STRUCTURE-FUNCTION OF NEUROPHYSIN HORMONE SYSTEMS

这项研究的长期目标是了解 神经生理素-激素系统通过 一系列神经生理素类激素和化合物的结晶学研究 神经物理素肽复合体。神经物理素(NP)及其相关基因 神经肽被发现存在于大脑中与 认知和情绪功能以及心血管调节。 已有研究表明，在衰老的早期，外源性加压素(VP) 可以提高唤醒能力和记忆力。此外，催产素(OT)有 最近被发现在协调社会和文化方面发挥着重要作用 性关系。因此，对结构功能的了解 这一制度的关系可能在 了解与老龄化、精神病和其他领域有关的问题 神经内分泌学。我们最近结晶了一个NP-OT络合物 并计划解决其结构问题。这是NP的第一个晶体 与一种适合于X射线分析的完整激素复合。这个 晶体的衍射率至少达到2.8埃。我们计划解决 用分子结构置换的方法研究结构 来自NP-二肽复合体的信息，最近被 由我们实验室用单波长的新用途测定的 异常散射数据与溶剂展平过程相结合。 NP-二肽复合体，这是第一个NP结构 下定决心，给了我们一些意想不到的结果；我们观察到五个 与NP四聚体结合的二肽分子及其二聚化方式 这与溶液研究中得出的结论有很大不同。 我们计划将结构优化到尽可能高的分辨率。我们 在过去结晶了5个其他NP-肽复合体，其中4个 在上述空间基团以外的空间基团中结晶。我们 计划解决它们的结构，以查看是否有混合 NP与多肽的相互作用，并寻找共同的结构 晶体中分子堆积的特点。这一点意义重大 由于这些络合物的浓度很高(1g/ml) 神经分泌颗粒(NSG)和我们的结构可能表明这些 复合体被包装在NSG中。我们计划生长NP-VP的晶体 复杂，求解其结构，并将其与在NP-OT中观察到的进行比较 很复杂。我们还计划在没有多肽的情况下生长NPs晶体。 或荷尔蒙，解决它们的结构，并将它们与 没有多肽或激素，解决它们的结构，并比较它们 与NP-激素或多肽复合体中的NPs。为了研究 在多肽结合上观察到构象变化。作为一个长期的 目的我们计划生长NPs和其他前体蛋白的晶体。 C-末端含有Copeptin的“大”NPs，并进行 一旦样品可用，就会进行结晶学研究。建议数 研究可以回答关于该结构的许多关键问题-- 神经内科药物的功能。它还将增加我们对以下方面的知识 神经物理素具后路特异性的分子基础 荷尔蒙、蛋白质-多肽相互作用和蛋白质动力学。 GRANT=R29DK44650 这项拟议研究的目的是阐明分子机制。 肝细胞将分泌和内吞的蛋白质运输到 确定了细胞质的位置。具体地说，我们将检验这个假设 微管细胞骨架及其相关的ATPase或马达 酶，在组织、运输和靶向方面起着重要作用 肝细胞内不同的囊泡群。无数的研究 微管与囊泡运输和肝脏病理有关。 然而，以前的方法在很大程度上是间接的，依赖于 关于药物在完整动物或灌流器官系统中的扰动 并产生了具有挑衅性但不完整和相互矛盾的结果。 因此，还没有确定微管和相关的 这些动作都需要ATPase。目前，这些机制由 肝细胞区别于分泌颗粒、内小体、 溶酶体和其他囊泡室将它们定向到 具有精度和效率的正弦或管状表面 完全没有定义。我们的目标是进行一项明确而新颖的研究 微管细胞骨架在囊泡中的作用 利用最先进的细胞生物学技术在肝细胞内进行转运 技巧。这项拟议的研究有三个具体目标。首先，我们将 检查微管是如何组织和极化的 肝细胞相对于肝窦和肝小管表面：a) 共聚焦免疫荧光和电子显微镜检查 结合计算机辅助重建形态计量学；b) 培养肝细胞的微管极性分析。第二，我们将 确定哪些内分泌成分和内吞成分在移动 联合微量注射在培养肝细胞联体中的微管 荧光探针和独特的抑制性抗体或药物 电脑增强，荧光，视频显微镜。第三，我们将测试 微管和相关的运动酶(动蛋白， 动力蛋白、动力蛋白)在囊泡运输中的作用 通透性和匀浆肝细胞的生化操作， 以及使用纯化的囊泡和细胞骨架的无细胞系统 组件。本建议书中所述的技术和实验 是肝细胞囊泡转运研究中所独有的。他们会 极大地扩展了我们对肝细胞如何分泌/排泄的理解 蛋白质以及这些关键过程是如何被药物或 疾病状态，如胆汁淤积症和酒精性肝硬变。

The long-term objective of this research is to understand the characteristics of the neurophysin-hormone system through crystallographic studies on a series of neurophysin-hormone and neurophysin-peptide complexes. Neurophysin (NP) and its associated neuropeptides have been found in areas of the brain associated with cognitive and emotional function as well as cardiovascular regulation. It has been suggested that during early aging, exogenous vasopressin (VP) could improve arousal and memory. In addition, oxytocin (OT) has recently been found to play an important role in orchestrating social and sexual relationships. Thus a knowledge of the structure-function relationship of this system may have long term significance in understanding problems related to aging, mental illness and other areas of neuroendocrinology. We have recently crystallized an NP-OT complex and plan to solve its structure. This is the first crystal of NP complexed with an intact hormone suitable for X-ray analysis. The crystals diffract to at least 2.8 Angstroms resolution. We plan to solve the structure by molecular replacement methods using structural information from a NP-dipeptide complex which has recently been determined in our laboratory by a novel use of the single-wavelength anomalous scattering data coupled with a solvent flattening procedure. The NP-dipeptide complex, which is the first NP structure to be determined, has given us some unexpected results; we observe five dipeptide molecules bound to the NP tetramer and a mode of dimerization which differs considerably from conclusions reached in solution studies. We plan to refine the structure to the highest possible resolution. We have in the past crystallized 5 other NP-peptide complexes, 4 of which were crystallized in space groups other than those described above. We plan to solve their structures in order to see whether there are mixed interactions between NP and peptides, and to find common structural features of the molecular packing in the