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Francesc X. Avilés - One of the best experts on this subject based on the ideXlab platform.
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synthesis and structural functional characterization of selective m14 metallocarboxypeptidase inhibitors based on phosphinic pseudopeptide scaffold implications on the design of specific optical probes
Journal of Medicinal Chemistry, 2019Co-Authors: Giovanni Covaleda, Josep Vendrell, Julia Lorenzo, Francesc X. Avilés, David Reverter, Pablo Gallego, Dimitris Georgiadis, Vincent Dive, L. DevelAbstract:Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the ...
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Synthesis and Structural/Functional Characterization of Selective M14 Metallocarboxypeptidase Inhibitors Based on Phosphinic Pseudopeptide Scaffold: Implications on the Design of Specific Optical Probes
2019Co-Authors: Giovanni Covaleda, Josep Vendrell, Julia Lorenzo, Francesc X. Avilés, David Reverter, Pablo Gallego, Dimitris Georgiadis, Vincent Dive, L. DevelAbstract:Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the first example of chemical probes useful to selectively report on type-A MCPs activity in complex media
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crystal structure and mechanism of human carboxypeptidase o insights into its specific activity for acidic residues
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Maria C Garciaguerrero, Peter J Lyons, Francesc X. Avilés, Javier Garciapardo, Robert Huber, Esther Berenguer, Roberto Fernandezalvarez, Gifty B Barfi, Julia LorenzoAbstract:Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic Metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1.85-A resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail Nerita versicolor . The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at “canonical” position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1′ substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.
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Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña.
International journal of molecular sciences, 2018Co-Authors: Juliana Cotabarren, Javier Garcia-pardo, Julia Lorenzo, Sebastian Tanco, Francesc X. Avilés, Mariana Edith Tellechea, Walter David ObregónAbstract:Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churquena. Our results demonstrated that chuPCI is a member of the A/B-type family of Metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.
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substrate specificity of human metallocarboxypeptidase d comparison of the two active carboxypeptidase domains
PLOS ONE, 2017Co-Authors: Javier Garciapardo, Julia Lorenzo, Sebastian Tanco, Lucia Diaz, Sayani Dasgupta, Juan Fernandezrecio, Francesc X. AvilésAbstract:Metallocarboxypeptidase D (CPD) is a membrane-bound component of the trans-Golgi network that cycles to the cell surface through exocytic and endocytic pathways. Unlike other members of the metallocarboxypeptidase family, CPD is a multicatalytic enzyme with three carboxypeptidase-like domains, although only the first two domains are predicted to be enzymatically active. To investigate the enzymatic properties of each domain in human CPD, a critical active site Glu in domain I and/or II was mutated to Gln and the protein expressed, purified, and assayed with a wide variety of peptide substrates. CPD with all three domains intact displays >50% activity from pH 5.0 to 7.5 with a maximum at pH 6.5, as does CPD with mutation of domain I. In contrast, the domain II mutant displayed >50% activity from pH 6.5–7.5. CPD with mutations in both domains I and II was completely inactive towards all substrates and at all pH values. A quantitative peptidomics approach was used to compare the activities of CPD domains I and II towards a large number of peptides. CPD cleaved C-terminal Lys or Arg from a subset of the peptides. Most of the identified substrates of domain I contained C-terminal Arg, whereas comparable numbers of Lys- and Arg-containing peptides were substrates of domain II. We also report that some peptides with C-terminal basic residues were not cleaved by either domain I or II, showing the importance of the P1 position for CPD activity. Finally, the preference of domain I for C-terminal Arg was validated through molecular docking experiments. Together with the differences in pH optima, the different substrate specificities of CPD domains I and II allow the enzyme to perform distinct functions in the various locations within the cell.
Lloyd D Fricker - One of the best experts on this subject based on the ideXlab platform.
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Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules
2016Co-Authors: Mark William Lopes, Matthew R. Sapio, Rodrigo B. Leal, Lloyd D FrickerAbstract:Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency.
