Mice homozygous for the fat mutation develop obesity and hyperglycaemia that can be suppressed by treatment with exogenous insulin. The fat mutation maps to mouse chromosome 8, very close to the gene for carboxypeptidase E (Cpe), which encodes an enzyme (CPE) that processes prohormone intermediates such as proinsulin. We now demonstrate a defect in proinsulin processing associated with the virtual absence of CPE activity in extracts of fat/fat pancreatic islets and pituitaries. A single Ser202Pro mutation distinguishes the mutant Cpe allele, and abolishes enzymatic activity in vitro. Thus, the fat mutation represents the first demonstration of an obesity-diabetes syndrome elicited by a genetic defect in a prohormone processing pathway.

Five novel peptides were identified in the brains of mice lacking active carboxypeptidase E, a neuropeptide-processing enzyme. These peptides are produced from a single precursor, termed proSAAS, which is present in human, mouse, and rat. ProSAAS mRNA is expressed primarily in brain and other neuroendocrine tissues (pituitary, adrenal, pancreas); within brain, the mRNA is broadly distributed among neurons. When expressed in AtT-20 cells, proSAAS is secreted via the regulated pathway and is also processed at paired-basic cleavage sites into smaller peptides. Overexpression of proSAAS in the AtT-20 cells substantially reduces the rate of processing of the endogenous prohormone proopiomelanocortin. Purified proSAAS inhibits prohormone convertase 1 activity with an IC 50 of 590 nM but does not inhibit prohormone convertase 2. Taken together, proSAAS may represent an endogenous inhibitor of prohormone convertase 1.

In neuropeptidomics, the degradation of a small fraction of abundant proteins overwhelms the low signals from neuropeptides, and many neuropeptides cannot be detected by mass spectrometry without extensive purification. Protein degradation was prevented when mice were sacrificed with focused microwave irradiation, permitting the detection of hypothalamic neuropeptides by mass spectrometry. Here we report an alternative and very simple method utilizing an ordinary microwave oven to inhibit enzymatic degradation. We used this technique to identify brain and pituitary neuropeptides. Quantitative analysis using mass spectrometry in combination with stable isotopic labeling was performed to determine the effect of microwave irradiation on relative levels of neuropeptides and protein degradation fragments. Microwave irradiation greatly reduced the levels of degradation fragments of proteins. In contrast, neuropeptide levels were increased about 2-3 times in hypothalamus by the microwave irradiation but not increased in pituitary. In a second experiment, three brain regions (hypothalamus, hippocampus, and striatum) from microwave-irradiated mice were analyzed. Altogether 41 neuropeptides or fragments of secretory pathway proteins were identified after microwave treatment; some of these are novel. These peptides were derived from 15 proteins: proopiomelanocortin, proSAAS, proenkephalin, preprotachykinins A and B, provasopressin, prooxytocin, melanin-concentrating hormone, proneurotensin, chromogranins A and B, secretogranin II, prohormone convertases 1 and 2, and peptidyl amidating monooxygenase. Although some protein degradation fragments were still found after microwave irradiation, these appear to result from protein breakdown during the extraction and not to an enzymatic reaction during the postmortem period. Two of the protein fragments corresponded to novel protein forms: VAP-33 with a 7-residue N-terminal extension and ␤ tubulin with a glutathione on the Cys near the N terminus. In conclusion, microwave irradiation with an ordinary microwave oven effectively inhibits enzymatic postmortem protein degradation, increases the recovery of neuropeptides, and makes it possible to conduct neuropeptidomic studies with mouse brain tissues. Peptides perform many important functions throughout the body as hormones and neurotransmitters. Neuropeptides are involved in a wide variety of systems, including reward mechanisms, pain, memory, food intake and body weight regulation, circadian rhythms, and many others (1, 2). A large number of studies have examined the changes in levels of various peptides upon different treatments or in different physiological states. These studies typically measured peptide levels using radioimmunoassays (RIAs).1 Although this approach is sensitive, most antisera are not specific for a single peptide and are able to cross-react with N-and/or C-terminally extended peptides and with peptides modified by post-translational modifications such as acetylation, phosphorylation, or sulfation (3). Also RIAs ...

Carboxypeptidase E appears to be involved in the biosynthesis of a wide range of peptide hormones and neurotransmitters. The evidence for this is: (a) CPE is present in tissues that produce bioactive peptides; (b) in tissues that have been subjected to subcellular fractionation, the CPE activity is associated with peptide-containing secretory granules; (c) CPE is able to remove C-terminal basic amino acids from a variety of synthetic peptides without further hydrolyzing the peptide; (d) CPE is active at pH 5.6, the internal pH of secretory granules. The CPE activities in various tissues have similar physical and enzymatic properties. Two forms of CPE, soluble and membrane-bound, are present in most tissues with CPE activity. These two forms differ slightly in molecular weight, but have identical enzymatic properties. Both forms arise from the same precursor, which is encoded by a single gene. This gene is a member of a carboxypeptidase gene family that includes CPA and CPB. At the amino acid level, CPE has approximately 20% homology with bovine CPA and 17% homology with bovine CPB. All of the amino acids in CPA and CPB that are thought to be essential for catalytic activity are present in CPE in comparable positions. The homology of CPE with CPA and CPB suggests a common evolutionary origin for the three enzymes. This relationship fits with the theory that certain peptide hormones may have evolved from serine proteases. Further studies are needed to investigate the processing of proCPE into CPE, and the regulation of CPE activity. While there is some evidence that CPE may be regulated, it does not appear that regulation of CPE activity plays an important role in controlling peptide biosynthesis. However, further studies are necessary before this possibility can be eliminated.

Peptides are known to play many important physiological roles in signaling. A large number of peptides have been detected in mouse brain extracts using mass spectrometry-based peptidomics studies, and 850 peptides have been identified. Half of these peptides are derived from secretory pathway proteins and many are known bioactive neuropeptides which activate G protein-coupled receptors; these are termed "classical neuropeptides." In addition, 427 peptides were identified that are derived from non-secretory pathway proteins; the majority are cystosolic, and the remainder are mitochondrial, nuclear, lysosomal, or membrane proteins. Many of these peptides represent the Nor C-terminus of the protein, rather than internal fragments, raising the possibility that they are formed by selective processing rather than protein degradation. In addition to consideration of the cleavage site required to generate the intracellular peptides, their potential functions are discussed. Several of the cytosolic peptides were previously found to interact with receptors and/or otherwise influence cellular activity; examples include hemophins, hemopressins, diazepam binding inhibitor, and hippocampal cholinergic neurostimulating peptide. The possibility that these peptides are secreted from cells and function in cell-cell signaling is discussed. If these intracellular peptides can be shown to be secreted in levels sufficient to produce a biological effect, they would appropriately be called "non-classical neuropeptides" by analogy with non-classical neurotransmitters such as nitric oxide and anandamide. It is also possible that intracellular peptides function as "microproteins" and modulate protein-protein interactions; evidence for this function is discussed, along with future directions that are needed to establish this and other possible functions for peptides.