ProSAAS-Derived Peptides are Colocalized with Neuropeptide Y and Function as Neuropeptides in the Regulation of Food Intake

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 Cpefat/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 Cpefat/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

Cytosolic carboxypeptidase 5 removes α- and γ-linked glutamates from tubulin

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 β-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 β-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 β3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides\u27 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

CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice

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.-Berezniuk, I., Sironi, J., Callaway, M. B., Castro, L. M., Hirata, I. Y., Ferro, E. S., Fricker, L. D. CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice. FASEB J. 24, 1813-1823 (2010). www.fasebj.orgU.S. National Institutes of Health (NIH)National Institutes of Health (NIH)[DK-51271]National Institutes of Health (NIH)[DA-04494]U.S. National Institutes of Health (NIH)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[04/04933-2]FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[04/14846-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Financiadora de Estudos e Projetos (FINEP)[A-03/134]Financiadora de Estudos e Projetos (FINEP)CNPq Conselho Nacional de Desenvolvimento Cientifico e TecnologicoConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade de Campinas (UNICAMP), Campinas, SP, BrazilUniversidade Estadual de Campinas (UNICAMP

Quantitative Peptidomics of <i>Purkinje Cell Degeneration</i> Mice

<div><p>Cytosolic carboxypeptidase 1 (CCP1) is a metallopeptidase that removes C-terminal and side-chain glutamates from tubulin. The <i>Purkinje cell degeneration</i> (<i>pcd</i>) mouse lacks CCP1 due to a mutation. Previously, elevated levels of peptides derived from cytosolic and mitochondrial proteins were found in adult <i>pcd</i> 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 <i>pcd</i> mice, examining adult heart, spleen, and brain, and presymptomatic 3 week-old amygdala and cerebellum. Contrary to adult mouse brain, young <i>pcd</i> 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 <i>pcd</i> mouse brain. The pattern of changes for the intracellular and secretory pathway peptides in <i>pcd</i> mice was generally similar to the pattern observed in mice lacking primary cilia. Collectively, these results suggest that intracellular peptide accumulation in adult <i>pcd</i> mouse brain is a secondary effect and is not due to a role of CCP1 in peptide turnover.</p> </div

Overview of intracellular peptides identified in analysis of <i>pcd</i> and WT mice, comparing adult amygdala, 3 week old amygdala, and 3 week old cerebellum.

<p>For this analysis, the intracellular peptides listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060981#pone.0060981.s003" target="_blank">Table S1</a> as conclusively identified by MS/MS were considered; unknowns and tentatively identified peptides in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060981#pone.0060981.s003" target="_blank">Table S1</a> were not used for this analysis. Each peptide was counted once, regardless of whether it was detected in both <i>pcd</i> and WT mice or in only one of these genotypes. Peptides listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060981#pone.0060981.s003" target="_blank">Table S1</a> with multiple charge states were counted only once.</p

Levels of intracellular peptides in the brain of young WT and <i>pcd</i> mice.

<p>The levels of peptides derived from intracellular proteins were studied using quantitative peptidomics. The relative levels of peptides in WT mice (black circles) and <i>pcd</i> mice (grey circles) are indicated for (<b>A</b>) amygdala of adult mice; (<b>B</b>) amygdala of 3-week old mice; and (<b>C</b>) cerebellum of 3-week old mice. Each dot in the graph shows the ratio between a peptide in one WT or <i>pcd</i> replicate versus the average level in the WT replicates. The x-axis reflects the number of peptides found in <i>pcd</i> mice. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060981#pone.0060981.s003" target="_blank">Table S1</a> for data.</p

Analysis of peptides derived from secretory pathway proteins.

<p>Secretory pathway peptides detected in <i>pcd</i> mouse amygdala and hypothalamus were divided into the same five groups as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060981#pone-0060981-g008" target="_blank">Figure 8</a>. The y-axes indicate the number of peptides in all LC/MS runs. <b>Lower panel</b>: Peptides were divided into two categories based on the cleavage sites required to release the peptide from the precursor, and plotted as above. Left: peptides generated by cleavage at prohormone convertase consensus sites and/or other sites containing basic amino acids (lysine, arginine). Right: peptides generated by cleavage at a non-basic site at either the N- or C-terminal side of the peptide.</p

Alterations of the Intracellular Peptidome in Response to the Proteasome Inhibitor Bortezomib

<div><p>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.</p> </div