The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process

Evidence for new C-terminally truncated variants of α- and β-tubulins

New C-terminally truncated α- and β-tubulin variants, both ending with an -EEEG sequence, are identified in vivo: αΔ3-tubulin, which has a specific neuronal distribution pattern (distinct from that of αΔ2-tubulin) and seems to be related to dynamic microtubules, and βΔ4-tubulin, corresponding to β2A/B-tubulin modified by truncation of four C-terminal residues, which is ubiquitously present in cells and tissues. Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the -EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same -EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with -EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development

Structural modeling and functional characterization of mammalian cytosolic carboxypeptidases 2 and 3

Aquesta tesis s’emmarca en el camp de les carboxipeptidases citosòliques de mamífers, centrant-­‐se particularment en els membres 2 i 3 d’aquesta subfamília de metalocarboxipeptidases. Aquest treball constitueix el primer estudi sobre la funció d’aquests enzims a través d’aproximacions in silico, in vitro i in vivo. Aquesta tesis consta de les parts descrites a continuació. El primer capítol introdueix el camp de les matalocarboxipeptidases, els microtúbuls, les modificacions post-­‐traduccionals de la tubulina i estableix els objectius de la tesi. El segon capítol és una compilació dels procediments experimentals i materials utilitzats en el treball experimental. El tercer capítol presenta la caracterització funcional de la carboxipeptidasa citosòlica 3 humana. Els resultats d’aquest estudi han estat la base pel quart capítol, on es descriu la funció de la CCP2. Ambdues CCP2 i CCP3 isoformes de ratolí s’estudien mitjançant enginyeria de proteïnes i utilitzant els models de ratolí knockout de CCP2 i CCP3. Els capítols III i IV es subdivideixen en un resum del capítol, els resultats que descriuen el detall dels descobriments i una discussió. A continuació es presenta una discussió general sobre CCP2 i CCP3, el seu rol en la cèl·∙lula i en el context de la subfamília de carboxipeptidases que, en mamífers, consta de sis gens codificants per enzims amb activitat deglutamilasa. La tesis finalitza amb un resum de les conclusions. El capítol IV juntament amb els experiments de modelat estructural del capítol III són la base de la següent publicació científica: Tort O, Tanco S, Rocha C, Bièche I, Seixas C, Bosc C, Andrieux A, Moutin MJ, Avilés FX, Lorenzo J, Janke C. The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids. MBoC. doi:10.1091/mbc.E14-­‐06-­‐1072This thesis is situated in the field of the mammalian cytosolic carboxypeptidases, particularly focusing in members 2 and 3 from this subfamily of metallocarboxypeptidases. This work gives insight for the first time into the role of those enzymes by in silico, in vitro and in vivo approaches. The thesis is divided in parts as described below. The first chapter introduces the fields of metallocarboxypeptidases, microtubules, tubulin posttranslational modifications and settle the objectives of the thesis. The second chapter is a compilation of the experimental procedures and materials used for the experimental work. The third chapter presents the functional characterization of human cytosolic carboxypeptidase 3. The results from this study were the basis for chapter four, where the function of CCP2 is described and both CCP2 and CCP3 mouse isoforms are studied by protein engineering and using CCP2 and CCP3 knock-­‐out mouse models. Chapters III and IV are subdivided into a summary of the chapter, the results describing the findings in detail and a discussion. The following chapter sets a general discussion on CCP2 and CCP3, their role in the cell and in the context of a family, in mammals, of six genes coding deglutamylating enzymes. A final section includes a summary of the conclusions. Chapter IV together with the modeling experiments form Chapter III is the basis of the following scientific paper: Tort O, Tanco S, Rocha C, Bièche I, Seixas C, Bosc C, Andrieux A, Moutin MJ, Avilés FX, Lorenzo J, Janke C. The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids. MBoC. doi:10.1091/mbc.E14-­‐06-­‐107

Structural modeling and functional characterization of mammalian cytosolic carboxypeptidases 2 and 3

