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1.
Mar Drugs ; 21(5)2023 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-37233473

RESUMO

Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Peptidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many biochemical and physiological processes. They are also involved in several pathophysiological processes. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids of proteins or peptide substrates. They are distributed in many phyla and play critical roles in physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development of therapeutic agents for human diseases, including cancer, hypertension, central nervous system disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as malaria. The relevance of aminopeptidases has driven the search and identification of potent and selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an important and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with foreseen biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further studies with inhibitors isolated from marine invertebrates in different biomedical models associated with the activity of these families of exopeptidases.


Assuntos
Aminopeptidases , Leucil Aminopeptidase , Humanos , Aminopeptidases/química , Aminopeptidases/metabolismo , Leucil Aminopeptidase/química , Peptídeos/química , Antígenos CD13
2.
Mar Drugs ; 21(2)2023 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-36827135

RESUMO

Metallocarboxypeptidases are zinc-dependent peptide-hydrolysing enzymes involved in several important physiological and pathological processes. They have been a target of growing interest in the search for natural or synthetic compound binders with biomedical and drug discovery purposes, i.e., with potential as antimicrobials or antiparasitics. Given that marine resources are an extraordinary source of bioactive molecules, we screened marine invertebrates for new inhibitory compounds with such capabilities. In this work, we report the isolation and molecular and functional characterization of NpCI, a novel strong metallocarboxypeptidase inhibitor from the marine snail Nerita peloronta. NpCI was purified until homogeneity using a combination of affinity chromatography and RP-HPLC. It appeared as a 5921.557 Da protein with 53 residues and six disulphide-linked cysteines, displaying a high sequence similarity with NvCI, a carboxypeptidase inhibitor isolated from Nerita versicolor, a mollusc of the same genus. The purified inhibitor was determined to be a slow- and tight-binding inhibitor of bovine CPA (Ki = 1.1·× 10-8 mol/L) and porcine CPB (Ki = 8.15·× 10-8 mol/L) and was not able to inhibit proteases from other mechanistic classes. Importantly, this inhibitor showed antiplasmodial activity against Plasmodium falciparum in an in vitro culture (IC50 = 5.5 µmol/L), reducing parasitaemia mainly by inhibiting the later stages of the parasite's intraerythrocytic cycle whilst having no cytotoxic effects on human fibroblasts. Interestingly, initial attempts with other related proteinaceous carboxypeptidase inhibitors also displayed similar antiplasmodial effects. Coincidentally, in recent years, a metallocarboxypeptidase named PfNna1, which is expressed in the schizont phase during the late intraerythrocytic stage of the parasite's life cycle, has been described. Given that NpCI showed a specific parasiticidal effect on P. falciparum, eliciting pyknotic/dead parasites, our results suggest that this and related inhibitors could be promising starting agents or lead compounds for antimalarial drug discovery strategies.


Assuntos
Antimaláricos , Carboxipeptidases , Plasmodium falciparum , Animais , Bovinos , Humanos , Antimaláricos/farmacologia , Carboxipeptidases/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Proteínas/farmacologia , Caramujos/química , Suínos
3.
Int J Mol Sci ; 24(2)2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36674791

RESUMO

The cytosolic carboxypeptidase 6 (CCP6) catalyzes the deglutamylation of polyglutamate side chains, a post-translational modification that affects proteins such as tubulins or nucleosome assembly proteins. CCP6 is involved in several cell processes, such as spermatogenesis, antiviral activity, embryonic development, and pathologies like renal adenocarcinoma. In the present work, the cellular role of CCP6 has been assessed by BioID, a proximity labeling approach for mapping physiologically relevant protein-protein interactions (PPIs) and bait proximal proteins by mass spectrometry. We used HEK 293 cells stably expressing CCP6-BirA* to identify 37 putative interactors of this enzyme. This list of CCP6 proximal proteins displayed enrichment of proteins associated with the centrosome and centriolar satellites, indicating that CCP6 could be present in the pericentriolar material. In addition, we identified cilium assembly-related proteins as putative interactors of CCP6. In addition, the CCP6 proximal partner list included five proteins associated with the Joubert syndrome, a ciliopathy linked to defects in polyglutamylation. Using the proximity ligation assay (PLA), we show that PCM1, PIBF1, and NudC are true CCP6 physical interactors. Therefore, the BioID methodology confirms the location and possible functional role of CCP6 in centrosomes and centrioles, as well as in the formation and maintenance of primary cilia.


