Sebastian Tanco

Vanadium polypyridyl compounds as potential antiparasitic and antitumoral agents: New achievements

In the search for new therapeutic tools against diseases produced by kinetoplastid parasites five vanadyl complexes, [V(IV)O(L-2H)(phen)], including 1,10-phenanthroline (phen) and tridentate salicylaldehyde semicarbazone derivatives as ligands have been synthesized and characterized in the solid state and in solution by using different techniques. EPR suggested a distorted octahedral geometry with the tridentate semicarbazone occupying three equatorial positions and phen coordinated in an equatorial/axial mode. The compounds were evaluated in vitro on epimastigotes of Trypanosoma cruzi, causative agent of Chagas disease, Leishmania panamensis and Leishmania chagasi and on tumor cells. The complexes showed higher in vitro anti-trypanosomal activities than the reference drug Nifurtimox (IC(50) values in the range 1.6-3.8 μM) and increased activities in respect to the free semicarbazone ligands. In vitro activity on promastigote and amastigote forms of Leishmania showed interesting results. The compounds [VO(L1-2H)(phen)] and [VO(L3-2H)(phen)], where L1 = 2-hydroxybenzaldehyde semicarbazone and L3 = 2-hydroxy-3-methoxybenzaldehyde semicarbazone, resulted active (IC(50) 2.74 and 2.75 μM, respectively, on promastigotes of L. panamensis; IC(50) 19.52 and 20.75 μM, respectively, on intracellular amastigotes of L. panamensis) and showed low toxicity on THP-1 mammalian cells (IC(50) 188.55 and 88.13 μM, respectively). In addition, the complexes showed cytotoxicity on human promyelocytic leukemia HL-60 cells with IC(50) values of the same order of magnitude as cisplatin. The interaction of the complexes with DNA was demonstrated by different techniques, suggesting that this biomolecule could be a potential target either in the parasites or in tumor cells.

Nna1-like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily

Nnal has some sequence similarity to metallocarboxypeptidases, but the biochemical characterization of Nnal has not previously been reported. In this work we performed a detailed genomic scan and found >100 Nnal homologues in bacteria, Protista, and Anima‐lia, including several paralogs in most eukaryotic species. Phylogenetic analysis of the Nnal‐like sequences demonstrates a major divergence between Nnal‐like peptidases and the previously known metallocarboxypeptidases subfamilies: M14A, M14B, and M14C. Conformational mod‐eling of representative Nnal‐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 Nnal‐like peptidases from Caenσrhabditis elegans and demonstrated that this protein is a fully functional metaUocarboxypeptidase 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 (1). Our findings demonstrate that the M14 family of metallocarboxypeptidases is more complex and diverse than expected, and that Nnal‐like peptidases are functional variants of such enzymes, representing a novel subfamily (we propose the name M14D) that contributes substantially to such diversity.—Rodriguez de la Vega, M., Sevilla, R. G., Hermoso, A., Lorenzo, J., Tanco, S., Diez, A., Fricker, L. D., Bautista, J. M., Avilés, F. X. Nna1‐like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily. FASEB J. 20, 851–865 (2007)

Characterization of the Substrate Specificity of Human Carboxypeptidase A4 and Implications for a Role in Extracellular Peptide Processing

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.

Proteome-derived peptide libraries to study the substrate specificity profiles of carboxypeptidases

Through processing peptide and protein C termini, carboxypeptidases participate in the regulation of various biological processes. Few tools are however available to study the substrate specificity profiles of these enzymes. We developed a proteome-derived peptide library approach to study the substrate preferences of carboxypeptidases. Our COFRADIC-based approach takes advantage of the distinct chromatographic behavior of intact peptides and the proteolytic products generated by the action of carboxypeptidases, to enrich the latter and facilitate its MS-based identification. Two different peptide libraries, generated either by chymotrypsin or by metalloendopeptidase Lys-N, were used to determine the substrate preferences of human metallocarboxypeptidases A1 (hCPA1), A2 (hCPA2), and A4 (hCPA4). In addition, our approach allowed us to delineate the substrate specificity profile of mouse mast cell carboxypeptidase (MC-CPA or mCPA3), a carboxypeptidase suggested to function in innate immune responses regulation and mast cell granule homeostasis, but which thus far lacked a detailed analysis of its substrate preferences. mCPA3 was here shown to preferentially remove bulky aromatic amino acids, similar to hCPA2. This was also shown by a hierarchical cluster analysis, grouping hCPA1 close to hCPA4 in terms of its P1 primed substrate specificity, whereas hCPA2 and mCPA3 cluster separately. The specificity profile of mCPA3 may further aid to elucidate the function of this mast cell carboxypeptidase and its biological substrate repertoire. Finally, we used this approach to evaluate the substrate preferences of prolylcarboxypeptidase, a serine carboxypeptidase shown to cleave C-terminal amino acids linked to proline and alanine.

