Neuropeptides biosynthesis

Additional endoproteases may be shown to play a role in neuropeptide biosynthesis. Leading candidates are the mammalian homolog of the yeast aspartyl protease-3 (YAP-3) and a thiol protease. The processing of proANF, which involves cleavage after a single Arg residue in proANF, cannot involve PCI or PC2 since there are negligible amounts of these PCs in the heart. 

The application of recombinant DNA techniques to the study of neuropeptide biosynthesis is essential because these techniques facilitate structural analyses and evolutionary studies, clarify biosynthetic pathways, provide the necessary background and probes for analysis of synthesis regulation at the levels of mRNA and gene transcription and assist in classical genetic experimentation. Examples of these benefits are cited and problems encountered in insect systems are discussed. 

Thus the application of recombinant DNA technology to the study of neuropeptide biosynthesis has enhanced, and will continue to enhance, our understanding of the structure, function, and evolution of these interesting modulatory agents. Some of the most exciting advances, such as the insights provided by genetic studies, are yet to come. 

Neuropeptides may also undergo post translational modification that modifies the biological activities of peptides. Activities of the neuropeptides may be altered by disulfide bond formation, glycosylation, COOH-terminal a-amidation, phosphoryla-ton, sulfation, and acetylation (6, 7). This article, however, will focus on protease mechanisms for neuropeptide biosynthesis. 

Recent achievements in the development of active-site directed affinity probes for proteases and other enzyme classes provide direct chemical labeling of proteases of interest in the biological system (24-27). These specific activity probes allow joint evaluation of selective protease inhibition concomitant with labeling of relevant protease enzymes for more analyses. Moreover, activity-based probes that selectively label the main protease subclasses—cysteine, serine, metallo, aspartic, and threonine—can provide advantageous chemical approaches for functional protease identification. Activity probe labeling of proteases allows direct identihcation of enzyme proteins by tandem mass spectrometry. Such chemical probes directed to cysteine proteases have been instrumental for identification of the new cathepsin L cysteine protease pathway for neuropeptide biosynthesis, as summarized in this article. 

Activity-based chemical profiling identifies the cathepsin L cysteine protease pathway in secretory vesicles that contributes to neuropeptide biosynthesis... 

Gene analyses of Cathepsin L in neuropeptide biosynthesis by protease gene knockout and gene expression approaches... 

Significantly, the approach of activity profiling for cysteine proteases has established cathepsin L as a new protease pathway for neuropeptide biosynthesis. Together with current knowledge in the field, these data demonstrate the existence of two distinct protease pathways for converting proneuropeptides into active peptide neurotransmitters and hormones. These dual pathways consist of the newly discovered cysteine protease pathway for proneuropeptide processing, which consists of cathepsin L followed by Arg/Lys aminopeptidase (aminopeptidase B), and the previously known proprotein convertase (PC) family of subtilisin-like proteases (15-17) that process proneuropeptides with carboxypeptidase E (Fig. 3). Elucidation of these two protease pathways resulted from the application of the biochemical criteria required for processing proteases. 

Proteomics of Secretory Vesicles for Defining Proteases and Related Systems for Neuropeptide Biosynthesis... 

Figure 5 Proteomics reveals functional secretory vesicle protein systems for neuropeptide biosynthesis, storage, and secretion. Chromaffin secretory vesicles (also known as chromaffin granules) were isolated and subjected to proteomic analyses of proteins in the soluble and membrane components of the vesicles. Protein systems in secretory vesicle function consisted of those for 1) production of hormones, neurotransmitters, and neuromodulatory factors, 2) generating selected internal vesicular conditions for reducing condition, acidic pH conditions maintained by ATPases, and chaperones for protein folding, and 3) vesicular trafficking mechanisms to allow the mobilization of secretory vesicles for exocytosis, which uses proteins for nucleotide-binding, calcium regulation, and vesicle exocytosis. These protein systems are coordinated to allow the secretory vesicle to synthesize and release neuropeptides for cell-cell communication in the control of neuroendocrine functions.

It is extremely important to apply knowledge of protease mechanisms for neuropeptide biosynthesis to small-molecule strategies for the development of therapeutic agents that can modulate the production of specific peptide neurotransmitters or hormones. 

