲人口的疾病状况造成重大影响。控制措施有助于减轻疟疾、利什曼病和南美锥虫病流行地区的疾病负担。然而,由于抗微生物药物耐药性的不断发展、疫苗相对不足或缺失,以及缺乏可信的发病结果标记物,因此出现了对新药物、创新疫苗接种策略以及疾病严重程度和结果分子标记物的需求。细胞外囊泡(EVs)自被发现以来,就被广泛报道在恶性疟原虫、利什曼原虫属和克鲁兹疟原虫的生物学和致病性中发挥作用。EVs是通过原生动物尚未完全了解的机制分泌到细胞外环境中的,其携带的各种分子反映了原生细胞的新陈代谢状态。尽管我们对 EVs 的生物发生和功能的理解在不断加深,但恶性疟原虫、利什曼原虫和克鲁斯原虫中的 EVs 如何作为转化为临床和公共卫生干预议程的目标,这一问题仍有待充分探讨。在此,作为一个原生动物研究人员联盟,我们为未来的研究人员概述了一项计划,并提出了三个问题,以指导恶性疟原虫、利什曼原虫属和克鲁斯原虫的 EV 转化议程。我们认为,从长远来看,执行这一蓝图将有助于满足撒哈拉以南非洲和拉丁美洲目前对这些具有重要医学意义的原生动物疾病尚未满足的需求。
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引用次数: 0
Inhibition of Autophagy Alters Intracellular Transport of APP Resulting in Increased APP Processing 抑制自噬会改变 APP 的细胞内转运,导致 APP 处理过程增加
IF 4.5 3区 生物学 Q3 CELL BIOLOGY Pub Date : 2024-04-13 DOI: 10.1111/tra.12934
Johanna Mayer, Dominik Boeck, Michelle Werner, Daniela Frankenhauser, Stephan Geley, Hesso Farhan, Makoto Shimozawa, Per Nilsson
Alzheimer's disease (AD) pathology is characterized by amyloid beta (Aβ) plaques and dysfunctional autophagy. Aβ is generated by sequential proteolytic cleavage of amyloid precursor protein (APP), and the site of intracellular APP processing is highly debated, which may include autophagosomes. Here, we investigated the involvement of autophagy, including the role of ATG9 in APP intracellular trafficking and processing by applying the RUSH system, which allows studying the transport of fluorescently labeled mCherry‐APP‐EGFP in a systematic way, starting from the endoplasmic reticulum. HeLa cells, expressing the RUSH mCherry‐APP‐EGFP system, were investigated by live cell imaging, immunofluorescence, and Western blot. We found that mCherry‐APP‐EGFP passed through the Golgi faster in ATG9 knockout cells. Furthermore, ATG9 deletion shifted mCherry‐APP‐EGFP from early endosomes and lysosomes toward the plasma membrane concomitant with reduced endocytosis. Importantly, this alteration in mCherry‐APP‐EGFP transport resulted in increased secreted mCherry‐soluble APP and C‐terminal fragment‐EGFP. These effects were also phenocopied by pharmacological inhibition of ULK1, indicating that autophagy is regulating the intracellular trafficking and processing of APP. These findings contribute to the understanding of the role of autophagy in APP metabolism and could potentially have implications for new therapeutic approaches for AD.
