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 ...
Nna1 is a recently described gene product that has sequence similarity with metallocarboxypeptidases. In the present study, five additional Nna1-like genes were identified in the mouse genome and named cytosolic carboxypeptidase (CCP) 2 through 6. Modeling suggests that the carboxypeptidase domain folds into a structure that resembles metallocarboxypeptidases of the M14 family, with all necessary residues for catalytic activity and broad substrate specificity. All CCPs are abundant in testis and also expressed in brain, pituitary, eye, and other mouse tissues. In brain, Nna1/CCP1, CCP5, and CCP6 are broadly distributed, whereas CCP2 and 3 exhibit restricted patterns of expression. Nna1/CCP1, CCP2, CCP5, and CCP6 were found to exhibit a cytosolic distribution, with a slight accumulation of CCP5 in the nucleus. Based on the above results, we hypothesized that Nna1/CCP1 and CCP2-6 function in the processing of cytosolic proteins such as alpha-tubulin, which is known to be modified by the removal of a C-terminal tyrosine. Analysis of the forms of alpha tubulin in the olfactory bulb of mice lacking Nna1/CCP1 showed the absence of the detyrosinylated form in the mitral cells. Taken together, these results are consistent with a role for Nna1/CCP1 and the related CCPs in the processing of tubulin.
Cpe(fat/fat) mice have a point mutation in the coding region of the carboxypeptidase E gene that renders the enzyme inactive. As a result, these mice have reduced levels of several neuropeptides and greatly increased levels of the peptide processing intermediates that contain C-terminal basic residues. However, previous studies examined a relatively small number of neuropeptides. In the present study, we used a quantitative peptidomics approach with stable isotopic labels to examine the levels of pituitary peptides in Cpe(fat/fat) mice relative to wild-type mice. Pituitary extracts from mutant and wild type mice were labeled with the stable isotopic label [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propyl]trimethylammonium chloride containing nine atoms of hydrogen or deuterium. Then, the two samples were pooled and analyzed by liquid chromatography/mass spectrometry (LC/MS). The relative abundance of peptides was determined from a comparison of the intensities of the heavy and light peaks. Altogether, 72 peptides were detected in the Cpe(fat/fat) and/or wild-type mouse pituitary extracts of which 53 were identified by MS/MS sequencing. Several peptides identified in this analysis represent previously undescribed post-translational processing products of known pituitary prohormones. Of the 72 peptides detected in pituitary, 17 were detected only in the Cpe(fat/fat) mouse extracts; these represent peptide processing intermediates containing C-terminal basic residues. The peptides common to both Cpe(fat/fat) and wild-type mice were generally present at 2-5-fold lower levels in the Cpe(fat/fat) mouse pituitary extracts, although some peptides were present at equal levels and one peptide (acetyl beta-endorphin 1-31) was increased approximately 7-fold in the Cpe(fat/fat) pituitary extracts. In contrast, acetyl beta-endorphin 1-26 was present at approximately 10-fold lower levels in the Cpe(fat/fat) pituitary, compared with wild-type mice. The finding that many peptides are substantially decreased in Cpe(fat/fat) pituitary is consistent with the broad role for carboxypeptidase E in the biosynthesis of numerous neuropeptides.
The biosynthesis of most neuropeptides and peptide hormones requires a carboxypeptidase such as carboxypeptidase E, which is inactive in Cpe fat/fat mice due to a naturally occurring point mutation. To assess the role of carboxypeptidase E in the processing of peptides in the prefrontal cortex, we used a quantitative peptidomics approach to examine the relative levels of peptides in Cpe fat/fat versus wild-type mice. Peptides representing internal fragments of prohormones and other secretory pathway proteins were decreased two-to 10-fold in the Cpe fat/fat mouse prefrontal cortex compared with wild-type tissue. Degradation fragments of cytosolic proteins showed no major differences between Cpe fat/fat and wild-type mice. Based on this observation, a search strategy for neuropeptides was performed by screening for peptides that decreased in the Cpe fat/fat mouse. Altogether, 32 peptides were identified, of which seven have not been previously reported. The novel peptides include fragments of VGF, procholecystokinin and prohormone convertase 2. Interestingly, several of the peptides do not fit with the consensus sites for prohormone convertase 1 and 2, raising the possibility that another endopeptidase is involved with their biosynthesis. Taken together, these findings support the proposal that carboxypeptidase E is the major, but not the only, peptideprocessing carboxypeptidase and also demonstrate the feasibility of searching for novel peptides based on their decrease in Cpe fat/fat mice. Keywords: carboxypeptidase, cholecystokinin, peptide processing, peptidomics, prohormone convertase, VGF. Neuropeptides perform many important functions in a variety of organisms (Strand 2003). For example, peptides have been reported to be involved in feeding and body weight regulation, sleep/wake cycles and arousal state, pain perception, reward mechanisms, memory, anxiety, reproduction and many other physiological processes. Neuropeptides are produced by the selective cleavage of precursors at specific sites that often contain basic amino acids (Eipper et al. 1986). Initially, an endopeptidase, such as prohormone convertase (PC) 1 (also called PC3) or PC2, cleaves to the C-terminal side of the basic residues and then a carboxypeptidase removes the C-terminal amino acids (Zhou et al. 1999;Fricker 2004; Seidah and Chretien 2004a,b). Carboxypeptidase E (CPE) was discovered as a carboxypeptidase in peptide-containing secretory vesicles and was proposed to function in the biosynthesis of numerous peptides (Fricker and Snyder 1982;Fricker 1988). This hypothesis was validated by studies on Cpe fat/fat mice, which contain a point mutation within the coding region of the CPE gene that causes the enzyme to be inactive and rapidly degraded (Naggert et al. 1995). In the absence of active CPE in the Cpe fat/fat mouse, there is a dramatic increase in the levels of C-terminally extended peptideprocessing intermediates (Naggert et al. 1995;Fricker et al. 1996;Rovere et al. 1996;Che et al. 2005a). However, studies on a handful of br...
Cpefat/fat mice have a point mutation in carboxypeptidase E (CPE), an exopeptidase that removes C-terminal basic amino acids from intermediates to produce bioactive peptides. The mutation renders the enzyme inactive and unstable. The absence of CPE activity in these mutants leads to abnormal processing of many peptides, with elevated levels of intermediates and greatly reduced levels of the mature peptides. Cpefat/fat mice develop obesity, diabetes, and infertility in adulthood. We examined whether anxiety- and/or depressive-like behaviors are also present. Anxiety-like responses are not evident in young Cpefat/fat mice (~60 days), but appear in older animals (>90 days). These behaviors are reversed by acute treatment with diazepam or fluoxetine. By contrast, increased immobilities in forced swim and tail suspension are evident in all age groups examined. These behaviors are reversed by acute administration of reboxetine. By comparison acute treatments with fluoxetine or bupropion are ineffective; however, immobility times are normalized with 2 wks of treatment. These data demonstrate that Cpefat/fat mice display depressive-like responses at ~60 days of age, whereas anxiety-like behaviors emerge ~1 month later. In tail suspension, the reboxetine findings show that noradrenergic actions of antidepressants are intact in Cpefat/fat mice. The ability of acute fluoxetine treatment to rescue anxiety-like while leaving depressive-like responses unaffected suggests that serotonin mechanisms underlying these behaviors are different. Since depressive-like responses in the Cpefat/fat mice are rescued by a 2 wk, but not acute, treatment with fluoxetine or buproprion, these mice may serve as a useful model that resembles human depression.
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