RESUMO
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.
Assuntos
Carboxipeptidases/metabolismo , Ácido Glutâmico/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Carboxipeptidases/química , Carboxipeptidases/genética , Ácido Glutâmico/química , Ácido Glutâmico/genética , Camundongos , Microtúbulos/química , Microtúbulos/genética , Paclitaxel/farmacologia , Células Sf9 , Spodoptera , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Moduladores de Tubulina/farmacologiaRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Degeneração Neural/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Neoplasias da Mama , Linhagem Celular Tumoral , Neoplasias do Colo , Citosol/enzimologia , Feminino , Proteínas de Ligação ao GTP/genética , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Degeneração Neural/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Estrutura Terciária de Proteína , Células de Purkinje/enzimologia , Células de Purkinje/patologia , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , Suínos , Tubulina (Proteína)/químicaRESUMO
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.
Assuntos
Mutação , Fragmentos de Peptídeos/metabolismo , Proteômica , Células de Purkinje/patologia , Sequência de Aminoácidos , Tonsila do Cerebelo/metabolismo , Animais , Cerebelo/metabolismo , Feminino , Proteínas de Ligação ao GTP/deficiência , Proteínas de Ligação ao GTP/genética , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Células de Purkinje/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/deficiência , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genéticaRESUMO
STAT1 (signal transducer and activator of transcription 1) has been implicated as a mediator of a variety of biological responses in response to stimulation by specific growth factors and cytokines. To understand better the role of STAT1 in the interferon-gamma (IFN-gamma)-induced phenotype, we generated an active form of STAT1 (STAT1C) by substituting Cys residues for both Arg-656 and Asn-658 within the C-terminal loop of the STAT1 SH2 domain. The IFN-gamma activation site element was stimulated and bound efficiently by STAT1C without IFN-gamma treatment. STAT1C was found to be tyrosine-phosphorylated in the nucleus for more than 30 h after IFN-gamma stimulation. STAT1-negative U3A cells reexpressing STAT1C showed retarded cell growth and underwent apoptosis when treated with IFN-gamma. Further analysis demonstrated that apoptosis was preceded by proteolytic cleavage of caspases 2, 3, and 7, and wild type STAT1 also induced cleavage of caspase 7 when expressed in STAT1-negative U3A cells, indicating that STAT1C augments potential activity of wild type STAT1. Studies with cycloheximide treatment showed that protein synthesis induced in the first 24 h after IFN-gamma treatment was required for apoptosis under these conditions. Finally, we found that STAT1C-induced apoptosis was, in part, mediated by caspase 2, 3, and 7 because benzyloxycarbonyl-valyl-aspartyl-valyl-alanyl-aspartic acid fluoromethyl ketone (Z-VDVAD-FMK) treatment partially blocked apoptosis. These results suggest that prolonged nuclear localization of activated STAT1 results in apoptosis involving specific regulation of caspase pathway.