ABSTRACT
To identify neuropeptides that are regulated by cocaine, we used a quantitative peptidomic technique to examine the relative levels of neuropeptides in several regions of mouse brain following daily intraperitoneal administration of 10 mg/kg cocaine or saline for 7 days. A total of 102 distinct peptides were identified in one or more of the following brain regions: nucleus accumbens, caudate putamen, frontal cortex, and ventral tegmental area. None of the peptides detected in the caudate putamen or frontal cortex were altered by cocaine administration. Three peptides in the nucleus accumbens and seven peptides in the ventral tegmental area were significantly decreased in cocaine-treated mice. Five of these ten peptides are derived from proSAAS, a secretory pathway protein and neuropeptide precursor. To investigate whether proSAAS peptides contribute to the physiological effects of psychostimulants, we examined acute responses to cocaine and amphetamine in the open field with wild-type (WT) and proSAAS knockout (KO) mice. Locomotion was stimulated more robustly in the WT compared to mutant mice for both psychostimulants. Behavioral sensitization to amphetamine was not maintained in proSAAS KO mice and these mutants failed to sensitize to cocaine. To determine whether the rewarding effects of cocaine were altered, mice were tested in conditioned place preference (CPP). Both WT and proSAAS KO mice showed dose-dependent CPP to cocaine that was not distinguished by genotype. Taken together, these results suggest that proSAAS-derived peptides contribute differentially to the behavioral sensitization to psychostimulants, while the rewarding effects of cocaine appear intact in mice lacking proSAAS.
Subject(s)
Cocaine/pharmacology , Dopamine Uptake Inhibitors/pharmacology , Hyperkinesis/chemically induced , Locomotion/drug effects , Nerve Tissue Proteins/metabolism , Amphetamine/pharmacology , Animals , Conditioning, Operant/drug effects , Dose-Response Relationship, Drug , Exploratory Behavior/drug effects , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Male , Mass Spectrometry , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/genetics , Neuropeptides , Nucleus Accumbens/drug effects , Ventral Tegmental Area/drug effectsABSTRACT
Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study, we examined the activity of purified CCP5 toward synthetic peptides as well as soluble α- and ß-tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain α- and ß-tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated α-tubulin. The enzyme also removed multiple glutamate residues from side chains and C termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of ß3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides' γ-linked residues. The rate of cleavage of α linkages by CCP5 is considerably slower than that of removal of a single γ-linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both α- and γ-linked glutamate from tubulin.
Subject(s)
Carboxypeptidases/metabolism , Glutamic Acid/metabolism , Microtubules/metabolism , Tubulin/metabolism , Animals , Carboxypeptidases/chemistry , Carboxypeptidases/genetics , Glutamic Acid/chemistry , Glutamic Acid/genetics , Mice , Microtubules/chemistry , Microtubules/genetics , Paclitaxel/pharmacology , Sf9 Cells , Spodoptera , Tubulin/chemistry , Tubulin/genetics , Tubulin Modulators/pharmacologyABSTRACT
Peptides represent a major class of cell-cell signaling molecules. Most peptidomic studies have focused on peptides present in brain or other tissues. For a peptide to function in intercellular signaling, it must be secreted. The present study was undertaken to identify the major peptides secreted from mouse brain slices that were cultured in oxygenated buffer for 3-4h. Approximately 75% of the peptides identified in extracts of cultured slices matched the previously reported peptide content of heat-inactivated mouse brain tissue, whereas only 2% matched the peptide content of unheated brain tissue; the latter showed a large number of postmortem changes. As found with extracts of heat-inactivated mouse brain, the extracts of cultured brain slices represented secretory pathway peptides as well as peptides derived from intracellular proteins such as those present in the cytosol and mitochondria. A subset of the peptides detected in the extracts of the cultured slices was detected in the culture media. The vast majority of secreted peptides arose from intracellular proteins and not secretory pathway proteins. The peptide RVD-hemopressin, a CB1 cannabinoid receptor agonist, was detected in culture media, which is consistent with a role for RVD-hemopressin as a non-classical neuropeptide. Taken together with previous studies, the present results show that short-term culture of mouse brain slices is an appropriate system to study peptide secretion, especially the non-conventional pathway(s) by which peptides produced from intracellular proteins are secreted. This article is part of a Special Issue entitled: An Updated Secretome.
