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Modifying Expression Modes of Human Neurotensin Receptor Type 1 Alters Sensing Capabilities for Agonists in Yeast Signaling Biosensor
Biotechnology Journal ( IF 3.2 ) Pub Date : 2017-11-30 , DOI: 10.1002/biot.201700522 Hiroki Hashi 1 , Yasuyuki Nakamura 1 , Jun Ishii 1 , Akihiko Kondo 1, 2
Biotechnology Journal ( IF 3.2 ) Pub Date : 2017-11-30 , DOI: 10.1002/biot.201700522 Hiroki Hashi 1 , Yasuyuki Nakamura 1 , Jun Ishii 1 , Akihiko Kondo 1, 2
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Neurotensin receptor type 1 (NTSR1), a member of the G-protein-coupled receptor (GPCR) family, is naturally activated by binding of a neurotensin peptide, leading to a variety of physiological effects. The budding yeast Saccharomyces cerevisiae is a proven host organism for assaying the agonistic activation of human GPCRs. Previous studies showed that yeast cells can functionally express human NTSR1 receptor, permitting the detection of neurotensin-promoted signaling using a ZsGreen fluorescent reporter gene. However, the fluorescence intensity (sensitivity) of NTSR1-expressing yeast cells is low compared to that of yeast cells expressing other human GPCRs (e.g., human somatostatin receptors). The present study sought to increase the sensitivity of the NTSR1-expressing yeast for use as a fluorescent biosensor, including modification of the expression of human NTSR1 in yeast. Changes in the transcription, translation, and transport of the receptor are attempted by altering the promoter, consensus Kozak-like sequence, and secretion signal sequences of the NTSR1-encoding gene. The resulting yeast cells exhibited increased sensitivity to exogenously added peptide. The cells are further engineered by using cell-surface display technology to ensure that the agonistic peptides are secreted and tethered to the yeast cell wall, yielding cells with enhanced NTSR1 activation. This yeast biosensor holds promise for the identification of agonists to treat NTSR1-related diseases. This study aims to increase the sensitivity of the human neurotensin receptor type 1 (NTSR1)-expressing yeast cells as a fluorescent biosensor, including modification of the expression modes of human NTSR1 in yeast Saccharomyces cerevisiae. To modify the transcription, translation, and transport of the receptor, changes in the promoter, yeast consensus Kozak-like sequence, and secretion signal sequences of the NTSR1-encoding gene are tested. The newly constructed yeast biosensor holds promise for the identification of agonists to treat NTSR1-related diseases.
中文翻译：
修改人类神经降压素受体1型的表达模式改变酵母信号生物传感器中激动剂的感知能力。
1型神经降压素受体（NTSR1）是G蛋白偶联受体（GPCR）家族的成员，通过与神经降压素肽的结合而自然激活，从而导致多种生理效应。出芽的酿酒酵母是一种经过验证的宿主生物，可用于测定人GPCR的激动性。先前的研究表明酵母细胞可以功能性表达人NTSR1受体，从而允许使用ZsGreen检测神经降压素促进的信号传导荧光报告基因。但是，与表达其他人GPCR（例如人生长抑素受体）的酵母细胞相比，表达NTSR1的酵母细胞的荧光强度（敏感性）低。本研究试图提高表达NTSR1的酵母用作荧光生物传感器的敏感性，包括修饰酵母中人NTSR1的表达。通过改变编码NTSR1的启动子，共有的Kozak样序列和分泌信号序列，尝试改变受体的转录，翻译和转运。所得的酵母细胞对外源添加的肽显示出增加的敏感性。通过使用细胞表面展示技术对细胞进行进一步工程设计，以确保将激动肽分泌并束缚在酵母细胞壁上，产生具有增强的NTSR1激活的细胞。该酵母生物传感器有望用于鉴定治疗NTSR1相关疾病的激动剂。这项研究旨在提高表达人类神经降压素受体1型（NTSR1）的酵母细胞作为荧光生物传感器的敏感性，包括修饰酵母中人类NTSR1的表达方式酿酒酵母。为了修饰受体的转录，翻译和转运，测试了启动子，酵母共有Kozak样序列和NTSR1编码基因的分泌信号序列的变化。新型酵母生物传感器有望用于鉴定治疗NTSR1相关疾病的激动剂。
更新日期：2017-12-14
中文翻译：
修改人类神经降压素受体1型的表达模式改变酵母信号生物传感器中激动剂的感知能力。
1型神经降压素受体（NTSR1）是G蛋白偶联受体（GPCR）家族的成员，通过与神经降压素肽的结合而自然激活，从而导致多种生理效应。出芽的酿酒酵母是一种经过验证的宿主生物，可用于测定人GPCR的激动性。先前的研究表明酵母细胞可以功能性表达人NTSR1受体，从而允许使用ZsGreen检测神经降压素促进的信号传导荧光报告基因。但是，与表达其他人GPCR（例如人生长抑素受体）的酵母细胞相比，表达NTSR1的酵母细胞的荧光强度（敏感性）低。本研究试图提高表达NTSR1的酵母用作荧光生物传感器的敏感性，包括修饰酵母中人NTSR1的表达。通过改变编码NTSR1的启动子，共有的Kozak样序列和分泌信号序列，尝试改变受体的转录，翻译和转运。所得的酵母细胞对外源添加的肽显示出增加的敏感性。通过使用细胞表面展示技术对细胞进行进一步工程设计，以确保将激动肽分泌并束缚在酵母细胞壁上，产生具有增强的NTSR1激活的细胞。该酵母生物传感器有望用于鉴定治疗NTSR1相关疾病的激动剂。这项研究旨在提高表达人类神经降压素受体1型（NTSR1）的酵母细胞作为荧光生物传感器的敏感性，包括修饰酵母中人类NTSR1的表达方式酿酒酵母。为了修饰受体的转录，翻译和转运，测试了启动子，酵母共有Kozak样序列和NTSR1编码基因的分泌信号序列的变化。新型酵母生物传感器有望用于鉴定治疗NTSR1相关疾病的激动剂。
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