Biosynthesis, Processing &Secretion Of Neuropeptides

Child Health And Human Development

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Abstract

The intracellular sorting of pro-neuropeptides, prohormones and neurotrophins to the regulated secretory pathway (RSP) is essential for processing, storage and release of active proteins and peptides in the neuroendocrine cell. The sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), pro-insulin and brain derived neurotrophic factor (BDNF) to the RSP was investigated. Such studies have led to the better understanding of diseases related to defects in hormone and neuropeptide targeting, obesity, diabetes, memory and learning.We show that these pro-proteins undergo homotypic oligomerization as a concentration step, as they traverse the cell from the site of synthesis in the endoplasmic reticulum to the trans-Golgi network (TGN), where they are sorted into dense-core granules of the regulated secretory pathway for processing and secretion. Site-directed mutagenesis studies identified a consensus sorting motif consisting of two acidic residues, 12-15? apart from each other, exposed on the surface of these molecules, and two hydrophobic residues, 5-7? away from the acidic residues which are necessary for sorting these pro-proteins to the RSP. A RSP sorting receptor that was specific for the sorting signal of POMC, pro-insulin and BDNF was identified as membrane carboxypeptidase E (CPE). The two acidic residues in the prohormone/pro-BDNF sorting motif specifically interact with two basic residues, R255 and K260, of the sorting receptor, CPE, to effect sorting to the RSP. Using a CPE knockout (KO) mouse model, we showed missorting of endogenous POMC and proinsulin in pituitary and pancreatic islet cells, respectively, in these animals. Furthermore BDNF which modulates synaptic plasticity was missorted in cortical and hippocampal neurons, which could account for the memory deficits observed in CPE KO mice. These studies provide evidence for a sorting signal/receptor mediated mechanism for targeting prohormones, neuropeptides and the neurotrophin, BDNF, to the regulated secretory pathway in endocrine cells and neurons. We have also used our knowledge of the sorting motif of hormones to engineer biologically active mutant hormones that are redirected to the constitutive pathway. Such mutant hormones are currently being expressed in salivary glands for systemic secretion, with the ultimate aim of applying such technology to gene therapeutics. Interestingly, CPE knockout mice exhibit impaired glucose clearance and were insulin resistant. High levels of leptin in plasma were present in the KO mice, however, they showed no circulating fully-processed cocaine-amphetamine regulated transcript (CART), a peptide that is responsive to leptin-induced feedback inhibition of feeding. Aside from obesity and diabetes phenotypes, the KO mice were subfertile and showed deficits in GnRH processing in hypothalamus. They also showed abnormal retinograms and behavioral analyses revealed that the KO animals have diminished reactivity to stimuli, and reduced muscle strength, coordination and visual placing and toe-pinch reflexes. Thus CPE KO mice display a wide-range of neural and endocrine abnormalities and will be a useful model for understanding diseases such diabetes, obesity and deficits in learning and memory. In another study, the sorting and processing of a mutant form of CART (CART Leu34Phe), found in a family of obese patients was investigated. CART, found in brain, is an anorexigenic peptide that has several physiological effects such as inhibiting feeding, regulating energy expenditure, and stress. CART acts downstream of leptin in the obesity controlling signaling pathway. We showed that all members of the family bearing the (Phe34Leu) mutation had only pro - and intermediate CART in their circulation, but no mature CART. In contrast , normal humans and the unaffected sibling have significant amounts of circulating CART. Cell biological studies demonstrated that mutant pro-CART was partially missorted, poorly processed to bioactive CART and was secreted via the constitutive pathway, in contrast to wild type proCART which was properly sorted, processed and secreted in the regulated secrtetory pathway. The reduction in levels of bioactive CART in humans is likely due to cellular missorting to the constitutive pathway which does not have the appropriate enzymes to process proCART. Thus the hyperphagia and obesity observed in humans bearing the CART (Leu34Phe) mutation is likely attributed to a putative defficiency of bioactive CART in the brain of these patients. Our recent studies showed that prohormone and neuropeptide processing enzymes, carboxypeptidase E (CPE) and prohormone convertases 1 and 2 (PC1 and PC2), are transmembrane proteins with an atypical membrane spanning domain at the C-terminus. They are sorted into granules of the RSP in neuroendocrine cells by a novel mechanism involving transmembrane association of their C-terminal domain into cholesterol-glycosphingolipid rich microdomains known as lipid rafts, at the TGN. Removal of cholesterol from secretory granule membranes resulted in the inability of CPE, the RSP sorting receptor to bind cargo; and cholesterol depletion by treatment of cells with lovastatin resulted in lack of sorting of CPE to the RSP. Thus membrane association with cholesterol-rich lipid rafts is essential for sorting of prohormones and their processing enzymes to the TGN. The importance of cholesterol in secretory granule biogenesis and in packaging of cargo into granules was analysed in vivo, in the pancreas of cholesterol-deficient mouse models of Smith-Lemli Opitz Syndrome (SLOS) and lathosterolosis (Sc5d-/-). SLOS and lathosterolosis are human disorders, respectively defective in 7-dehydrocholesterol reductase and lathosterol 5-desaturase, enzymes necessary for the final steps of cholesterol synthesis. Morphological analysis by light and electron microscopy of neonatal pancreas zymogen granules showed a marked decrease in the number of granules, as well as aberant granule morphology in both SLOS and Sc5d-/- versus control mice. Furthermore, in primary cultures of cholesterol deficient secretory cells in the exocrine pancreas, protein synthesis and regulated secretion were impaired as compared to control cells. Thus cholesterol defficiency in SLOS and Sc5d-/- impairs regulated secretory pathway granule biogenesis and maturation, leading to deficits in the secretory function in the exocrine pancreas, and possibly also in the endocrine and nervous systems. In another project we have studied the protein factors governing the formation of large dense-core granules (LDCG) at the TGN, which is essential for regulated secretion of hormones and neuropeptides from neuroendocrine cells. Our studies demonstrate that chromogranin A (CgA) controls the formation of LDCG in neuroendocrine cells ex vivo and in vivo. We showed that an antisense mRNA transgenic mouse model deficient in CgA had severe aberrant granule formation quantitatively and qualitatively in the adrenal medulla.Depletion of CgA in rat PC12 cells using antisense technology resulted in the loss of LDCG, regulated secretion, and degradation of granule proteins including CgB and synaptotagmin. Overexpression of bovine CgA in these cells rescued the wild type phenotype. In a mutant endocrine cell line, 6T3, lacking CgA, there was a deficit in LDCGs, regulated hormone secretion and degradation of granule proteins. We recently identified the Golgi as the site of degradation of the secretory granule proteins in these cells. Transfection of CgA into the 6T3 cells rescued the protein degradation and promoted granule biogenesis. Thus we propose that regulation of the stability of granule proteins at the Golgi by CgA is a mechanism f