Subject(s)
Brain/metabolism , Neuropeptides/analysis , Neuropeptides/metabolism , Secretory Pathway , Amino Acid Sequence , Animals , Brain Chemistry , Female , Male , Mice , Molecular Sequence Data , Proteomics , Tissue Culture TechniquesABSTRACT
The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and ß-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and ß-tubulin. The hyperglutamylation of α- and ß-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and ß-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from ß- as well as α-tubulin in vitro and in vivo.
Subject(s)
GTP-Binding Proteins/metabolism , Nerve Degeneration/metabolism , Serine-Type D-Ala-D-Ala Carboxypeptidase/metabolism , Tubulin/metabolism , Animals , Breast Neoplasms , Cell Line, Tumor , Colonic Neoplasms , Cytosol/enzymology , Female , GTP-Binding Proteins/genetics , Glutamic Acid/metabolism , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Mutant Strains , Nerve Degeneration/genetics , Peptide Synthases/genetics , Peptide Synthases/metabolism , Protein Structure, Tertiary , Purkinje Cells/enzymology , Purkinje Cells/pathology , Serine-Type D-Ala-D-Ala Carboxypeptidase/genetics , Swine , Tubulin/chemistryABSTRACT
Cpe(fat/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. Cpe(fat/fat) mice develop obesity, diabetes and infertility in adulthood. We examined whether anxiety- and/or depressive-like behaviours are also present. Anxiety-like responses are not evident in young Cpe(fat/fat) mice (â¼60 d), but appear in older animals (>90 d). These behaviours are reversed by acute treatment with diazepam or fluoxetine. In contrast, increased immobilities in forced swim and tail suspension are evident in all age groups examined. These behaviours are reversed by acute administration of reboxetine. In comparison acute treatments with fluoxetine or bupropion are ineffective; however, immobility times are normalized with 2 wk treatment. These data demonstrate that Cpe(fat/fat) mice display depressive-like responses aged â¼60 d, whereas anxiety-like behaviours emerge â¼1 month later. In tail suspension, the reboxetine findings show that noradrenergic actions of antidepressants are intact in Cpe(fat/fat) mice. The ability of acute fluoxetine treatment to rescue anxiety-like while leaving depressive-like responses unaffected suggests that serotonin mechanisms underlying these behaviours are different. Since depressive-like responses in the Cpe(fat/fat) mice are rescued by 2 wk, but not acute, treatment with fluoxetine or bupropion, these mice may serve as a useful model that resembles human depression.
Subject(s)
Anxiety/etiology , Carboxypeptidase H/genetics , Depression/etiology , Obesity/complications , Age Factors , Analysis of Variance , Animals , Antidepressive Agents/therapeutic use , Anxiety/drug therapy , Anxiety/genetics , Depression/drug therapy , Depression/genetics , Diazepam/therapeutic use , Exploratory Behavior/drug effects , Hindlimb Suspension/methods , Maze Learning/drug effects , Mice , Mice, Transgenic , Morpholines/therapeutic use , Motor Activity/drug effects , Obesity/genetics , Point Mutation/genetics , Reboxetine , Swimming/psychologyABSTRACT
Acute myeloid leukemia (AML) is characterized by poor clinical outcomes due to high rates of relapse following standard-of-care induction chemotherapy. While many pathogenic drivers have been described in AML, our understanding of the molecular mechanisms mediating chemotherapy resistance remains poor. Therefore, we sought to identify resistance genes to induction therapy in AML and elucidated ALOX5 as a novel mediator of resistance to anthracycline-based therapy. ALOX5 is transcriptionally upregulated in AML patient blasts in comparison to normal hematopoietic stem/progenitor cells (HSPCs) and ALOX5 mRNA, and protein expression is increased in response to induction therapy. In vitro, and in vivo genetic, and pharmacologic perturbation studies confirm that ALOX5 positively regulates the leukemogenic potential of AML LSCs, and its loss does not significantly affect the function of normal HSPCs. ALOX5 mediates resistance to daunorubicin (DNR) and promotes AML cell survival and maintenance through its leukotriene (LT) synthetic capacity, specifically via modulating the synthesis of LTB4 and its binding to LTB receptor (BLTR). Our study reveals a previously unrecognized role of LTs in AML pathogenesis and chemoresistance, whereby inhibition of ALOX5 mediated LTB4 synthesis and function could be combined with standard chemotherapy, to enhance the overall therapeutic efficacy in AML.