阿尔茨海默病(AD)的病理特征是淀粉样 beta(Aβ)斑块和自噬功能障碍。淀粉样β是由淀粉样前体蛋白(APP)的连续蛋白水解产生的,而细胞内APP的加工部位还存在很大争议,其中可能包括自噬体。RUSH系统可以从内质网开始系统地研究荧光标记的mCherry-APP-EGFP的转运,我们在此研究了自噬的参与,包括ATG9在APP胞内转运和处理中的作用。我们通过活细胞成像、免疫荧光和 Western 印迹对表达 RUSH mCherry-APP-EGFP 系统的 HeLa 细胞进行了研究。我们发现,在 ATG9 基因敲除的细胞中,mCherry-APP-EGFP 通过高尔基体的速度更快。此外,ATG9 基因缺失会使 mCherry-APP-EGFP 从早期内体和溶酶体转移到质膜,同时内吞作用也会减弱。重要的是,mCherry-APP-EGFP转运的这种改变导致分泌的mCherry-可溶性APP和C-末端片段-EGFP增加。药物抑制ULK1也会产生这些效应,这表明自噬正在调节APP的胞内转运和处理。这些发现有助于人们了解自噬在APP代谢中的作用,并有可能对AD的新治疗方法产生影响。
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引用次数: 0
ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism ATG7(2) 与代谢蛋白相互作用并调节中枢能量代谢
IF 4.5 3区 生物学 Q3 CELL BIOLOGY Pub Date : 2024-04-11 DOI: 10.1111/tra.12933
Kevin Ostacolo, Adrián López García de Lomana, Clémence Larat, Valgerdur Hjaltalin, Kristrun Yr Holm, Sigríður S. Hlynsdóttir, Margaret Soucheray, Linda Sooman, Ottar Rolfsson, Nevan J. Krogan, Eirikur Steingrimsson, Danielle L. Swaney, Margret H. Ogmundsdottir
Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity‐purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein–protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice‐dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform‐dependent expression of a key autophagy gene.
大自噬/自噬是一种重要的分解代谢过程,它以包括蛋白质、细胞器和病原体在内的多种细胞成分为目标。ATG7是一种参与自噬过程的蛋白质,它在维持细胞稳态方面起着至关重要的作用,并可能导致癌症等疾病的发生。ATG7 通过促进 ATG8 蛋白在不断生长的自噬体膜上脂化来启动自噬。非规范异构体 ATG7(2) 无法进行 ATG8 脂化,但其细胞调控和功能尚不清楚。在这里,我们发现了 ATG7(2) 与 ATG7(1)(典型异构体)不同的调控和功能。首先,亲和纯化质谱分析表明,ATG7(2)与代谢蛋白建立了直接的蛋白-蛋白相互作用(PPIs),而ATG7(1)主要与自噬机制蛋白相互作用。此外,我们还发现 ATG7(2) 能介导代谢活性的降低,这突显了这一重要自噬蛋白的一种新的剪接依赖性功能。然后,我们发现 ATG7(1) 和 ATG7(2) 在人体组织中的表达模式存在差异。最后,我们的研究揭示了 ATG7(2) 与 ATG7(1) 在表达、蛋白相互作用和功能方面的不同模式。这些发现表明,主要分解代谢过程之间的分子转换是通过一个关键自噬基因的同工酶依赖性表达实现的。
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引用次数: 0
Spatial-Temporal Mapping Reveals the Golgi as the Major Processing Site for the Pathogenic Swedish APP Mutation: Familial APP Mutant Shifts the Major APP Processing Site. 空间-时间图谱显示高尔基体是致病性瑞典APP突变的主要加工部位:家族性APP突变体转移了APP的主要加工部位
IF 4.5 3区 生物学 Q3 CELL BIOLOGY Pub Date : 2024-03-01 DOI: 10.1111/tra.12932
Jingqi Wang, Paul A Gleeson, Lou Fourriere

Alzheimer's disease is associated with increased levels of amyloid beta (Aβ) generated by sequential intracellular cleavage of amyloid precursor protein (APP) by membrane-bound secretases. However, the spatial and temporal APP cleavage events along the trafficking pathways are poorly defined. Here, we use the Retention Using Selective Hooks (RUSH) to compare in real time the anterograde trafficking and temporal cleavage events of wild-type APP (APPwt) with the pathogenic Swedish APP (APPswe) and the disease-protective Icelandic APP (APPice). The analyses revealed differences in the trafficking profiles and processing between APPwt and the APP familial mutations. While APPwt was predominantly processed by the β-secretase, BACE1, following Golgi transport to the early endosomes, the transit of APPswe through the Golgi was prolonged and associated with enhanced amyloidogenic APP processing and Aβ secretion. A 20°C block in cargo exit from the Golgi confirmed β- and γ-secretase processing of APPswe in the Golgi. Inhibition of the β-secretase, BACE1, restored APPswe anterograde trafficking profile to that of APPwt. APPice was transported rapidly through the Golgi to the early endosomes with low levels of Aβ production. This study has revealed different intracellular locations for the preferential cleavage of APPwt and APPswe and Aβ production, and the Golgi as the major processing site for APPswe, findings relevant to understand the molecular basis of Alzheimer's disease.