crystals. This is significant because of the high concentration of these complexes (1g/ml) found in the neurosecretory granules (NSG), and our structures may suggest how these complexes are packaged in the NSG. We plan to grow crystals of the NP-VP complex, solve its structure and compare it to that observed in the NP-OT complex. We also plan to grow crystals of NPs in the absence of peptides or hormone, solve their structures, and compare them with the NPs in the absence of peptides or hormone, solve their structures, and compare them with the NPs in the NP-hormone or peptide complexes. to study the conformational changes observed on peptide binding. As a long-term objective we plan to grow crystals of precursor proteins of NPs and other "big" NPs which contain copeptin at the C-terminal, and to carry out crystallographic studies once the samples become available. The proposed research could answer many of the key questions about the structure- function of neurophysins. It will also increase our knowledge concerning the molecular basis of the specificity of neurophysin for posterior hormones, protein-peptide interactions, and protein dynamics in general. GRANT=R29DK44650 The goal of this proposed study is to elucidate the molecular mechanisms by which hepatocytes transport secretory and endocytosed proteins to defined cytoplasmic locations. Specifically, we will test the hypothesis that the microtubule cytoskeleton and its associated ATPases, or motor enzymes, play a major role in the organization, transport, and targeting of different vesicle populations within the hepatocyte. Numerous studies have implicated microtubules in vesicular transport and liver pathology. However, previous approaches have been largely indirect and have relied on drug perturbation either in intact animals or perfused organ systems and have yielded provocative but incomplete and conflicting results. Thus, it has not been established that microtubules and associated ATPases are required for these movements. At present, the mechanisms by which the hepatocyte discriminates between secretory granules, endosomes, lysosomes and other vesicular compartments to direct them to either sinusoidal or canalicular surfaces with precision and efficiency are totally undefined. Our goal is to conduct a definitive and novel study on the role of the microtubule-based cytoskeleton in vesicular trafficking within the hepatocyte using state of the art cell biological techniques. The proposed study has three specific aims. First, we will examine how microtubules are organized and polarized within the hepatocyte in respect to the sinusoidal and canalicular surfaces by: a) confocal-immunofluorescent and electron microscopic examination in conjunction with computer-aided reconstruction morphometry; b) microtubule polarity assays of cultured hepatocytes. Second, we will determine what secretory and endocytotic components move along microtubules in cultured hepatocyte couplets by combining microinjection of fluorescent probes and unique inhibitory antibodies or drugs with computer-enhanced, fluorescent, video microscopy. Third, we will test the participation of microtubules and associated motor enzymes (kinesin, dynein, dynamin) in vesicle transport through morphological and biochemical manipulation of permeabilized and homogenized hepatocytes, and cell-free systems using purified vesicular and cytoskeletal components. The technology and experiments described in this proposal are unique to the study of vesicular transport in hepatocytes. They will expand greatly our understanding of how liver cells secrete/excrete proteins and how these critical processes are disrupted by drugs or diseased states such as cholestasis and alcohol-induced cirrhosis.