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carboxypeptidase o is a glycosylphosphatidylinositol anchored intestinal peptidase with acidic amino acid specificity
Journal of Biological Chemistry, 2011Co-Authors: Peter J Lyons, Lloyd D FrickerAbstract:Abstract The first metallocarboxypeptidase (CP) was identified in pancreatic extracts more than 80 years ago and named carboxypeptidase A (CPA; now known as CPA1). Since that time, seven additional mammalian members of the CPA subfamily have been described, all of which are initially produced as proenzymes, are activated by endoproteases, and remove either C-terminal hydrophobic or basic amino acids from peptides. Here we describe the enzymatic and structural properties of carboxypeptidase O (CPO), a previously uncharacterized and unique member of the CPA subfamily. Whereas all other members of the CPA subfamily contain an N-terminal prodomain necessary for folding, bioinformatics and expression of both human and zebrafish CPO orthologs revealed that CPO does not require a prodomain. CPO was purified by affinity chromatography, and the purified enzyme was able to cleave proteins and synthetic peptides with greatest activity toward acidic C-terminal amino acids unlike other CPA-like enzymes. CPO displayed a neutral pH optimum and was inhibited by common metallocarboxypeptidase inhibitors as well as citrate. CPO was modified by attachment of a glycosylphosphatidylinositol membrane anchor to the C terminus of the protein. Immunocytochemistry of Madin-Darby canine kidney cells stably expressing CPO showed localization to vesicular membranes in subconfluent cells and to the plasma membrane in differentiated cells. CPO is highly expressed in intestinal epithelial cells in both zebrafish and human. These results suggest that CPO cleaves acidic amino acids from dietary proteins and peptides, thus complementing the actions of well known digestive carboxypeptidases CPA and CPB.
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Individual carboxypeptidase D domains have both redundant and unique functions in Drosophila development and behavior
Cellular and Molecular Life Sciences, 2010Co-Authors: Galyna Sidyelyeva, Christian Wegener, Nicholas E Baker, Brian P. Schoenfeld, Aaron J. Bell, Sean M. J. Mcbride, Lloyd D FrickerAbstract:Metallocarboxypeptidase D (CPD) functions in protein and peptide processing. The Drosophila CPD svr gene undergoes alternative splicing, producing forms containing 1–3 active or inactive CP domains. To investigate the function of the various CP domains, we created transgenic flies expressing specific forms of CPD in the embryonic-lethal svr ^PG33 mutant. All constructs containing an active CP domain rescued the lethality with varying degrees, and full viability required inactive CP domain-3. Transgenic flies overexpressing active CP domain-1 or -2 were similar to each other and to the viable svr mutants, with pointed wing shape, enhanced ethanol sensitivity, and decreased cold sensitivity. The transgenes fully compensated for a long-term memory deficit observed in the viable svr mutants. Overexpression of CP domain-1 or -2 reduced the levels of Lys/Arg-extended adipokinetic hormone intermediates. These findings suggest that CPD domains-1 and -2 have largely redundant functions in the processing of growth factors, hormones, and neuropeptides.
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characterization of the substrate specificity of human carboxypeptidase a4 and implications for a role in extracellular peptide processing
Journal of Biological Chemistry, 2010Co-Authors: Sebastian Tanco, Julia Lorenzo, Francesc X. Avilés, Xin Zhang, Cain Morano, Lloyd D FrickerAbstract:CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment.
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ccp1 nna1 functions in protein turnover in mouse brain implications for cell death in purkinje cell degeneration mice
The FASEB Journal, 2010Co-Authors: Iryna Berezniuk, Juan Sironi, Myrasol Callaway, Leandro M Castro, Izaura Y Hirata, Emer S Ferro, Lloyd D FrickerAbstract: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.
Julia Lorenzo - One of the best experts on this subject based on the ideXlab platform.
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Characterization, Recombinant Production and Structure-Function Analysis of NvCI, A Picomolar Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita versicolor.
