CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death inPurkinje cell degenerationmice

Abstract

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.

Similar Intracellular Peptide Profile of TAP1/β2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

“…In the present study, a semi-quantitative peptidomic assay using stable isotopic labels and mass spectrometry that has been extensively characterized (18,19,35,36) was used to examine possible differences between the intracellular peptide profile of whole brain tissue from TAP1/β2m −/− mice and C57BL/6 wild-type mice. We chose to investigate the peptide profile of the whole brain instead of specific brain areas because our main goal was to identify possible differences in the peptide diversity of TAP1/β2m −/− mice compared with C57BL/6 wild-type mice rather than to quantitate differences related to specific brain regions.…”

“…On the other hand, a large number of intracellular peptides, apparently not related to antigen presentation, have been recently identified in mammalian cells and tissues (18)(19)(20). Thus, intracellular peptides isolated from rat brain homogenates using the inactive thimet oligopeptidase (EC 3.4.24.15; EP24.15) "substrate capture" assay (21) can efficiently interfere with G-protein-coupled receptor signal transduction in both Chinese hamster ovarian-S cells expressing the angiotensin type 1 receptor and stimulated with angiotensin II, as well as in human embryonic kidney 293 cells stimulated with isoproterenol (22).…”

A missense mutation in AGTPBP1 was identified in sheep with a lower motor neuron disease

“…This protein has a role in protein turnover by cleaving proteasome-generated peptides into amino acids (Berezniuk et al, 2010), enzymatically removing geneencoded glutamic acids or tyrosine from the C-terminus of proteins (Rogowski et al, 2010, Kalinina et al, 2007 and also has a role in neuronal bioenergetics at least in mouse brains (Chakrabarti et al, 2010). Similar to other cytosolic carboxypeptidases, the mouse Nna1 protein is broadly expressed in the brain, pituitary, eye, testis and other tissues (Kalinina et al, 2007).…”

Limitations of Mass Spectrometry-Based Peptidomic Approaches

“…These three databases were composed of published studies [20,22,24,25,28,30,[33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48] and a few additional studies that have not yet been published; these unpublished studies used identical methods, differing only in the treatment groups (which is not relevant to the present meta-analysis). The mouse database contains all peptidomic results from studies using mouse tissues-mainly brain regions, but also whole brain as well as heart, spleen, and testis.…”

“…The TMAB tags can be produced in five distinct isotopic forms, allowing for multivariate analysis [32]. Using a combination of heat inactivation of the sample and TMAB labels for quantitative peptidomics, we have performed hundreds of LC/MS runs of samples and MS/MS analyses of peptides [20,22,24,25,28,30,[33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48]. In each LC/MS run, up to several hundred peptides were detected and identified.…”