阿尔茨海默病与淀粉样蛋白前体蛋白(APP)在细胞内被膜结合分泌酶依次裂解而产生的淀粉样β(Aβ)水平升高有关。然而,APP沿转运途径的空间和时间裂解事件还不十分明确。在这里,我们利用选择性挂钩保留(RUSH)实时比较了野生型APP(APPwt)与致病性瑞典APP(APPswe)和疾病保护性冰岛APP(APPice)的前向运输和时间裂解事件。分析结果表明,APPwt 和 APP 家族突变体之间的转运特征和加工过程存在差异。APPwt在高尔基体转运到早期内体后主要由β分泌酶BACE1处理,而APPswe在高尔基体的转运时间延长,并与淀粉样蛋白生成APP处理和Aβ分泌增强有关。20°C阻断货物从高尔基体的出口证实了APPswe在高尔基体中的β和γ分泌酶加工。抑制β分泌酶BACE1可使APPswe的前向运输曲线恢复到APPwt的曲线。APPice 通过高尔基体快速转运至早期内体,Aβ生成水平较低。这项研究揭示了 APPwt 和 APPswe 在细胞内优先裂解和产生 Aβ 的不同位置,以及高尔基体是 APPswe 的主要加工场所,这些发现与了解阿尔茨海默病的分子基础有关。
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引用次数: 0
Exploring Retrograde Trafficking: Mechanisms and Consequences in Cancer and Disease. 探索逆向贩运:癌症和疾病的机制与后果》。
IF 4.5 3区 生物学 Q3 CELL BIOLOGY Pub Date : 2024-02-01 DOI: 10.1111/tra.12931
Rachel Bingham, Helen McCarthy, Niamh Buckley

Retrograde trafficking (RT) orchestrates the intracellular movement of cargo from the plasma membrane, endosomes, Golgi or endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) in an inward/ER-directed manner. RT works as the opposing movement to anterograde trafficking (outward secretion), and the two work together to maintain cellular homeostasis. This is achieved through maintaining cell polarity, retrieving proteins responsible for anterograde trafficking and redirecting proteins that become mis-localised. However, aberrant RT can alter the correct location of key proteins, and thus inhibit or indeed change their canonical function, potentially causing disease. This review highlights the recent advances in the understanding of how upregulation, downregulation or hijacking of RT impacts the localisation of key proteins in cancer and disease to drive progression. Cargoes impacted by aberrant RT are varied amongst maladies including neurodegenerative diseases, autoimmune diseases, bacterial and viral infections (including SARS-CoV-2), and cancer. As we explore the intricacies of RT, it becomes increasingly apparent that it holds significant potential as a target for future therapies to offer more effective interventions in a wide range of pathological conditions.