Subject(s)
Antineoplastic Agents , Leukemia, Myeloid, Acute , Antineoplastic Agents/therapeutic use , Cell Self Renewal/genetics , Daunorubicin/pharmacology , Daunorubicin/therapeutic use , Drug Resistance, Neoplasm , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Leukotriene B4/metabolism , Leukotriene B4/therapeutic use , Neoplastic Stem Cells/pathologyABSTRACT
Purkinje cell degeneration (pcd) mice have a mutation within the gene encoding cytosolic carboxypeptidase 1 (CCP1/Nna1), which has homology to metallocarboxypeptidases. To assess the function of CCP1/Nna1, quantitative proteomics and peptidomics approaches were used to compare proteins and peptides in mutant and wild-type mice. Hundreds of peptides derived from cytosolic and mitochondrial proteins are greatly elevated in pcd mouse hypothalamus, amygdala, cortex, prefrontal cortex, and striatum. However, the major proteins detected on 2-D gel electrophoresis were present in mutant and wild-type mouse cortex and hypothalamus at comparable levels, and proteasome activity is normal in these brain regions of pcd mice, suggesting that the increase in cellular peptide levels in the pcd mice is due to reduced degradation of the peptides downstream of the proteasome. Both nondegenerating and degenerating regions of pcd mouse brain, but not wild-type mouse brain, show elevated autophagy, which can be triggered by a decrease in amino acid levels. Taken together with previous studies on CCP1/Nna1, these data suggest that CCP1/Nna1 plays a role in protein turnover by cleaving proteasome-generated peptides into amino acids and that decreased peptide turnover in the pcd mice leads to cell death.
Subject(s)
GTP-Binding Proteins/physiology , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Nerve Tissue Proteins/metabolism , Purkinje Cells/pathology , Serine-Type D-Ala-D-Ala Carboxypeptidase/physiology , Amino Acids/metabolism , Animals , Autophagy , Cell Death , Electrophoresis, Gel, Two-Dimensional , Female , Male , Mice , Mice, Inbred BALB C , Peptide Fragments/metabolism , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Despite significant efforts to improve therapies for acute myeloid leukemia (AML), clinical outcomes remain poor. Understanding the mechanisms that regulate the development and maintenance of leukemic stem cells (LSCs) is important to reveal new therapeutic opportunities. We have identified CD97, a member of the adhesion class of G protein-coupled receptors (GPCRs), as a frequently up-regulated antigen on AML blasts that is a critical regulator of blast function. High levels of CD97 correlate with poor prognosis, and silencing of CD97 reduces disease aggressiveness in vivo. These phenotypes are due to CD97's ability to promote proliferation, survival, and the maintenance of the undifferentiated state in leukemic blasts. Collectively, our data credential CD97 as a promising therapeutic target on LSCs in AML.
Subject(s)
Antigens, CD/biosynthesis , Blast Crisis/metabolism , Gene Expression Regulation, Leukemic , Leukemia, Myeloid, Acute/metabolism , Neoplasm Proteins/biosynthesis , Neoplastic Stem Cells/metabolism , Receptors, G-Protein-Coupled/biosynthesis , Up-Regulation , Animals , Antigens, CD/genetics , Blast Crisis/genetics , Blast Crisis/pathology , Cell Proliferation , Cell Survival , Female , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Male , Mice , Mice, Knockout , Neoplasm Proteins/genetics , Neoplastic Stem Cells/pathology , Receptors, G-Protein-Coupled/geneticsABSTRACT
Quantitative peptidomics was used to compare levels of peptides in wild type (WT) and Cpe(fat/fat) mice, which lack carboxypeptidase E (CPE) activity because of a point mutation. Six different brain regions were analyzed: amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, and thalamus. Altogether, 111 neuropeptides or other peptides derived from secretory pathway proteins were identified in WT mouse brain extracts by tandem mass spectrometry, and another 47 peptides were tentatively identified based on mass and other criteria. Most secretory pathway peptides were much lower in Cpe(fat/fat) mouse brain, relative to WT mouse brain, indicating that CPE plays a major role in their biosynthesis. Other peptides were only partially reduced in the Cpe(fat/fat) mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpe(fat/fat) mouse brain at levels similar to those in WT mouse brain. Many peptides were greatly elevated in the Cpe(fat/fat) mice; these peptide processing intermediates with C-terminal Lys and/or Arg were generally not detectable in WT mice. Taken together, these results indicate that CPE contributes, either directly or indirectly, to the production of the majority of neuropeptides.