Aquesta tesis s'emmarca en el camp de les carboxipeptidases citosòliques de mamífers, centrant-­-se particularment en els membres 2 i 3 d'aquesta subfamília de metalocarboxipeptidases. Aquest treball constitueix el primer estudi sobre la funció d'aquests enzims a través d'aproximacions in silico, in vitro i in vivo. Aquesta tesis consta de les parts descrites a continuació. El primer capítol introdueix el camp de les matalocarboxipeptidases, els microtúbuls, les modificacions post-­-traduccionals de la tubulina i estableix els objectius de la tesi. El segon capítol és una compilació dels procediments experimentals i materials utilitzats en el treball experimental. El tercer capítol presenta la caracterització funcional de la carboxipeptidasa citosòlica 3 humana. Els resultats d'aquest estudi han estat la base pel quart capítol, on es descriu la funció de la CCP2. Ambdues CCP2 i CCP3 isoformes de ratolí s'estudien mitjançant enginyeria de proteïnes i utilitzant els models de ratolí knockout de CCP2 i CCP3. Els capítols III i IV es subdivideixen en un resum del capítol, els resultats que descriuen el detall dels descobriments i una discussió. A continuació es presenta una discussió general sobre CCP2 i CCP3, el seu rol en la cèl·∙lula i en el context de la subfamília de carboxipeptidases que, en mamífers, consta de sis gens codificants per enzims amb activitat deglutamilasa. La tesis finalitza amb un resum de les conclusions. El capítol IV juntament amb els experiments de modelat estructural del capítol III són la base de la següent publicació científica: Tort O, Tanco S, Rocha C, Bièche I, Seixas C, Bosc C, Andrieux A, Moutin MJ, Avilés FX, Lorenzo J, Janke C. The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids. MBoC. doi:10.1091/mbc.E14-­-06-­-1072This thesis is situated in the field of the mammalian cytosolic carboxypeptidases, particularly focusing in members 2 and 3 from this subfamily of metallocarboxypeptidases. This work gives insight for the first time into the role of those enzymes by in silico, in vitro and in vivo approaches. The thesis is divided in parts as described below. The first chapter introduces the fields of metallocarboxypeptidases, microtubules, tubulin posttranslational modifications and settle the objectives of the thesis. The second chapter is a compilation of the experimental procedures and materials used for the experimental work. The third chapter presents the functional characterization of human cytosolic carboxypeptidase 3. The results from this study were the basis for chapter four, where the function of CCP2 is described and both CCP2 and CCP3 mouse isoforms are studied by protein engineering and using CCP2 and CCP3 knock-­-out mouse models. Chapters III and IV are subdivided into a summary of the chapter, the results describing the findings in detail and a discussion. The following chapter sets a general discussion on CCP2 and CCP3, their role in the cell and in the context of a family, in mammals, of six genes coding deglutamylating enzymes. A final section includes a summary of the conclusions. Chapter IV together with the modeling experiments form Chapter III is the basis of the following scientific paper: Tort O, Tanco S, Rocha C, Bièche I, Seixas C, Bosc C, Andrieux A, Moutin MJ, Avilés FX, Lorenzo J, Janke C. The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids. MBoC. doi:10.1091/mbc.E14-­-06-­-107

Versatile iron-catechol-based nanoscale coordination polymers with antiretroviral ligand functionalization and their use as efficient carriers in HIV/AIDS therapy

A novel chemical approach integrating the benefits of nanoparticles with versatility of coordination chemistry is reported herein to increase the effectiveness of well-known HIV antiretroviral drugs. The novelty of our approach is illustrated using a catechol ligand tethered to the known antiretroviral azidothymidine (AZT) as a constitutive building block of the nanoparticles. The resulting nanoscale coordination polymers (NCPs) ensure good encapsulation yields and equivalent antiretroviral activity while significantly diminishing its cytotoxicity. Moreover, this novel family of nanoparticles also offers (i) long-lasting drug release that is dissimilar inside and outside the cells depending on pH, (ii) triggered release in the presence of esterases, activating the antiviral activity in an on-off manner due to a proper chemical design of the ligand and (iii) improved colloidal stabilities and cellular uptakes (up to 50-fold increase). The presence of iron nodes also adds multifunctionality as possible contrast agents. The present study demonstrates the suitability of NCPs bearing pharmacologically active ligands as an alternative to conventional antiretroviral treatments

Versatile iron-catechol-based nanoscale coordination polymers with antiretroviral ligand functionalization and their use as efficient carriers in HIV/AIDS therapy

A novel chemical approach integrating the benefits of nanoparticles with versatility of coordination chemistry is reported herein to increase the effectiveness of well-known HIV antiretroviral drugs. The novelty of our approach is illustrated using a catechol ligand tethered to the known antiretroviral azidothymidine (AZT) as a constitutive building block of the nanoparticles. The resulting nanoscale coordination polymers (NCPs) ensure good encapsulation yields and equivalent antiretroviral activity while significantly diminishing its cytotoxicity. Moreover, this novel family of nanoparticles also offers (i) long-lasting drug release that is dissimilar inside and outside the cells depending on pH, (ii) triggered release in the presence of esterases, activating the antiviral activity in an on-off manner due to a proper chemical design of the ligand and (iii) improved colloidal stabilities and cellular uptakes (up to 50-fold increase). The presence of iron nodes also adds multifunctionality as possible contrast agents. The present study demonstrates the suitability of NCPs bearing pharmacologically active ligands as an alternative to conventional antiretroviral treatments

The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process