Assuntos
Centríolos , Cílios , Masculino , Humanos , Cílios/metabolismo , Células HEK293 , Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas/metabolismo
4.
Dev Comp Immunol ; 127: 104273, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34619175

RESUMO

Metallo-carboxypeptidases are exopeptidases with diverse expression and function, found in all kingdoms of life from bacteria to mammals. One of them, the carboxypeptidase A3 (CPA3), has become an important component of the mammalian immune system by its expression in mast cells. Mast cells (MCs) are highly specialized sentinel cells, which store large amounts of bioactive mediators, including CPA3, in very abundant cytoplasmic granules. Clinical studies have found an increased CPA3 expression in asthma but the physiological role as well as the evolutionary origin of CPA3 remains largely unexplored. CPA3 belongs to the M14A subfamily of metallo-carboxypeptidases, which among others also includes the digestive enzymes CPA1, CPA2, CPB1 and CPO. To study the appearance of CPA3 during vertebrate evolution, we here performed bioinformatic analyses of homologous genes and gene loci from a broad panel of metazoan animals from invertebrates to mammals. The phylogenetic analysis indicated that CPA3 appeared at the base of tetrapod evolution in a branch closer to CPB1 than to other CPAs. Indeed, CPA3 and CPB1 are also located in the same locus, on chromosome 3 in humans. The presence of CPA3 only in tetrapods and not in fishes, suggested that CPA3 could have appeared by a gene duplication from CPB1 during early tetrapod evolution. However, the apparent loss of CPA3 in several tetrapod lineages, e.g. in birds and monotremes, indicates a complex evolution of the CPA3 gene. Interestingly, in the lack of CPA3 in fishes, zebrafish MCs express instead CPA5 for which the most closely related human carboxypeptidase is CPA1, which has a similar cleavage specificity as CPA3. Collectively, these findings clarify and add to our understanding of the evolution of hematopoietic proteases expressed by mast cells.


Assuntos
Mastócitos , Animais , Carboxipeptidases A/genética , Carboxipeptidases A/metabolismo , Mamíferos , Filogenia , Peixe-Zebra
5.
Int J Mol Sci ; 21(22)2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33217972

RESUMO

Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1' position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions.


Assuntos
Carboxipeptidases/química , Humanos , Domínios Proteicos , Especificidade por Substrato
6.
Chem Biol Interact ; 306: 123-130, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30958995

RESUMO

Aldehyde dehydrogenases (ALDHs) are enzymes catalyzing the NAD(P)+-dependent oxidation of aldehydes to their corresponding carboxylic acids. High ALDH activity has been related to some important features of cancer stem cells. ALDH1A enzymes, involved in the retinoic acid signaling pathway, are promising drug targets for cancer therapy, and the design of selective ALDH1A inhibitors has a growing pharmacological interest. In the present work, two already known compounds (DEAB and WIN 18,446) and novel thiazolidinedione and pyrimido quinoline acetic acid derivatives (compounds 5a and 64, formerly described as aldo-keto reductase inhibitors) were tested as inhibitors of the ALDH1A enzymes (namely, ALDH1A1, ALDH1A2 and ALDH1A3) as a first step to develop some potential drugs for cancer therapy. The inhibitory capacity of these compounds against the ALDH1A activity was characterized in vitro by using purified recombinant proteins. The IC50 values of each compound were determined indicating that the most potent inhibitors against ALDH1A1, ALDH1A2 and ALDH1A3 were DEAB, WIN 18,446 and compound 64, respectively. Type of inhibition and Ki values were determined for DEAB against ALDH1A1 (competitive, Ki = 0.13 µM) and compound 64 against ALDH1A3 (non-competitive, Ki = 1.77 µM). The effect of these inhibitors on A549 human lung cancer cell viability was assessed, being compound 64 the only inhibitor showing an important reduction of cell survival. We also tested the effect of the ALDH substrate, retinaldehyde, which was cytotoxic above 10 µM. This toxicity was enhanced in the presence of DEAB. Both DEAB and compound 64 were able to inhibit the ALDH1A activity in A549 cells. The current work suggests that, by blocking ALDH activity, drug inactivation may be avoided. Thus these results may be relevant to design novel combination therapies to fight cancer cell chemoresistance, using both enzyme inhibitors and chemotherapeutic agents.