Subject(s)
Brain/metabolism , Carboxypeptidase H/metabolism , Neuropeptides/metabolism , Animals , Carboxypeptidase H/deficiency , Carboxypeptidase H/genetics , Chromatography, High Pressure Liquid , Mass Spectrometry/methods , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Obese , Models, Biological , Neuropeptides/analysis , Peptide Fragments/chemistry , Physiological Phenomena/physiology , Point Mutation , Secretory PathwayABSTRACT
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.
Subject(s)
Carboxypeptidases/genetics , Amino Acid Sequence , Animals , Base Sequence , Carboxypeptidases/isolation & purification , Carboxypeptidases/metabolism , Cytoplasm/enzymology , DNA, Complementary/analysis , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , Sequence Alignment , Tissue DistributionABSTRACT
Bortezomib is an antitumor drug that competitively inhibits proteasome beta-1 and beta-5 subunits. While the impact of bortezomib on protein stability is known, the effect of this drug on intracellular peptides has not been previously explored. A quantitative peptidomics technique was used to examine the effect of treating human embryonic kidney 293T (HEK293T) cells with 5-500 nM bortezomib for various lengths of time (30 minutes to 16 hours), and human neuroblastoma SH-SY5Y cells with 500 nM bortezomib for 1 hour. Although bortezomib treatment decreased the levels of some intracellular peptides, the majority of peptides were increased by 50-500 nM bortezomib. Peptides requiring cleavage at acidic and hydrophobic sites, which involve beta-1 and -5 proteasome subunits, were among those elevated by bortezomib. In contrast, the proteasome inhibitor epoxomicin caused a decrease in the levels of many of these peptides. Although bortezomib can induce autophagy under certain conditions, the rapid bortezomib-mediated increase in peptide levels did not correlate with the induction of autophagy. Taken together, the present data indicate that bortezomib alters the balance of intracellular peptides, which may contribute to the biological effects of this drug.
Subject(s)
Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Neuroblastoma/drug therapy , Peptides/metabolism , Proteasome Inhibitors/pharmacology , Pyrazines/pharmacology , Amino Acid Sequence , Bortezomib , Cell Line, Tumor , HEK293 Cells , Humans , Molecular Sequence Data , Neuroblastoma/pathology , Peptides/analysis , ProteomicsABSTRACT
Cytosolic carboxypeptidase 1 (CCP1) is a metallopeptidase that removes C-terminal and side-chain glutamates from tubulin. The Purkinje cell degeneration (pcd) mouse lacks CCP1 due to a mutation. Previously, elevated levels of peptides derived from cytosolic and mitochondrial proteins were found in adult pcd mouse brain, raising the possibility that CCP1 functions in the degradation of intracellular peptides. To test this hypothesis, we used a quantitative peptidomics technique to compare peptide levels in wild-type and pcd mice, examining adult heart, spleen, and brain, and presymptomatic 3 week-old amygdala and cerebellum. Contrary to adult mouse brain, young pcd brain and adult heart and spleen did not show a large increase in levels of intracellular peptides. Unexpectedly, levels of peptides derived from secretory pathway proteins were altered in adult pcd mouse brain. The pattern of changes for the intracellular and secretory pathway peptides in pcd mice was generally similar to the pattern observed in mice lacking primary cilia. Collectively, these results suggest that intracellular peptide accumulation in adult pcd mouse brain is a secondary effect and is not due to a role of CCP1 in peptide turnover.
