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Identification and functional analysis of new chromogranin-derived peptides.

机译：新的嗜铬粒蛋白衍生肽的鉴定和功能分析。

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摘要

Chromogranin (Cg) is an acidic, heat-stable protein family including chromogranin A, chromogranin B/secretogranin I, secretogranin II, 7B2 and NESP 55. They are mainly located in the chromaffin granules and large dense-cored vesicles of endocrine and neuroendocrine cells and neurons. Chromogranin A and B have been suggested to play an important role in the biogenesis of secretory vesicles and to be precursors of small peptides. This dissertation deals with the identification of novel chromogranin-derived peptides and the investigation of their functions.; Size-exclusion, affinity and reverse-phase chromatographies were performed to purify peptides from porcine chromaffin granule lysate and the peptides were characterized by N-terminal sequencing and mass spectrometry.; A new CgA-derived peptide (GN-17) and two longer forms were identified. GN-17 is located between pancreastatin and WE-14 and was found to inhibit CK2 activity dose-dependently. The apparent Km increased due to the addition of GN-17 to the reaction system. However, the Vmax was not significant changed. Therefore, GN-17 is a competitive inhibitor of CK2 activity.; Porcine CgB cDNA was sequenced and three novel CgB-derived peptides (SR-17, HR-34 and KR-11) were identified from porcine chromaffin granule lysate. These peptides are located at the C-terminal region of CgB. We also found that SR-17 is phosphorylated at two serine residues (Ser595 and Ser600). Furthermore, we demonstrated that SR-17 and KR-11 are not only produced in chromaffin cells but also in peripheral noradrenergic nerves. Both peptides were shown to be co-released from the spleen with noradrenaline and/or NPY upon electric stimulation.; Functional investigation of CgB-derived peptides was undertaken. A major finding is that a binding motif for heat shock cognate protein 70 (Hsc70) is contained in SR-17 and that phosphorylation of SR-17 at Serine595 significantly enhances its association with Hsc70. Moreover, co-immunoprecipitation experiments showed that Hsc70 and intact CgB are associated in the chromaffin cells of the adrenal medulla. Immunohistochemical data and protein analysis of subcellular fractions indicate that CgB and Hsc70 co-localize, besides in the cytoplasm, also in the nucleus of chromaffin cells. The present result suggests that Hsc70 participates in the process of nuclear transportation of CgB. Phosphorylation of CgB is likely to mediate the regulation of this process.
机译：嗜铬粒蛋白（Cg）是一种酸性，热稳定的蛋白家族，包括嗜铬粒蛋白A，嗜铬粒蛋白B /分泌素I，分泌素II，7B2和NESP55。它们主要位于内分泌和神经内分泌细胞的嗜铬粒和大而稠密的囊泡中和神经元。已建议嗜铬粒蛋白A和B在分泌囊泡的生物发生中起重要作用，并且是小肽的前体。本文主要研究嗜铬粒蛋白新肽的鉴定及其功能研究。进行大小排阻，亲和层析和反相色谱法纯化猪嗜铬粒蛋白裂解液中的肽，并通过N端测序和质谱对肽进行表征。确定了一种新的CgA衍生肽（GN-17）和两种更长的形式。 GN-17位于胰腺抑素和WE-14之间，并被发现可剂量依赖性地抑制CK2活性。由于向反应体系中添加了GN-17，表观Km增加。但是，V max 没有显着变化。因此，GN-17是CK2活性的竞争性抑制剂。对猪CgB cDNA进行测序，并从猪嗜铬粒蛋白裂解物中鉴定出三种新颖的CgB衍生肽（SR-17，HR-34和KR-11）。这些肽位于CgB的C末端区域。我们还发现，SR-17在两个丝氨酸残基（Ser595和Ser600）处被磷酸化。此外，我们证明了SR-17和KR-11不仅在嗜铬细胞中产生，而且在周围的去甲肾上腺素能神经中产生。在电刺激后，两种肽均被证明与去甲肾上腺素和/或NPY从脾脏中共同释放。进行了CgB衍生肽的功能研究。一个主要发现是在SR-17中包含了热休克同源蛋白70（Hsc70）的结合基序，并且在Serine595上SR-17的磷酸化显着增强了其与Hsc70的结合。此外，共同免疫沉淀实验表明，Hsc70和完整的CgB与肾上腺髓质的嗜铬细胞相关。免疫组织化学数据和亚细胞级分的蛋白质分析表明，CgB和Hsc70除在细胞质中外，还在嗜铬细胞的细胞核中共定位。目前的结果表明Hsc70参与CgB的核运输过程。 CgB的磷酸化可能介导该过程的调节。

著录项

  • 作者

    Zesheng, Wang.;

  • 作者单位

    Universitaire Instelling Antwerpen (Belgium).;

  • 授予单位 Universitaire Instelling Antwerpen (Belgium).;
  • 学科 Chemistry Biochemistry.; Biology Neuroscience.
  • 学位 Ph.D.
  • 年度 2003
  • 页码 191 p.
  • 总页数 191
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;神经科学;
  • 关键词

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