REGULATION OF HISTIDINE DECARBOXYLASE

组氨酸脱羧酶的调节

• 批准号: 6011671
• 负责人: Timothy Cragin Wang
• 金额: $ 28.52万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-30 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6011671
• 关键词: DNA binding protein; cell line; chromaffin cells; chromogranins; gastric mucosa; gastrins; genetic library; genetic mapping; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; histidine decarboxylase; laboratory mouse; posttranslational modifications; transfection

Histidine decarboxylase (HDC) is highly expressed in enterochromaffin-like (ECL) cells, and carries out the conversion of L-histidine to histamine. In preliminary studies, we have demonstrated that the HDC gene is regulated both transcriptionally and post-transcriptionally by gastrin in transfected gastric cell lines. The promoter elements mediating gastrin responsiveness in the HDC gene have been identified as two novel cis-acting elements (GAS-RE1 and GAS-RE2) which are located in tandem downstream of the transcriptional start site, and bind to 52 kD and 35 kD proteins, respectively. The activation of HDC transcription by gastrin occurs through a MAP kinase-dependent pathway leading to increased promoter binding by GAS-REBP1 and GAS-REBP2. A candidate for GAS-REBP2 has been cloned from a human stomach cDNA library, and appears to represent a novel 35 kD DNA binding protein. Gastrin stimulation also leads to post-transitional HDC enzymatic activity, most likely through increases in translation. HDC activation also occurs through post-translational cleavage of the 74 kDa HDC precursor protein to a more active 54 kD enzymatic form. In preliminary studies, we have localized this cleavage site to an 8 amino acid region, and demonstrated that deletion of this region inhibits cleavage and enzymatic activation. Further, we have shown that expression of HDC protein leads to feedback inhibition of HDC promoter activity, and that this inhibition is independent of enzyme activity and mediated by N-terminal peptide sequences. Finally, we have shown that 4.8 kb of the mouse chromogranin A promoter are sufficient for targeting ECL cells and mediating gastrin responsiveness in transgenic mice. The proposed studies are aimed at investigating further the regulation of the HDC gene, using both in vitro and in vivo approaches. (1) The candidate GAS-REBP2 protein will be further characterized, and its role in HDC gene regulation investigated. (2) The HDC enzymatic cleavage site will be precisely defined, and translational regulation of HDC by gastrin will be analyzed. (3) The N-terminal HDC sequences mediating transcriptional inhibition will be mapped, and mechanisms of inhibition explored. (4) Finally, the cis-acting DNA sequences which are necessary for ECL cell-specific expression will be analyzed for the HDC gene using HDC-GFP reporter gene constructs. Overall, these studies will provide greater understanding of the mechanisms involved in the regulation of HDC gene expression and enzymatic activity by gastrin.

组氨酸脱羧酶（HDC）在肠嗜铬样细胞（ECL）中高度表达，并将L-组氨酸转化为组胺。 在初步的研究中，我们已经证明，HDC基因的转录和转录后调节的胃泌素在转染的胃细胞系。 HDC基因中调控胃泌素应答的启动子元件被鉴定为两个新的顺式作用元件（GAS-RE 1和GAS-RE 2），它们位于转录起始位点的下游串联，分别与52 kD和35 kD蛋白结合。 胃泌素对HDC转录的激活通过MAP激酶依赖性途径发生，导致GAS-REBP 1和GAS-REBP 2的启动子结合增加。 GAS-REBP 2的候选者已从人胃cDNA文库中克隆，并且似乎代表新的35 kD DNA结合蛋白。 胃泌素刺激还导致过渡后HDC酶活性，最有可能是通过增加翻译。 HDC活化还通过将74 kDa HDC前体蛋白翻译后切割成更有活性的54 kD酶形式而发生。 在初步研究中，我们已经将该切割位点定位于8个氨基酸的区域，并证明该区域的缺失抑制切割和酶促活化。 此外，我们已经表明，HDC蛋白的表达导致HDC启动子活性的反馈抑制，并且这种抑制不依赖于酶活性并且由N-末端肽序列介导。最后，我们已经表明，4.8 kb的小鼠嗜铬粒蛋白A启动子足以靶向ECL细胞和介导的胃泌素反应在转基因小鼠。 拟议的研究旨在进一步研究HDC基因的调控，使用体外和体内方法。 (1)候选GAS-REBP 2蛋白将被进一步表征，并研究其在HDC基因调控中的作用。 (2)将精确定义HDC酶切位点，并分析胃泌素对HDC的翻译调节。 (3)将绘制介导转录抑制的N-末端HDC序列，并探索抑制机制。 (4)最后，将使用HDC-GFP报告基因构建体分析HDC基因的ECL细胞特异性表达所必需的顺式作用DNA序列。 总体而言，这些研究将提供更多的了解的机制，参与调控的HDC基因表达和酶活性的胃泌素。