Marine Drugs, 2019Co-Authors: Giovanni Covaleda-cortés, Manuel Mansur, Sergi Rodríguez-calado, Javier Garcia-pardo, Martha Hernandez, Josep Vendrell, Julia Lorenzo, Sebastián A. Trejo, Maria A Chavez, Maday Alonso-del-riveroAbstract:A very powerful proteinaceous inhibitor of Metallocarboxypeptidases has been isolated from the marine snail Nerita versicolor and characterized in depth. The most abundant of four, very similar isoforms, NvCla, was taken as reference and N-terminally sequenced to obtain a 372-nucleotide band coding for the protein cDNA. The mature protein contains 53 residues and three disulphide bonds. NvCIa and the other isoforms show an exceptionally high inhibitory capacity of around 1.8 pM for human Carboxypeptidase A1 (hCPA1) and for other A-like members of the M14 CPA subfamily, whereas a twofold decrease in inhibitory potency is observed for carboxypeptidase B-like members as hCPB and hTAFIa. A recombinant form, rNvCI, was produced in high yield and HPLC, mass spectrometry and spectroscopic analyses by CD and NMR indicated its homogeneous, compact and thermally resistant nature. Using antibodies raised with rNvCI and histochemical analyses, a preferential distribution of the inhibitor in the surface regions of the animal body was observed, particularly nearby the open entrance of the shell and gut, suggesting its involvement in biological defense mechanisms. The properties of this strong, small and stable inhibitor of Metallocarboxypeptidases envisage potentialities for its direct applicability, as well as leading or minimized forms, in biotechnological/biomedical uses.
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synthesis and structural functional characterization of selective m14 metallocarboxypeptidase inhibitors based on phosphinic pseudopeptide scaffold implications on the design of specific optical probes
Journal of Medicinal Chemistry, 2019Co-Authors: Giovanni Covaleda, Josep Vendrell, Julia Lorenzo, Francesc X. Avilés, David Reverter, Pablo Gallego, Dimitris Georgiadis, Vincent Dive, L. DevelAbstract:Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the ...
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Synthesis and Structural/Functional Characterization of Selective M14 Metallocarboxypeptidase Inhibitors Based on Phosphinic Pseudopeptide Scaffold: Implications on the Design of Specific Optical Probes
2019Co-Authors: Giovanni Covaleda, Josep Vendrell, Julia Lorenzo, Francesc X. Avilés, David Reverter, Pablo Gallego, Dimitris Georgiadis, Vincent Dive, L. DevelAbstract:Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the first example of chemical probes useful to selectively report on type-A MCPs activity in complex media
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crystal structure and mechanism of human carboxypeptidase o insights into its specific activity for acidic residues
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Maria C Garciaguerrero, Peter J Lyons, Francesc X. Avilés, Javier Garciapardo, Robert Huber, Esther Berenguer, Roberto Fernandezalvarez, Gifty B Barfi, Julia LorenzoAbstract:Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic Metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1.85-A resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail Nerita versicolor . The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at “canonical” position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1′ substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.
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Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña.
International journal of molecular sciences, 2018Co-Authors: Juliana Cotabarren, Javier Garcia-pardo, Julia Lorenzo, Sebastian Tanco, Francesc X. Avilés, Mariana Edith Tellechea, Walter David ObregónAbstract:Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churquena. Our results demonstrated that chuPCI is a member of the A/B-type family of Metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.
Josep Vendrell - One of the best experts on this subject based on the ideXlab platform.
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Characterization, Recombinant Production and Structure-Function Analysis of NvCI, A Picomolar Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita versicolor.
Marine Drugs, 2019Co-Authors: Giovanni Covaleda-cortés, Manuel Mansur, Sergi Rodríguez-calado, Javier Garcia-pardo, Martha Hernandez, Josep Vendrell, Julia Lorenzo, Sebastián A. Trejo, Maria A Chavez, Maday Alonso-del-riveroAbstract:A very powerful proteinaceous inhibitor of Metallocarboxypeptidases has been isolated from the marine snail Nerita versicolor and characterized in depth. The most abundant of four, very similar isoforms, NvCla, was taken as reference and N-terminally sequenced to obtain a 372-nucleotide band coding for the protein cDNA. The mature protein contains 53 residues and three disulphide bonds. NvCIa and the other isoforms show an exceptionally high inhibitory capacity of around 1.8 pM for human Carboxypeptidase A1 (hCPA1) and for other A-like members of the M14 CPA subfamily, whereas a twofold decrease in inhibitory potency is observed for carboxypeptidase B-like members as hCPB and hTAFIa. A recombinant form, rNvCI, was produced in high yield and HPLC, mass spectrometry and spectroscopic analyses by CD and NMR indicated its homogeneous, compact and thermally resistant nature. Using antibodies raised with rNvCI and histochemical analyses, a preferential distribution of the inhibitor in the surface regions of the animal body was observed, particularly nearby the open entrance of the shell and gut, suggesting its involvement in biological defense mechanisms. The properties of this strong, small and stable inhibitor of Metallocarboxypeptidases envisage potentialities for its direct applicability, as well as leading or minimized forms, in biotechnological/biomedical uses.