C-terminomics: Targeted analysis of natural and posttranslationally modified protein and peptide C-termini

The C‐terminus (where C is carboxyl) of a protein can serve as a recognition signature for a variety of biological processes, including protein trafficking and protein complex formation. Hence, the identity of the in vivo protein C‐termini provides valuable information about biological processes. Analysis of protein C‐termini is also crucial for the study of C‐terminal PTMs, particularly for monitoring proteolytic processing by endopeptidases and carboxypeptidases. Although technical difficulties have limited the study of C‐termini, a range of technologies have been proposed in the last couple of years. Here, we review the current proteomics technologies for C‐terminal analysis, with a focus on the biological information that can be derived from C‐terminomics studies.

C-terminomics screen for natural substrates of cytosolic carboxypeptidase 1 reveals processing of acidic protein C-termini

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.

The Novel Structure of a Cytosolic M14 Metallocarboxypeptidase (Ccp) from Pseudomonas Aeruginosa: A Model for Mammalian Ccps.

PaCCP is a metallocarboxypeptidase (MCP) of the M14 family from Pseudomonas aeruginosa, which belongs to a bacterial clade of carboxypeptidases that are homologous to the recently discovered M14D subfamily of human nonsecretory cytosolic carboxypeptidases (CCPs). CCPs are intracellular peptidases involved, among other roles, in the post‐translational modifications of tubulin. Here we report the crystal structure of PaCCP at high resolution (1.6 Å). Its 375 residues are folded in a novel β‐sandwich N‐terminal domain followed by the classical carboxypeptidase α/β‐hydrolase domain, this one in a shorter and more compact form. The former is unique in the whole family and does not have sequential or structural homology with other domains that are usually flanking the latter, like the prodomain of the M14A subfamily or the C‐terminal transthyretin/prealbumin‐like domains of the M14B subfamily. PaCCP does not display activity against small carboxypeptidase substrates, so in this form it might constitute an inactive precursor of the protease. Structural results derived from cocrystallization with well‐known inhibitors of MCPs indicate that the enzyme might only possess C‐terminal hydrolase activity against cellular substrates of particular specificity and/or when undergoes structural rearrangements. The derived PaCCP structure allows a first structural insight into the more complex and largely unknown mammalian CCP subfamily.—Otero, A., Rodríguez de la Vega, M., Tanco, S., Lorenzo, J., Avilés, F. X., Reverter, D. The novel structure of a cytosolic M14 metallocarboxypeptidase (CCP) from Pseudomonas aeruginosa: a model for mammalian CCPs. FASEB J. 26, 3754–3764 (2012). www.fasebj.org

Structure-function analysis of the short splicing variant carboxypeptidase encoded by Drosophila melanogaster silver.

Abstract Drosophila melanogaster silver gene is the ortholog of the coding gene of mammalian carboxypeptidase D (CPD). The silver gene gives rise to eight different splicing variants of differing length that can contain up to three homologous repeats. Among the protein variants encoded, the short form 1B alias DmCPD1Bs (D . melanogaster CPD variant 1B short) is necessary and sufficient for viability of the fruit fly. It has one single repeat, it is active against standard peptide substrates, and it is localized to the secretory pathway. In this work, the enzyme was found as a monomer in solution and as a homodimer in the crystal structure, which features a protomer with an N-terminal 311-residue catalytic domain of α/β-hydrolase fold and a C-terminal 84-residue all-β transthyretin-like domain. Overall, DmCPD1Bs conforms to the structure of N/E-type funnelins/M14B metallopeptidases, but it has two unique structural elements potentially involved in regulation of its activity: (i) two contiguous surface cysteines that may become palmitoylated and target the enzyme to membranes, thus providing control through localization, and (ii) a surface hot spot targetable by peptidases that would provide a regulatory mechanism through proteolytic inactivation. Given that the fruit fly possesses orthologs of only two out of the five proteolytically competent N/E-type funnelins found in higher vertebrates, DmCPD1Bs may represent a functional analog of at least one of the missing mammalian CPs.

Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes

Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2-4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B was demonstrated.

Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis

Summary Interneurons navigate along multiple tangential paths to settle into appropriate cortical layers. They undergo a saltatory migration paced by intermittent nuclear jumps whose regulation relies on interplay between extracellular cues and genetic-encoded information. It remains unclear how cycles of pause and movement are coordinated at the molecular level. Post-translational modification of proteins contributes to cell migration regulation. The present study uncovers that carboxypeptidase 1, which promotes post-translational protein deglutamylation, controls the pausing of migrating cortical interneurons. Moreover, we demonstrate that pausing during migration attenuates movement simultaneity at the population level, thereby controlling the flow of interneurons invading the cortex. Interfering with the regulation of pausing not only affects the size of the cortical interneuron cohort but also impairs the generation of age-matched projection neurons of the upper layers.