逆行运输(RT)以内向/外向的方式协调来自质膜、内体、高尔基体或内质网(ER)-高尔基体中间区室(ERGIC)的货物在细胞内的移动。RT是逆向运输(向外分泌)的对立运动,两者共同作用以维持细胞平衡。这是通过维持细胞极性、检索负责逆向运输的蛋白质以及重新定向定位错误的蛋白质来实现的。然而,异常 RT 会改变关键蛋白的正确位置,从而抑制或改变它们的典型功能,可能导致疾病。本综述将重点介绍在了解 RT 的上调、下调或劫持如何影响癌症和疾病中关键蛋白的定位从而推动疾病进展方面取得的最新进展。受异常 RT 影响的货物种类繁多,包括神经退行性疾病、自身免疫性疾病、细菌和病毒感染(包括 SARS-CoV-2)以及癌症。随着我们对 RT 复杂性的探索,我们越来越清楚地认识到,RT 作为未来疗法的一个靶点具有巨大的潜力,可为各种病理状况提供更有效的干预。
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引用次数: 0
Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD 调节 CLN5 和 CTSD 细胞内贩运和释放的机制
IF 4.5 3区 生物学 Q3 CELL BIOLOGY Pub Date : 2024-01-21 DOI: 10.1111/tra.12925
Robert J. Huber, William D. Kim, Morgan L. D. M. Wilson-Smillie
Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal-lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP-3 complex mu subunit, the lysosomal-trafficking regulator LYST, mucopilin-1, and the Wiskott–Aldrich syndrome-associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation-independent mannose-6-phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans.
类神经细胞色素脂褐质沉着病神经元 5(CLN5)和凝血酶 D(CTSD)是可溶性溶酶体酶,也可在细胞外定位。在人类中,CLN5 和 CTSD 的同基因突变分别导致 CLN5 病和 CLN10 病,这是神经细胞类脂质硬化症(俗称巴顿病)的两个亚型。CLN5和CTSD在细胞内的转运及其从细胞中释放的调控机制尚不十分清楚。在这里,我们以社会阿米巴盘基变形虫为模型系统,研究了调节人类 CLN5(Cln5)和 CTSD(CtsD)的盘基变形虫同源物的胞内运输和释放的途径和细胞成分。我们的研究表明,Cln5 和 CtsD 都含有促进它们从细胞中释放的分泌信号肽。与 Cln5 一样,细胞外的 CtsD 也是糖基化的。此外,Cln5的释放受细胞外CtsD数量的调节。自噬诱导可促进 Cln5 的释放,在较小程度上也可促进 CtsD 的释放。Cln5 的释放需要自噬蛋白 Atg1、Atg5 和 Atg9 以及自噬体-溶酶体融合。CtsD的释放需要Atg1和Atg5。总之,这些数据支持这样一个模型,即 Cln5 和 CtsD 通过其分泌信号肽以及与自噬相关的途径从细胞中主动释放出来。Cln5和CtsD从细胞中释放出来还需要微丝和盘状核虫同源的人类AP-3复合体μ亚基、溶酶体贩运调节因子LYST、粘蛋白-1和Wiskott-Aldrich综合征相关蛋白WASH,它们在该模式生物中都能调节溶酶体的外泌。这些研究结果表明,溶酶体外泌也有助于 Cln5 和 CtsD 从细胞中释放出来。此外,我们还报告了ABC转运体、微管、渗透压以及人类索氏蛋白和阳离子依赖性甘露糖-6-磷酸受体的D. Discoideum推测同源物在调节Cln5和CtsD的胞内/胞外分布中的作用。总之,这项研究确定了调节盘形盘虫细胞中Cln5和CtsD释放的细胞机制,并深入探讨了CLN5和CTSD的贩运改变如何导致人类疾病。
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引用次数: 0
Endoglin mutants retained in the endoplasmic reticulum exacerbate loss of function in hereditary hemorrhagic telangiectasia type 1 (HHT1) by exerting dominant negative effects on the wild type allele 保留在内质网中的内胚层蛋白突变体通过对野生型等位基因产生显性负效应而加剧遗传性出血性毛细血管扩张症1型(HHT1)的功能丧失
IF 4.5 3区 生物学 Q3 CELL BIOLOGY Pub Date : 2024-01-16 DOI: 10.1111/tra.12928
Nesrin Gariballa, Sally Badawi, Bassam R. Ali
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder affecting 1 in 5000–8000 individuals. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is the most common HHT and manifests as diverse vascular malformations ranging from mild symptoms such as epistaxis and mucosal and cutaneous telangiectases to severe arteriovenous malformations (AVMs) in the lungs, brain or liver. HHT1 is caused by heterozygous mutations in the ENG gene, which encodes endoglin, the TGFβ homodimeric co-receptor. It was previously shown that some endoglin HHT1-causing variants failed to traffic to the plasma membrane due to their retention in the endoplasmic reticulum (ER) and consequent degradation by ER-associated degradation (ERAD). Endoglin is a homodimer formed in the ER, and we therefore hypothesized that mixed heterodimers might form between ER-retained variants and WT protein, thus hampering its maturation and trafficking to the plasma membrane causing dominant negative effects. Indeed, HA-tagged ER-retained mutants formed heterodimers with Myc-tagged WT endoglin. Moreover, variants L32R, V105D, P165L, I271N and C363Y adversely affected the trafficking of WT endoglin by reducing its maturation and plasma membrane localization. These results strongly suggest dominant negative effects exerted by these ER-retained variants aggravating endoglin loss of function in patients expressing them in the heterozygous state with the WT allele. Moreover, this study may help explain some of the variability observed among HHT1 patients due to the additional loss of function exerted by the dominant negative effects in addition to that due to haploinsufficiency. These findings might also have implications for some of the many conditions impacted by ERAD.
