Iryna Berezniuk

A novel subfamily of mouse cytosolic carboxypeptidases

Nnal is a recently described gene product that has sequence similarity with metallocar‐boxypeptidases. In the present study, five additional Nnal‐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, Nnal/CCPl, CCP5, and CCP6 are broadly distributed, whereas CCP2 and 3 exhibit restricted patterns of expression. Nnal/CCPl, 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 Nnal/CCPl and CCP2‐6 function in the processing of cytosolic proteins such as alpha‐tubuHn, 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 Nnal/CCPl showed the absence of the detyrosinylated form in the mitral cells. Taken together, these results are consistent with a role for Nnal/CCPl and the related CCPs in the processing of tubulin.—Kalinina, E., Biswas, R., Berezniuk, I., Hermoso, A., Aviles, F. X., Fricker, L. D. A novel subfamily of mouse cytosolic carboxypeptidases. FASEB J. 21, 836–850 (2007)

Peptidomics of Cpefat/fat mouse brain regions: implications for neuropeptide processing

Quantitative peptidomics was used to compare levels of peptides in wild type (WT) and Cpefat/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 Cpefat/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 Cpefat/fat mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpefat/fat mouse brain at levels similar to those in WT mouse brain. Many peptides were greatly elevated in the Cpefat/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.

Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin

Background: Several cellular functions for cytosolic carboxypeptidase 1 (CCP1) have been proposed. Results: Various experimental approaches support a role for CCP1 in the removal of Glu residues from both α- and β-tubulin. Conclusion: CCP1 functions in tubulin processing and is not involved in intracellular peptide degradation. Significance: Neurodegeneration in mice lacking CCP1 is a result of altered tubulin processing. 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 β-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 β-tubulin. The hyperglutamylation of α- and β-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 β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.

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.org

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

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.

Altered neuropeptide processing in prefrontal cortex of Cpefat/fat mice: implications for neuropeptide discovery

The biosynthesis of most neuropeptides and peptide hormones requires a carboxypeptidase such as carboxypeptidase E, which is inactive in Cpefat/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 Cpefat/fat versus wild‐type mice. Peptides representing internal fragments of prohormones and other secretory pathway proteins were decreased two‐ to 10‐fold in the Cpefat/fat mouse prefrontal cortex compared with wild‐type tissue. Degradation fragments of cytosolic proteins showed no major differences between Cpefat/fat and wild‐type mice. Based on this observation, a search strategy for neuropeptides was performed by screening for peptides that decreased in the Cpefat/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 Cpefat/fat mice.

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.

Analysis of peptides secreted from cultured mouse brain tissue

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.

Emergence of anxiety-like behaviours in depressive-like Cpe fat/fat mice

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 (∼60 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 ∼60 d, whereas anxiety-like behaviours emerge ∼1 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.

A defect in cytosolic carboxypeptidase 1 (Nna1) causes autophagy in Purkinje cell degeneration mouse brain

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.