REGULATION OF HISTIDINE DECARBOXYLASE

组氨酸脱羧酶的调节

• 批准号: 6380879
• 负责人: Timothy Cragin Wang
• 金额: $ 28.73万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 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/6380879
• 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）在肠嗜铬细胞（ECL）中高表达，负责l -组氨酸向组胺的转化。在初步研究中，我们已经证明了HDC基因在转染的胃细胞系中受到胃泌素的转录和转录后调控。HDC基因中介导胃泌素反应的启动子元件被鉴定为两个新的顺式作用元件（GAS-RE1和GAS-RE2），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。胃泌素通过MAP激酶依赖性途径激活HDC转录，导致GAS-REBP1和GAS-REBP2启动子结合增加。从人胃cDNA文库中克隆出一个GAS-REBP2候选蛋白，它似乎代表了一种新的35kd DNA结合蛋白。胃泌素刺激也会导致转化后HDC酶活性，最有可能是通过增加翻译。HDC激活也可以通过翻译后74 kDa的HDC前体蛋白裂解成更活跃的54 kD酶的形式发生。在初步研究中，我们将该裂解位点定位在一个8个氨基酸的区域，并证明该区域的缺失会抑制裂解和酶的激活。此外，我们已经证明HDC蛋白的表达导致HDC启动子活性的反馈抑制，并且这种抑制是独立于酶活性的，由n端肽序列介导。最后，我们已经证明4.8 kb的小鼠嗜铬粒蛋白A启动子足以靶向ECL细胞并介导转基因小鼠的胃泌素反应。提出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)进一步对GAS-REBP2候选蛋白进行表征，研究其在HDC基因调控中的作用。(2)精确定位HDC酶解位点，分析胃泌素对HDC的翻译调控作用。(3)定位介导转录抑制的n端HDC序列，探索其抑制机制。(4)最后，利用HDC- gfp报告基因构建体分析HDC基因在ECL细胞特异性表达所需的顺式作用DNA序列。总的来说，这些研究将为胃泌素调节HDC基因表达和酶活性的机制提供更好的理解。