Date Published: April 8, 2013
Publisher: Public Library of Science
Author(s): Iryna Berezniuk, Juan J. Sironi, Jonathan Wardman, Raymond C. Pasek, Nicolas F. Berbari, Bradley K. Yoder, Lloyd D. Fricker, Hubert Vaudry. http://doi.org/10.1371/journal.pone.0060981
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.
In the 1970s, a spontaneous mutant mouse was discovered and named Purkinje cell degeneration (pcd) due to the loss of cerebellar Purkinje cells starting around 3 weeks after birth . A small number of other cell types undergo degeneration in pcd mice, including olfactory bulb mitral cells, retinal photoreceptor cells, and spermatocytes . The mutation responsible for the pcd phenotype was mapped to the gene encoding cytosolic carboxypeptidase 1 (CCP1, also known as Nna1), and the gene was named Agtpbp1 because the protein was initially considered to be an ATP/GTP binding protein . CCP1 was discovered in a search for mRNAs upregulated in spinal motor neurons during regeneration after axotomy . Thus, CCP1 is linked to both degeneration and regeneration. CCP1 has sequence homology to metallocarboxypeptidases, including conservation of critical active site residues, but lacks a signal peptide and is expressed in the cytosol . Five additional members of the CCP1 subfamily were discovered and named CCP2 through CCP6 , . CCP1 is the most abundant of the CCPs in mouse brain .
If CCP1 functions in peptide degradation in adult mouse brain, we reasoned that because protein/peptide degradation is such a fundamental process, CCP1 would also function in this capacity in young mouse brain, and therefore a peptidomics analysis of young pcd mouse brain would also show marked changes in levels of intracellular peptides. In contrast, if the previously observed increase in levels of many brain peptides was a consequence of the neurodegeneration in the adult pcd mice, then young mice analyzed prior to the onset of neurodegeneration would not show the increase in intracellular peptide levels. Purkinje cells start to degenerate in pcd mice when animals are 3 weeks old ; this was confirmed in our colony of pcd mice by immunohistochemistry (Figure S1). To test if young presymptomatic mice show greatly altered levels of the brain peptidome, a quantitative peptidomics approach involving TMAB isotopic labels was used to compare amygdala and cerebellum of 3 week old WT mice versus pcd mice. The five distinct masses of the TMAB labels allow 2–3 mutant mice to be compared to 2–3 WT mice in the same experiment; this allows for variation among replicates of WT animals to be determined along with the ratio of peptides in mutant versus WT mice. Representative MS data are shown in Figure 1, comparing relative levels of the same peptide in three different LC/MS runs. Panel A shows the LC/MS run for adult amygdala in which two pcd mice were compared to three WT mice. Panel B shows the LC/MS run for 3 week old amygdala in which three pcd mice were compared to two WT mice. Panel C shows the LC/MS run for 3 week old cerebellum in which three pcd mice were compared to two WT mice. For this peptide, levels are generally comparable between the pcd and WT mice when tested at 3 weeks of age, but clearly much higher in the adult pcd mice than the WT mice. Altogether, 146 distinct peptides arising from intracellular proteins were identified in the young amygdala and 177 distinct intracellular peptides identified in the young cerebellum; there was considerable overlap between the two sets of peptides (Figure 2). These two sets of peptides also showed considerable overlap with the intracellular peptides previously identified in the study comparing the amygdala of adult WT and pcd mice . Data are provided in Table S1.
The overall goal of the present study was to determine whether CCP1 plays a role in peptide turnover within the cell. Previously, some of the authors of the present study proposed that CCP1 contributed to peptide degradation based on the dramatic increase in levels of intracellular peptides observed in pcd mice . While these authors had proposed that CCP1 could also play a role in tubulin processing, the simplest explanation to account for the increase in peptides derived from intracellular proteins was that these peptides represented substrates of CCP1. This was found to be the case for peptides that accumulate in the brains of Cpefat/fat mice, which lack carboxypeptidase E activity due to a point mutation . However, the Cpefat/fat mice showed both an increase in levels of substrates and a decrease in levels of products, as expected for a normal substrate-product relationship . In contrast, the adult pcd mouse brain peptides were mainly elevated, which was interpreted to indicate that CCP1 degraded all peptides that it came into contact with . However, the peptides elevated in adult pcd mouse brains contain a wide range of C-terminal amino acids; if these peptides represent substrates, then CCP1 has a very broad substrate specificity. This was a surprising observation; even the metallocarboxypeptidases present in the digestive system show marked preference towards particular amino acids –. Additional problems with this hypothesized function for CCP1 were found from studies overexpressing and knocking down the protein in HEK293 cells; only a small number of peptides were significantly elevated by the knock-down of CCP1, and no peptides were significantly altered by the overexpression of the protein . Because protein turnover is a fundamental process that occurs in every cell of the body, it was expected that if CCP1 performs this role in adult mouse brain, it would also perform this role in cultured cells as well as in young mouse brain and other tissues. Therefore, the goal of the present study was to address the function of CCP1 by examining peptide levels in pcd mouse tissues that do not undergo degeneration, and young brain, prior to the degeneration of Purkinje cells.