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.
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