Hwang SR, Garza C, Mosier C, Toneff T, Wunderlich E, et al. Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion. J. Biol. Chem. 2007 282 9556-9563. 

Biosynthesis. The biosynthesis of neuropeptides is much more complex and involves the multistep process of transcription of specific mRNA from specific genes, formation of a high molecular weight protein product by translation, post-translational processing of the protein precursor to allow for... 

Biosynthesis. Two closely related genes encode the three mammalian tachykinins. The preprotachykinin A gene encodes both substance P and substance K, while the preprotachykinin B gene encodes neuromedin K (45—47). The active sequences are flanked by the usual double-basic amino acid residues, and the carboxy-terrninal amino acid is a glycine residue which is decarboxylated to an amide. As with most neuropeptide precursors, intermediates in peptide processing can be detected, but their biological activities are not clear (ca 1994). 

Abstract Pheromones are utilized by many insects in a complex chemical communication system. This review will look at the biosynthesis of sex and aggregation pheromones in the model insects, moths, flies, cockroaches, and beetles. The biosynthetic pathways involve altered pathways of normal metabolism of fatty acids and isoprenoids. Endocrine regulation of the biosynthetic pathways will also be reviewed for the model insects. A neuropeptide named pheromone biosynthesis activating neuropeptide regulates sex pheromone biosynthesis in moths. Juvenile hormone regulates pheromone production in the beetles and cockroaches, while 20-hydroxyecdysone regulates pheromone production in the flies. 

PBAN Pheromone biosynthesis activating neuropeptide PGN PBAN-encoding gene neuropeptides SEG Subesophageal ganglion yne Triple bond... 

The biosynthesis of neuropeptides is fundamentally different from that of conventional neurotransmitters 321 Many of the enzymes involved in peptide biogenesis have been identified 321... 

Probably the most striking difference between neuropeptides and conventional neurotransmitters is in their biosynthesis (Fig. 18-2). Neuropeptides are derived from larger, inactive precursors that are generally at least 90 amino acid residues in length [2-4]. The simplest example is prolactin, a pituitary product. The signal sequence for... 

FIGURE 1 8-3 Intracellular pathway of bioactive peptide biosynthesis, processing and storage. Neuropeptide precursors are synthesized on ribosomes at the endoplasmic reticulum and processed through the Golgi. Axonal transport of the large dense-core vesicle to the synaptic site of release precedes the actual secretion. 

The biosynthesis of neuropeptides is fundamentally different from that of conventional neurotransmitters. 

The study of peptidergic neurons requires a number of special tools. These tools include methods to detect the neuropeptides both in cells and after release, the enzymes specific to their biosynthesis and their cognate receptors. Since the actions of peptides require secretion, measurements of cell content (e.g. immunostaining) can be deceptive, with a decrease in content reflecting increased release. 

In moths, it was discovered in Helicoverpa zea that a peptide produced in the subesophageal ganglion portion of the brain complex regulates pheromone production in female moths (19). This factor has been purified and characterized in three species, Helicoverpa zea (20), Bombyx mori (21, 22), and Lymantria dispar (23). They are all a 33- or 34-amino acid peptide (named pheromone biosynthesis activating neuropeptide, PBAN) and have in common an amidated C-terminal 5-amino acid sequence (FXPRL-amide), which is the minimum peptide fragment required for pheromon-tropic activity. In the redbanded leafroller moth, it was shown that PBAN from the brain stimulates the release of a different peptide from the bursae copulatrix that is used to stimulate pheromone production in the pheromone gland found at the posterior tip of the abdomen (24). 

Using the C-terminal hexapeptide of substance P as a model compound, each one of cycloscan diversity parameters have been shown to affect the conformation and hence the biological activity of the peptide.1417,470,4711 This approach has been successfully applied to various peptides such as somatostatin,14191 the insect neuropeptide pheromone biosynthesis activating neuropeptide (PBAN),1420,4311 BPTI,14351 and on the nuclear localization signal (NLS) of the HIV-1 matrix protein (MA),14291 and HIV-1 Tat/Rev.14301... 

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