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synthesis and structural functional characterization of selective m14 metallocarboxypeptidase inhibitors based on phosphinic pseudopeptide scaffold implications on the design of specific optical probes
Journal of Medicinal Chemistry, 2019Co-Authors: Giovanni Covaleda, Josep Vendrell, Julia Lorenzo, Francesc X. Avilés, David Reverter, Pablo Gallego, Dimitris Georgiadis, Vincent Dive, L. DevelAbstract:Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the ...
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Synthesis and Structural/Functional Characterization of Selective M14 Metallocarboxypeptidase Inhibitors Based on Phosphinic Pseudopeptide Scaffold: Implications on the Design of Specific Optical Probes
2019Co-Authors: Giovanni Covaleda, Josep Vendrell, Julia Lorenzo, Francesc X. Avilés, David Reverter, Pablo Gallego, Dimitris Georgiadis, Vincent Dive, L. DevelAbstract:Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the first example of chemical probes useful to selectively report on type-A MCPs activity in complex media
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Discovery of Mechanism-Based Inactivators for Human Pancreatic Carboxypeptidase A from a Focused Synthetic Library
2017Co-Authors: Sebastián A. Testero, Josep Vendrell, Giovanni Covaleda, David Reverter, Pablo Gallego, Carla Granados, Daniel Fernández, Francesc X. Avilés, Irantzu Pallarès, Shahriar MobasheryAbstract:Metallocarboxypeptidases (MCPs) are involved in many biological processes such as fibrinolysis or inflammation, development, Alzheimer’s disease, and various types of cancer. We describe the synthesis and kinetic characterization of a focused library of 22 thiirane- and oxirane-based potential mechanism-based inhibitors, which led to discovery of an inhibitor for the human pro-carboxypeptidase A1. Our structural analyses show that the thiirane-based small-molecule inhibitor penetrates the barrier of the pro-domain to bind within the active site. This binding leads to a chemical reaction that covalently modifies the catalytic Glu270. These results highlight the importance of combined structural, biophysical, and biochemical evaluation of inhibitors in design strategies for the development of spectroscopically nonsilent probes as effective beacons for in vitro, in cellulo, and/or in vivo localization in clinical and industrial applications
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the crystal structure of thrombin activable fibrinolysis inhibitor tafi provides the structural basis for its intrinsic activity and the short half life of tafia
Journal of Biological Chemistry, 2008Co-Authors: Kanchan Anand, Josep Vendrell, Irantzu Pallares, Zuzana Valnickova, Trine Christensen, Ulrich K Wendt, Herman Schreuder, Jan J Enghild, Francesc X. AvilésAbstract:Thrombin-activable fibrinolysis inhibitor (TAFI)6 (1), also known as plasma carboxypeptidase B (CPB), carboxypeptidase B2, carboxypeptidase R, or procarboxypeptidase U (2-4), is a member of the M14A subfamily of Metallocarboxypeptidases (MEROPS data base; Ref. 5). It has been described as a fibrinolysis inhibitor that circulates in plasma as a plasminogen-binding protein. TAFI is involved in the fibrinolytic pathway and therefore seems to be highly regulated (6). It is a 60-kDa glyco-protein secreted by the liver with an isoelectric point of 5.0 (2). The glycans are mainly located on the pro-domain. They account for around 20% of the human TAFI mass and are assumed to play a role in stability and solubility (7). The pro-domain consists of a 92-residue N-terminal peptide, which can be removed by proteolysis, generating a 304-residue catalytic region called TAFIa (1-3). The removal of this peptide takes place by proteolytic cleavage of a specific Arg-Ala peptide bond at the loop between the pro-domain and the catalytic moiety. These two residues are Arg-92-Ala-93, numbered according to the N terminus of TAFI. The scission and dissociation of the two moieties causes the pI to shift from ∼5 to 8 for mature TAFIa, rendering the protein more basic and less soluble than TAFI (7). Several proteases, including trypsin, thrombin, and plasmin, are able to cleave the Arg-92-Ala-93 bond. In vivo, the thrombin/thrombomodulin complex is most likely responsible for generation of TAFIa, although plasmin may also be able to function in this manner (8). TAFIa down-regulates fibrinolysis by removing C-terminal lysine residues from fibrin that has already been partially degraded by plasmin. Those lysine residues act as ligands for the lysine-binding sites of plasminogen and tissue-type plasminogen activator (9). Removal of the lysine residues attenuates the fibrin cofactor function in the activation of plasminogen by tissue-type plasminogen activator, resulting in the prevention of accelerated plasmin formation and subsequent down-regulation of fibrinolysis (10). Consequently, inhibition of TAFIa may potentiate