Assuntos
Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Oxirredutases/antagonistas & inibidores , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Retinal Desidrogenase/antagonistas & inibidores , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Aldeído Oxirredutases/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Cinética , Estrutura Molecular , Retinal Desidrogenase/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
7.
J Med Chem ; 62(4): 1917-1931, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30688452

RESUMO

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.


Assuntos
Carboxipeptidases A/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Oligopeptídeos/farmacologia , Ácidos Fosfínicos/farmacologia , Carboxipeptidases A/química , Carboxipeptidases A/metabolismo , Domínio Catalítico , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Células HEK293 , Células HeLa , Humanos , Indóis/síntese química , Indóis/farmacologia , Cinética , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Ácidos Fosfínicos/síntese química , Ácidos Fosfínicos/metabolismo , Ligação Proteica
8.
Int J Mol Sci ; 19(3)2018 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-29495576

RESUMO

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. Churqueña. 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.


Assuntos
Miniproteínas Nó de Cistina/química , Proteínas de Plantas/química , Proteômica , Proteínas Recombinantes , Solanum tuberosum/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Animais , Carboxipeptidases/antagonistas & inibidores , Bovinos , Clonagem Molecular , Miniproteínas Nó de Cistina/análise , Miniproteínas Nó de Cistina/genética , Miniproteínas Nó de Cistina/isolamento & purificação , Ativação Enzimática/efeitos dos fármacos , Cinética , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Inibidores de Proteases/análise , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Proteômica/métodos , Análise de Sequência de DNA , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Suínos
9.
Protein Expr Purif ; 144: 55-61, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29223927

RESUMO

The cystine-knot metallocarboxypeptidase inhibitors (MCPIs) are peptides that contribute to control proteolytic activity, involved in storage, growth and maintenance of plants. Lately studies reported several MCPIs with potential use in biomedical applications; as anti-cancer, anti-thrombotic, anti-malaric and anti-angiogenic agents. We report the isolation, purification, chemical stability and biochemical characterization of a novel carboxypeptidase A inhibitor (YBPCI) isolated from Capsicum annuum L. var. Yellow Bell Pepper, the first cystine-knot miniprotein (CKM) of the species. We demonstrate the stability of YBPCI (IC50 = 0.90 µg/ml) to high temperatures, high salt concentration and extreme pH values. MALDI-TOF/MS analysis detected a molecular weight of 4057 Da, and peptide mass fingerprint resulted in no matches with other protease inhibitors. In vitro gastrointestinal digestion subjecting YBPCI to pH 2 incubation and proteolytic attack resulted in complete inhibitory activity. To summarize, there are no reports to date of carboxypeptidase inhibitors in C. annuum species, giving our report much more relevance.


Assuntos
Carboxipeptidases/antagonistas & inibidores , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Inibidores de Proteases/farmacologia , Animais , Capsicum/química , Bovinos , Extratos Vegetais/química , Proteínas de Plantas/análise , Inibidores de Proteases/análise , Inibidores de Proteases/isolamento & purificação
10.
Mar Drugs ; 15(4)2017 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-28430158