遗传性出血性毛细血管扩张症(HHT)是一种常染色体显性遗传疾病,每 5000-8000 人中就有 1 人患病。遗传性出血性毛细血管扩张症 1 型(HHT1)是最常见的遗传性出血性毛细血管扩张症,表现为各种血管畸形,从鼻衄、粘膜和皮肤毛细血管扩张症等轻微症状到肺部、脑部或肝部严重的动静脉畸形(AVM)不等。HHT1是由ENG基因的杂合突变引起的,ENG基因编码TGFβ同源二聚体共受体endoglin。以前的研究表明,一些内胚叶蛋白 HHT1 致病变体由于滞留在内质网(ER)中,因此无法转运到质膜,从而被ER相关降解(ERAD)降解。Endoglin 是一种在 ER 中形成的同源二聚体,因此我们假设 ER 保留的变体与 WT 蛋白之间可能会形成混合的异源二聚体,从而阻碍其成熟和向质膜的迁移,造成显性的负面影响。事实上,HA 标记的 ER 保留突变体与 Myc 标记的 WT endoglin 形成了异二聚体。此外,变体 L32R、V105D、P165L、I271N 和 C363Y 通过降低 WT endoglin 的成熟度和质膜定位,对其贩运产生了不利影响。这些结果强烈表明,这些保留ER的变体会产生显性负效应,加剧与WT等位基因杂合表达这些变体的患者的内切蛋白功能丧失。此外,这项研究可能有助于解释在 HHT1 患者中观察到的一些变异性,这些变异除了单倍体功能缺失造成的功能丧失外,显性负效应还造成了额外的功能丧失。这些发现还可能对受ERAD影响的许多疾病产生影响。
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引用次数: 0
Glucocorticoids rescue cell surface trafficking of R451C Neuroligin3 and enhance synapse formation 糖皮质激素可挽救 R451C Neuroligin3 的细胞表面贩运并增强突触的形成
IF 4.5 3区 生物学 Q3 CELL BIOLOGY Pub Date : 2024-01-14 DOI: 10.1111/tra.12930
Tamara Diamanti, Laura Trobiani, Lorenza Mautone, Federica Serafini, Roberta Gioia, Laura Ferrucci, Clotilde Lauro, Sara Bianchi, Camilla Perfetto, Stefano Guglielmo, Raimondo Sollazzo, Ezio Giorda, Andrea Setini, Davide Ragozzino, Elena Miranda, Davide Comoletti, Silvia Di Angelantonio, Emanuele Cacci, Antonella De Jaco
Neuroligins are synaptic cell adhesion proteins with a role in synaptic function, implicated in neurodevelopmental disorders. The autism spectrum disorder-associated substitution Arg451Cys (R451C) in NLGN3 promotes a partial misfolding of the extracellular domain of the protein leading to retention in the endoplasmic reticulum (ER) and the induction of the unfolded protein response (UPR). The reduced trafficking of R451C NLGN3 to the cell surface leads to altered synaptic function and social behavior. A screening in HEK-293 cells overexpressing NLGN3 of 2662 compounds (FDA-approved small molecule drug library), led to the identification of several glucocorticoids such as alclometasone dipropionate, desonide, prednisolone sodium phosphate, and dexamethasone (DEX), with the ability to favor the exit of full-length R451C NLGN3 from the ER. DEX improved the stability of R451C NLGN3 and trafficking to the cell surface, reduced the activation of the UPR, and increased the formation of artificial synapses between HEK-293 and hippocampal primary neurons. The effect of DEX was validated on a novel model system represented by neural stem progenitor cells and differentiated neurons derived from the R451C NLGN3 knock-in mouse, expressing the endogenous protein. This work shows a potential rescue strategy for an autism-linked mutation affecting cell surface trafficking of a synaptic protein.