thrombolysis and thrombolytic therapies (11). TAFIa has also been implicated in the regulation of the inflammatory response by inactivating complement-derived inflammatory peptides (12). This enzyme is therefore considered an important biomedical target (13). TAFI shows around 40% sequence identity to several Metallocarboxypeptidases of the M14A subfamily (Fig. 1), sharing with them the most important residues implicated in catalysis, zinc binding, and substrate binding (2, 14). It displays carboxypeptidase B (CPB)-like specificity, although, as mentioned above, it is designed to trim a polymeric natural protein substrate (fibrin) and not peptides like most of the CPB-like enzymes. FIGURE 1. Sequence alignment of human and bovine TAFI with secondary structure elements corresponding to bovine TAFI indicated below. The blue arrow indicates the limit between the pro-domain and the catalytic domain. The sequences of bovine proCPA1 and human ... Following the removal of the pro-domain, the activity of TAFIa exhibits a short half-life, both in vivo and in vitro, with reported half-lives at 37 °C from 8 to 15 min (15). Due to its physiological role and the need for a very tight regulation of the blood coagulation cascade, it is likely that TAFIa has evolved to acquire its intrinsic instability, which ensures rapid inactivation at the site of action. The irreversible activity decay has been shown to be accompanied by structural changes of the enzyme (16-18). TAFIa can also be inactivated in vivo by further proteolytic degradation, indicating that other parts of the molecule become flexible and accessible. It was therefore suggested that the instability of the activity of TAFIa is due to intrinsic structural lability of the enzyme (19). This is quite unusual among Metallocarboxypeptidases, which are generally very stable enzymes in the mature form (14). The three-dimensional structure of TAFI described here is an advance in the long pursued structural knowledge. It will help to understand the instability of the mature enzyme and predict its ligands and ligand-binding sites. Significantly, these results reinforce a recent report of one of our groups (20) describing the significant intrinsic catalytic activity of TAFI and its potential capability to act in fibrinolysis, a function until recently exclusively assigned to its mature form, TAFIa.
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Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña.
International journal of molecular sciences, 2018Co-Authors: Juliana Cotabarren, Javier Garcia-pardo, Julia Lorenzo, Sebastian Tanco, Francesc X. Avilés, Mariana Edith Tellechea, Walter David ObregónAbstract:Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churquena. Our results demonstrated that chuPCI is a member of the A/B-type family of Metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.
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substrate specificity of human metallocarboxypeptidase d comparison of the two active carboxypeptidase domains
PLOS ONE, 2017Co-Authors: Javier Garciapardo, Julia Lorenzo, Sebastian Tanco, Lucia Diaz, Sayani Dasgupta, Juan Fernandezrecio, Francesc X. AvilésAbstract:Metallocarboxypeptidase D (CPD) is a membrane-bound component of the trans-Golgi network that cycles to the cell surface through exocytic and endocytic pathways. Unlike other members of the metallocarboxypeptidase family, CPD is a multicatalytic enzyme with three carboxypeptidase-like domains, although only the first two domains are predicted to be enzymatically active. To investigate the enzymatic properties of each domain in human CPD, a critical active site Glu in domain I and/or II was mutated to Gln and the protein expressed, purified, and assayed with a wide variety of peptide substrates. CPD with all three domains intact displays >50% activity from pH 5.0 to 7.5 with a maximum at pH 6.5, as does CPD with mutation of domain I. In contrast, the domain II mutant displayed >50% activity from pH 6.5–7.5. CPD with mutations in both domains I and II was completely inactive towards all substrates and at all pH values. A quantitative peptidomics approach was used to compare the activities of CPD domains I and II towards a large number of peptides. CPD cleaved C-terminal Lys or Arg from a subset of the peptides. Most of the identified substrates of domain I contained C-terminal Arg, whereas comparable numbers of Lys- and Arg-containing peptides were substrates of domain II. We also report that some peptides with C-terminal basic residues were not cleaved by either domain I or II, showing the importance of the P1 position for CPD activity. Finally, the preference of domain I for C-terminal Arg was validated through molecular docking experiments. Together with the differences in pH optima, the different substrate specificities of CPD domains I and II allow the enzyme to perform distinct functions in the various locations within the cell.