Subject(s)
Mutation , Peptide Fragments/metabolism , Proteomics , Purkinje Cells/pathology , Amino Acid Sequence , Amygdala/metabolism , Animals , Cerebellum/metabolism , Female , GTP-Binding Proteins/deficiency , GTP-Binding Proteins/genetics , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Purkinje Cells/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Serine-Type D-Ala-D-Ala Carboxypeptidase/deficiency , Serine-Type D-Ala-D-Ala Carboxypeptidase/geneticsABSTRACT
ProSAAS is the precursor of a number of peptides that have been proposed to function as neuropeptides. Because proSAAS mRNA is highly expressed in the arcuate nucleus of the hypothalamus, we examined the cellular localization of several proSAAS-derived peptides in the mouse hypothalamus and found that they generally colocalized with neuropeptide Y (NPY), but not α-melanocyte stimulating hormone. However, unlike proNPY mRNA, which is upregulated by food deprivation in the mediobasal hypothalamus, neither proSAAS mRNA nor proSAAS-derived peptides were significantly altered by 1-2 days of food deprivation in wild-type mice. Furthermore, while proSAAS mRNA levels in the mediobasal hypothalamus were significantly lower in Cpe(fat/fat) mice as compared to wild-type littermates, proNPY mRNA levels in the mediobasal hypothalamus and in other subregions of the hypothalamus were not significantly different between wild-type and Cpe(fat/fat) mice. Intracerebroventricular injections of antibodies to two proSAAS-derived peptides (big LEN and PEN) significantly reduced food intake in fasted mice, while injections of antibodies to two other proSAAS-derived peptides (little LEN and little SAAS) did not. Whole-cell patch clamp recordings of parvocellular neurons in the hypothalamic paraventricular nucleus, a target of arcuate NPY projections, showed that big LEN produced a rapid and reversible inhibition of synaptic glutamate release that was spike independent and abolished by blocking postsynaptic G protein activity, suggesting the involvement of a postsynaptic G protein-coupled receptor and the release of a retrograde synaptic messenger. Taken together with previous studies, these findings support a role for proSAAS-derived peptides such as big LEN as neuropeptides regulating food intake.
Subject(s)
Eating/genetics , Gene Expression Regulation , Neuropeptide Y/chemistry , Peptides/chemistry , Animals , Arcuate Nucleus of Hypothalamus/metabolism , Feeding Behavior , GTP-Binding Proteins/metabolism , Green Fluorescent Proteins/metabolism , Hypothalamus/metabolism , Immunohistochemistry/methods , Infusions, Intraventricular , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence/methods , Nerve Tissue Proteins/genetics , Neuropeptides , Patch-Clamp Techniques , RNA, Messenger/metabolismABSTRACT
Purkinje cell degeneration (pcd) is a mouse mutant which is characterized by postnatal degeneration of selective cell types. The pcd mutation was mapped to a gene encoding a cytosolic carboxypeptidase-like protein (CCP), named CCP1/Nna1. Many neurons in pcd mice show increased levels of autophagy, including cell types which do not undergo neurodegeneration. These brain regions have greatly elevated levels of many intracellular peptides, suggesting that CCP1/Nna1 functions in protein turnover by degrading peptides to amino acids. We propose that the lack of CCP1/Nna1 leads to decreases in cellular levels of amino acids, which leads to elevated autophagy as a protective response to cellular amino acid starvation.
Subject(s)
Autophagy , GTP-Binding Proteins/metabolism , Nerve Degeneration/enzymology , Nerve Degeneration/pathology , Purkinje Cells/enzymology , Purkinje Cells/pathology , Serine-Type D-Ala-D-Ala Carboxypeptidase/metabolism , Animals , Mice , Mice, Neurologic Mutants , Models, BiologicalABSTRACT
Sample preparation for neuropeptidomic studies is a critical issue since protein degradation can produce high levels of peptides that obscure the endogenous neuropeptides. We compared different extraction conditions for the recovery of neuropeptides and the formation of protein breakdown fragments from mouse hypothalami. Sonication and heating in water (70 degrees C for 20 min) followed by cold acid and centrifugation enabled the efficient extraction of many neuropeptides without the formation of protein degradation fragments seen with hot acid extractions. The hot water/cold acid extraction procedure resulted in the reproducible recovery of many hypothalamic peptides, including several novel peptides.
Subject(s)
Hypothalamus/chemistry , Hypothalamus/metabolism , Neuropeptides/isolation & purification , Amino Acid Sequence , Animals , Chromatography, Liquid , Female , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Neuropeptides/metabolism , Tandem Mass SpectrometryABSTRACT
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, peptide-processing carboxypeptidase and also demonstrate the feasibility of searching for novel peptides based on their decrease in Cpe(fat/fat) mice.