Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.
Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.
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DOI：
10.1371/journal.ppat.1004794
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发表时间：
2015-04
期刊：
影响因子：
6.7
通讯作者：
Komuniecki R
中科院分区：
文献类型：
--
作者：
Law W;Wuescher LM;Ortega A;Hapiak VM;Komuniecki PR;Komuniecki R
单胺类药物，如5-羟色胺和酪胺（TA），在外源应用时可使自由线虫和寄生线虫瘫痪，血清素能激动剂已被用于清除体内扭曲血蜱感染。由于没有线虫细胞系，动物筛选选择有限，我们开发了一个筛选平台来鉴定单胺受体激动剂。关键受体在嵌合的、基因工程的秀丽隐杆线虫中异种表达，在激动剂刺激下可能产生强大的表型。这种方法潜在地保留了受体的独特药理学，同时包括线虫特异性辅助蛋白和线虫角质层。重要的是，单胺依赖性麻痹的敏感性可以通过低渗培养或使用表皮通透性增加的bus突变体而显著增加。我们已经证明单胺依赖性抑制关键中间神经元、胆碱能运动神经元或体壁肌抑制运动并导致瘫痪。具体来说，5-HT麻痹了线虫5-HT受体缺失的动物，这些动物表达了线虫、昆虫或人类在胆碱能运动神经元中关键的g αo偶联5- ht1样受体的同源物。重要的是，8-OH-DPAT和PAPP， 5-HT受体激动剂，不同程度地使转基因动物瘫痪，8-OH-DPAT使表达人类受体的突变动物瘫痪，其浓度远低于影响秀丽隐杆线虫或昆虫的浓度。同样，5-HT和TA分别麻痹秀丽隐杆线虫5-HT或TA受体缺失的动物，在秀丽隐杆线虫的胆碱能运动神经元或体壁肌肉中表达5-HT或TA门控的Cl-通道。总之，这些数据表明，这种异源、异位表达筛选方法将有助于从寄生虫中鉴定关键单胺受体的激动剂，并可广泛应用于鉴定许多潜在的驱虫靶点的配体。单胺类药物，如5-羟色胺（5-HT）和酪胺（TA），在外源应用时可以使自由线虫和寄生线虫瘫痪。由于没有线虫细胞系，动物筛选选择有限，我们开发了一个筛选平台来鉴定单胺受体激动剂，该激动剂涉及寄生线虫在嵌合基因工程突变的秀丽隐杆线虫中关键受体的异源表达，在激动剂刺激后可能产生强大的表型。具体来说，我们已经证明，在关键的中间神经元、胆碱能运动神经元或体壁肌中，g αo偶联5-HT受体或单胺门控Cl-通道的激动剂依赖激活会抑制运动并导致瘫痪。这种方法包括线虫特异性辅助蛋白和线虫角质层，并且似乎保留了个体受体的独特药理学。总之，这些数据突出了这些转基因秀丽隐杆线虫在激动剂鉴定和驱虫筛选方面的潜力。
Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets. Monoamines, such as serotonin (5-HT) and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists that involves the heterologous expression of key receptors from parasitic nematodes in chimeric, genetically-engineered mutant C. elegans, at sites likely to yield robust phenotypes upon agonist stimulation. Specifically, we have demonstrated that agonist dependent activation of Gαo-coupled 5-HT receptors or monoamine-gated Cl- channels in key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. This approach includes nematode-specific accessory proteins and the nematode cuticle, and appears to preserve the unique pharmacologies of the individual receptors. Together these data highlight the utility of these transgenic C. elegans for agonist identification and their potential for anthelmintic screening.
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影响因子：
9.8
作者：
Donnelly JL;Clark CM;Leifer AM;Pirri JK;Haburcak M;Francis MM;Samuel AD;Alkema MJ
通讯作者：
Alkema MJ
影响因子：
3.8
作者：
Hotez PJ;Kamath A
通讯作者：
Kamath A
影响因子：
2.4
作者：
Buckingham SD;Sattelle DB
通讯作者：
Sattelle DB
DOI：
10.1073/pnas.0409009101
发表时间：
2005-03-01
影响因子：
11.1
作者：
Gray, JM;Hill, JJ;Bargmann, CI
通讯作者：
Bargmann, CI
DOI：
10.1073/pnas.0806933105
发表时间：
2008-11-25
影响因子：
11.1
作者：
Boulin, Thomas;Gielen, Marc;Bessereau, Jean-Louis
通讯作者：
Bessereau, Jean-Louis