神经胶质蛋白是突触细胞粘附蛋白,在突触功能中发挥作用,并与神经发育障碍有关。NLGN3 中与自闭症谱系障碍相关的置换 Arg451Cys(R451C)会促进蛋白质胞外结构域的部分错误折叠,导致其滞留在内质网(ER)中并诱发未折叠蛋白反应(UPR)。R451C NLGN3向细胞表面的贩运减少导致突触功能和社会行为的改变。在过表达 NLGN3 的 HEK-293 细胞中筛选了 2662 种化合物(FDA 批准的小分子药物库),最终确定了几种糖皮质激素,如二丙酸阿氯米松、地索奈德、泼尼松龙磷酸钠和地塞米松(DEX),它们都能促进全长 R451C NLGN3 从 ER 退出。DEX提高了R451C NLGN3的稳定性并使其向细胞表面迁移,减少了UPR的激活,增加了HEK-293和海马初级神经元之间人工突触的形成。DEX的作用在一个新的模型系统中得到了验证,该系统由神经干祖细胞和分化的神经元组成,这些神经元来自表达内源性蛋白的R451C NLGN3基因敲入小鼠。这项工作显示了一种潜在的拯救策略,可以挽救与自闭症有关的突变,这种突变会影响突触蛋白的细胞表面转运。
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引用次数: 0
Rescue of secretion of rare-disease-associated misfolded mutant glycoproteins in UGGT1 knock-out mammalian cells 在 UGGT1 基因被敲除的哺乳动物细胞中挽救与罕见疾病相关的错误折叠突变糖蛋白的分泌
IF 4.5 3区 生物学 Q3 CELL BIOLOGY Pub Date : 2024-01-14 DOI: 10.1111/tra.12927
Gabor Tax, Kevin P. Guay, Ludovica Pantalone, Martina Ceci, Tatiana Soldà, Charlie J. Hitchman, Johan C. Hill, Snežana Vasiljević, Andrea Lia, Carlos P. Modenutti, Kees R. Straatman, Angelo Santino, Maurizio Molinari, Nicole Zitzmann, Daniel N. Hebert, Pietro Roversi, Marco Trerotola
Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER-localized eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re-glucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity (“responsive mutant”). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop-2-Q118E, E227K and L186P mutants, which cause gelatinous drop-like corneal dystrophy (GDLD). Compared with the wild-type Trop-2, which is correctly localized at the plasma membrane, these Trop-2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop-2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop-2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1−/−cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad-spectrum rescue-of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.