RESUMO

Natural products from marine origin constitute a very promising and underexplored source of interesting compounds for modern biotechnological and pharmaceutical industries. However, their evaluation is quite challenging and requires specifically designed assays to reliably identify the compounds of interest in a highly heterogeneous and interfering context. In the present study, we describe a general strategy for the confident identification of tight-binding protease inhibitors in the aqueous extracts of 62 Cuban marine invertebrates, using Plasmodium falciparum hemoglobinases Plasmepsin II and Falcipain 2 as model enzymes. To this end, we first developed a screening strategy that combined enzymatic with interaction-based assays and then validated screening conditions using five reference extracts. Interferences were evaluated and minimized. The results from the massive screening of such extracts, the validation of several hits by a variety of interaction-based assays and the purification and functional characterization of PhPI, a multifunctional and reversible tight-binding inhibitor for Plasmepsin II and Falcipain 2 from the gorgonian Plexaura homomalla, are presented.


Assuntos
Organismos Aquáticos/química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Cisteína Endopeptidases/metabolismo , Invertebrados/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Cisteína Endopeptidases/química , Plasmodium falciparum/metabolismo , Ligação Proteica
11.
Methods Mol Biol ; 1574: 115-133, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28315247

RESUMO

We here present a detailed procedure for studying protein C-termini and their posttranslational modifications by C-terminal COFRADIC. In fact, this procedure can enrich for both C-terminal and N-terminal peptides through a combination of a strong cation exchange fractionation step at low pH, which removes the majority of nonterminal peptides in whole-proteome digests, while the actual COFRADIC step segregates C-terminal peptides from N-terminal peptides. When used in a differential mode, C-terminal COFRADIC allows for the identification of neo-C-termini generated by the action of proteases, which in turn leads to the identification of protease substrates. More specifically, this technology can be applied to determine the natural substrate repertoire of carboxypeptidases on a proteome-wide scale.


Assuntos
Carboxipeptidases/metabolismo , Peptídeos , Proteoma , Proteômica/métodos , Carboxipeptidases/química , Linhagem Celular , Cromatografia/métodos , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Humanos , Peptídeos/química , Peptídeos/isolamento & purificação , Espectrometria de Massas em Tandem
12.
Phytochemistry ; 120: 36-45, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26521146

RESUMO

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.


Assuntos
Carboxipeptidases/antagonistas & inibidores , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Solanum tuberosum/química , Sequência de Aminoácidos , Animais , Argentina , Sequência de Bases , Sítios de Ligação , Bovinos , Humanos , Dados de Sequência Molecular , Peso Molecular , Pâncreas/enzimologia , Proteínas de Plantas/química , Inibidores de Proteases/farmacologia , Solanum tuberosum/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade
13.
Eur J Med Chem ; 83: 374-88, 2014 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-24980119

RESUMO

The present study describes the synthesis, anticancer activity and SAR studies of novel platinum(IV) complexes having 1,2-bis(aminomethyl)carbobicyclic or oxabicyclic carrier ligands, bearing chlorido and/or hydroxido ligands in axial position and chlorido or malonato ligands in equatorial position (labile ligands). These complexes were synthetized with the aim of obtaining new anticancer principles more soluble in water and therefore more bioavailable. Several substitution patterns on the platinum atom have been designed in order to evaluate their antiproliferative activity and to establish structure-activity relationship rules. The synthesis of platinum(IV) complexes with axial hydroxyl ligands on the platinum(IV) were carried out by reaction of K2Pt(OH)2Cl4 with the corresponding diamines. The complexes with axial chlorido ligands on the platinum(IV) atom were synthesized by direct reaction of diamines with K2PtCl6. Carboxylated complexes were synthesized by the substitution reaction of equatorial chlorido ligands by silver dicarboxylates. The most actives complexes were those having malonate as a labile ligand, no matter of the structure of the carrier ligand. Regarding the influence of the structure of the non-labile 1,4-diamine carrier ligand on the cytotoxicity, it was found that the complexes having the more lipophilic and symmetrical bicyclo[2.2.2]octane framework were much more active than those having an oxygen or methylene bridge.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos Organoplatínicos/química , Compostos Organoplatínicos/farmacologia , Aminas/química , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Proliferação de Células/efeitos dos fármacos , Técnicas de Química Sintética , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Humanos , Ligantes , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/farmacocinética , Relação Estrutura-Atividade
14.
Planta ; 236(5): 1471-84, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22790602