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C-terminomics Screen for Natural Substrates of Cytosolic Carboxypeptidase 1 Reveals Processing of Acidic Protein C termini
Molecular & cellular proteomics : MCP, 2014Co-Authors: Sebastian Tanco, Kris Gevaert, Petra Van Damme, Olivia Tort, Hans Demol, Francesc X. Avilés, Julia LorenzoAbstract:Cytosolic carboxypeptidases (CCPs) constitute a new subfamily of M14 Metallocarboxypeptidases associated to axonal regeneration and neuronal degeneration, among others. CCPs are deglutamylating enzymes, able to catalyze the shortening of polyglutamate side-chains and the gene-encoded C termini of tubulin, telokin, and myosin light chain kinase. The functions of these enzymes are not entirely understood, in part because of the lack of information about C-terminal protein processing in the cell and its functional implications. By means of C-terminal COFRADIC, a positional proteomics approach, we searched for cellular substrates targets of CCP1, the most relevant member of this family. We here identified seven new putative CCP1 protein substrates, including ribosomal proteins, translation factors, and high mobility group proteins. Furthermore, we showed for the first time that CCP1 processes both glutamates as well as C-terminal aspartates. The implication of these C termini in molecular interactions furthermore suggests that CCP1-mediated shortening of acidic protein tails might regulate protein-protein and protein-DNA interactions.
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characterization of the substrate specificity of human carboxypeptidase a4 and implications for a role in extracellular peptide processing
Journal of Biological Chemistry, 2010Co-Authors: Sebastian Tanco, Julia Lorenzo, Francesc X. Avilés, Xin Zhang, Cain Morano, Lloyd D FrickerAbstract:CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment.
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nna1 like proteins are active Metallocarboxypeptidases of a new and diverse m14 subfamily
The FASEB Journal, 2007Co-Authors: Monica Rodriguez De La Vega, Julia Lorenzo, Lloyd D Fricker, Sebastian Tanco, Rafael G Sevilla, Antoni Hermoso, Amalia Diez, Jose M Bautista, Francesc X. AvilésAbstract:Nna1 has some sequence similarity to Metallocarboxypeptidases, but the biochemical characterization of Nna1 has not previously been reported. In this work we performed a detailed genomic scan and found >100 Nna1 homologues in bacteria, Protista, and Animalia, including several paralogs in most eukaryotic species. Phylogenetic analysis of the Nna1-like sequences demonstrates a major divergence between Nna1-like peptidases and the previously known Metallocarboxypeptidases subfamilies: M14A, M14B, and M14C. Conformational modeling of representative Nna1-like proteins from a variety of species indicates an unusually open active site, a property that might facilitate its action on a wide variety of peptide and protein substrates. To test this, we expressed a recombinant form of one of the Nna1-like peptidases from Caenorhabditis elegans and demonstrated that this protein is a fully functional metallocarboxypeptidase that cleaves a range of C-terminal amino acids from synthetic peptides. The enzymatic activity is activated by ATP/ADP and salt-inactivated, and is preferentially inhibited by Z-Glu-Tyr dipeptide, which is without precedent in Metallocarboxypeptidases and resembles tubulin carboxypeptidase functioning; this hypothesis is strongly reinforced by the results depicted in Kalinina et al. published as accompanying paper in this journal. Our findings demonstrate that the M14 family of Metallocarboxypeptidases is more complex and diverse than expected, and that Nna1-like peptidases are functional variants of such enzymes, representing a novel subfamily (we propose the name M14D) that contributes substantially to such diversity.