内质网(ER)保留折叠错误的糖蛋白是由位于ER的真核糖蛋白分泌检查点--UDP-葡萄糖糖蛋白葡萄糖基转移酶(UGGT)--介导的。这种酶能识别折叠错误的糖蛋白,并通过对其中一个 N-连接的糖基进行再糖基化,将其标记为保留在 ER 中。在分泌型糖蛋白基因先天突变的背景下,UGGT 介导的 ER 保留可导致罕见疾病,即使突变型糖蛋白仍具有活性("反应突变体")。利用激光共聚焦扫描显微镜,我们研究了人Trop-2-Q118E、E227K和L186P突变体的亚细胞定位,这些突变体会导致胶样滴状角膜营养不良症(GDLD)。与野生型 Trop-2 正确定位于质膜相比,这些 Trop-2 突变体保留在 ER 中。我们在通过 CRISPR/Cas9 介导抑制 UGGT1 和/或 UGGT2 基因的哺乳动物细胞中研究了 Trop-2 Q118E、E227K 和 L186P 突变体的荧光嵌合体。在 UGGT1-/ 细胞中,Trop-2 Q118E、E227K 和 L186P 突变体的膜定位被成功挽救。UGGT1 还能在细胞中有效地对 Trop-2-Q118E-EYFP 进行再葡糖基化。这项研究支持这样的假设,即调节 UGGT1 将成为一种新的治疗策略,用于治疗与折叠错误的膜糖蛋白相关的病理状况(只要突变会损害但不会削弱功能),它还鼓励将 ER 糖蛋白折叠质量控制调节剂作为广谱的分泌拯救药物,用于测试由反应性分泌糖蛋白突变体引起的罕见疾病。
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引用次数: 0
The emerging functions of intraflagellar transport 52 in ciliary transport and ciliopathies 纤毛运输和纤毛疾病中纤毛内运输 52 的新功能
IF 4.5 3区 生物学 Q3 CELL BIOLOGY Pub Date : 2024-01-11 DOI: 10.1111/tra.12929
Prajna Udupa, Debasish Kumar Ghosh
Ciliary transport in eukaryotic cells is an intricate and conserved process involving the coordinated assembly and functioning of a multiprotein intraflagellar transport (IFT) complex. Among the various IFT proteins, intraflagellar transport 52 (IFT52) plays a crucial role in ciliary transport and is implicated in various ciliopathies. IFT52 is a core component of the IFT-B complex that facilitates movement of cargoes along the ciliary axoneme. Stable binding of the IFT-B1 and IFT-B2 subcomplexes by IFT52 in the IFT-B complex regulates recycling of ciliary components and maintenance of ciliary functions such as signal transduction and molecular movement. Mutations in the IFT52 gene can disrupt ciliary trafficking, resulting in dysfunctional cilia and affecting cellular processes in ciliopathies. Such ciliopathies caused by IFT52 mutations exhibit a wide range of clinical features, including skeletal developmental abnormalities, retinal degeneration, respiratory failure and neurological abnormalities in affected individuals. Therefore, IFT52 serves as a promising biomarker for the diagnosis of various ciliopathies, including short-rib thoracic dysplasia 16 with or without polydactyly. Here, we provide an overview of the IFT52-mediated molecular mechanisms underlying ciliary transport and describe the IFT52 mutations that cause different disorders associated with cilia dysfunction.
真核细胞中的纤毛转运是一个复杂而保守的过程,涉及多蛋白鞘内转运(IFT)复合体的协调组装和运作。在各种 IFT 蛋白中,鞘内转运 52(IFT52)在纤毛转运中起着至关重要的作用,并与各种纤毛疾病有关。IFT52 是 IFT-B 复合物的核心成分,可促进货物沿纤毛轴丝移动。IFT-B 复合物中的 IFT52 与 IFT-B1 和 IFT-B2 亚复合物的稳定结合调节着纤毛成分的循环以及信号转导和分子运动等纤毛功能的维持。IFT52 基因突变会破坏纤毛的运输,导致纤毛功能障碍,影响纤毛疾病的细胞过程。由 IFT52 基因突变引起的纤毛疾病具有多种临床特征,包括骨骼发育异常、视网膜变性、呼吸衰竭和神经系统异常。因此,IFT52 是诊断各种纤毛症(包括伴有或不伴有多指畸形的短肋胸廓发育不良 16)的一种很有前景的生物标记物。在此,我们概述了 IFT52 介导的纤毛运输分子机制,并描述了导致与纤毛功能障碍相关的不同疾病的 IFT52 基因突变。
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