RESUMO

Vasconcellea quercifolia (Caricaceae) latex contains several cysteine endopeptidases with high proteolytic activity. Cysteine endopeptidases are the main active compounds used by the plant as a defense mechanism. A proteolytic preparation from V. quercifolia ("oak leaved papaya") latex was purified by cation exchange chromatography. From SDS-PAGE and blotting of the selected fractions, the N-terminal amino acid sequences of polypeptides were determined by Edman's degradation. The analysis by peptide mass fingerprinting (PMF) of the enzymes allowed their characterization and confirmed the presence of seven different cysteine proteinases in the latex of V. quercifolia. Moreover, the comparison between the tryptic maps with those deposited in databases using the MASCOT tool showed that none of the isolated proteases matched with another plant protease. Notably, a propeptidase was detected in the plant latex, which is being the first report in this sense. Furthermore, the cDNA of one of the cysteine proteases that is expressed in the latex of V. quercifolia was cloned and sequenced. The consensus sequence was aligned using the ClustalX web server, which allowed detecting a high degree of identity with cysteine proteases of the Caricaceae family and establishing the evolutionary relationship between them. We also observed a high conservation degree for those amino acid residues which are essential for the catalytic activity and tridimensional structure of the plant proteases belonging to the subfamily C1A. The PMF analysis strongly suggests that the sequence obtained corresponds to the VQ-III peptidase.


Assuntos
Caricaceae/química , Látex/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Caricaceae/metabolismo , Clonagem Molecular , Sequência Conservada , Cisteína Proteases/química , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Dados de Sequência Molecular , Peptídeo Hidrolases/isolamento & purificação , Mapeamento de Peptídeos , Filogenia , Proteínas de Plantas/análise , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estrutura Terciária de Proteína , Proteólise
15.
Appl Biochem Biotechnol ; 165(2): 583-93, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21584778

RESUMO

Fruits of Bromelia hieronymi, a tropical South American plant, possess a high content of peptidases with potential biotechnological uses. Total RNA was extracted from unripe fruits and peptidase cDNA was obtained by 3'RACE-PCR. The consensus sequence of the cysteine peptidase cDNA contained 875 bp, the 690 first ones codifying for a hypothetical polypeptide chain of the mature peptidase, named Bh-CP1 (molecular mass 24.773 kDa, pI 8.6, extinction molar coefficient 58,705 M(-1) cm(-1)). Bh-CP1 sequence shows a high percentage of identity with those of other cysteine plant proteases. The presence of highly preserved residues is observed, like those forming the catalytic site (Gln19, Cys25, His159, and Asn175, papain numbering), as well as other six Cys residues, involved in the formation of disulfide bounds. Molecular modeling results suggest the enzyme belongs to the α + ß class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the α domain, while the ß domain is stabilized by another disulfide bridge (Cys153-Cys203). Additionally, peptide mass fingerprints (PMFs) of the three peptidases previously isolated from B. hieronymi fruits (namely hieronymain I, II, and III) were performed and compared with the theoretical fingerprint of PMF of Bh-CP1, showing a partial matching between the in silico-translated protein and hieronymain II.


Assuntos
Bromelia/enzimologia , Sequência Conservada/genética , Cisteína Endopeptidases/química , Proteínas de Plantas/química , Proteômica/métodos , Proteínas Recombinantes/química , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , Clonagem Molecular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Escherichia coli , Frutas/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Plasmídeos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transformação Bacteriana
16.
J Biol Chem ; 285(24): 18385-96, 2010 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-20385563

RESUMO

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.


Assuntos
Carboxipeptidases A/química , Animais , Encéfalo/metabolismo , Carboxipeptidases/química , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Células HeLa , Humanos , Cinética , Camundongos , Peptídeos/química , Pichia/metabolismo , Estrutura Terciária de Proteína , Proteômica/métodos , Especificidade por Substrato
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