RESUMO
The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma/tratamento farmacológico , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Sequência de Aminoácidos , Anexina A5/análise , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/uso terapêutico , Receptores ErbB/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma/tratamento farmacológico , Dados de Sequência Molecular , Regulação para Cima/efeitos dos fármacosRESUMO
Human kallikrein 8 (hK8), whose gene was originally cloned as the human ortholog of a mouse brain protease, is known to be associated with diseases such as ovarian cancer and Alzheimer's disease. Recombinant human pro-kallikrein 8 was activated with lysyl endopeptidase-conjugated beads. Amino-terminal sequencing of the activated enzyme demonstrated the cleavage of a 9-aa propeptide from the pro-enzyme. The substrate specificity of activated hK8 was characterized using synthetic fluorescent substrates. hK8 showed trypsin-like specificity, as predicted from sequence analysis and enzymatic characterization of the mouse ortholog. All synthetic substrates tested containing either arginine or lysine at P1 position were cleaved by hK8. The highest kcat/Km value of 20x10(3)M-1 s-1 was observed with Boc-Val-Pro-Arg-7-amido-4-methylcoumarin. The activity of hK8 was inhibited by antipain, chymostatin, and leupeptin. The concentration for 50% inhibition by the best inhibitor, antipain, was 0.46 microM. The effect of different metal ions on the enzyme activity was analyzed. Whereas Na+ had no effect on hK8 activity, Ni2+ and Zn2+ decreased the activity and Ca2+, Mg2+, and K+ had a stimulatory effect. Ca2+ was the best activator, with an optimal concentration of approximately 10 microM.
Assuntos
Calicreínas/efeitos dos fármacos , Sequência de Aminoácidos , Cálcio/farmacologia , Cumarínicos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Metais Pesados/farmacologia , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Proteínas Recombinantes , Especificidade por SubstratoRESUMO
The reactive center loop (RCL) of serpins plays an essential role in the inhibition mechanism acting as a substrate for their target proteases. Changes within the RCL sequence modulate the specificity and reactivity of the serpin molecule. Recently, we reported the construction of alpha1-antichymotrypsin (ACT) variants with high specificity towards human kallikrein 2 (hK2) [Cloutier SM, Kündig C, Felber LM, Fattah OM, Chagas JR, Gygi CM, Jichlinski P, Leisinger HJ & Deperthes D (2004) Eur J Biochem271, 607-613] by changing amino acids surrounding the scissile bond of the RCL and obtained specific inhibitors towards hK2. Based on this approach, we developed highly specific recombinant inhibitors of human kallikrein 14 (hK14), a protease correlated with increased aggressiveness of prostate and breast cancers. In addition to the RCL permutation with hK14 phage display-selected substrates E8 (LQRAI) and G9 (TVDYA) [Felber LM, Borgoño CA, Cloutier SM, Kündig C, Kishi T, Chagas JR, Jichlinski P, Gygi CM, Leisinger HJ, Diamandis EP & Deperthes D (2005) Biol Chem386, 291-298], we studied the importance of the scaffold, serpins alpha1-antitrypsin (AAT) or ACT, to confer inhibitory specificity. All four resulting serpin variants ACT(E8), ACT(G9), AAT(E8) and AAT(G9) showed hK14 inhibitory activity and were able to form covalent complex with hK14. ACT inhibitors formed more stable complexes with hK14 than AAT variants. Whereas E8-based inhibitors demonstrated a rather relaxed specificity reacting with various proteases with trypsin-like activity including several human kallikreins, the two serpins variants containing the G9 sequence showed a very high selectivity for hK14. Such specific inhibitors might prove useful to elucidate the biological role of hK14 and/or its implication in cancer.
Assuntos
Calicreínas/antagonistas & inibidores , Serpinas/farmacologia , Sequência de Bases , Primers do DNA , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
The human KLK14 gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members. KLK14 , like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated that KLK14 gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin alpha-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.
Assuntos
Calicreínas/metabolismo , Bacteriófagos/genética , Clonagem Molecular , Colágeno Tipo IV/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Hidrólise , Calicreínas/genética , Cinética , Laminina/metabolismo , Especificidade por SubstratoRESUMO
Although the cellular steps required for metastasis are similar for all cancer cells, proteases involved in this process and their expression levels vary greatly between different cancer types. Thus, the identification of these proteolytic activities represents a crucial issue in the understanding of cancer development. Until now, phage display substrate technology has been successfully employed for the characterization of purified proteases but was never used with a mix of proteases. In the present work, we report an easy protocol to identify multiple proteolytic activities secreted by cancer cells. We selected substrates from a phage display library of high diversity using secreted media of three established prostate cancer cell lines (DU-145, LNCaP and PC-3) with variable degrees of invasive capability. Some of these selected peptide substrates were hydrolyzed by the secreted proteins of all three prostatic cancer cell lines, demonstrating similarities in their proteolytic activities. On the other hand, a few substrates were cancer cell specific, indicating differences in the phenotypes of protease expression in prostate cancer. This work reports for the first time the selection of substrates from a mix of proteases using phage display technology and opens a new avenue for the direct identification of proteolytic activities for tumor extracts.
Assuntos
Endopeptidases/farmacologia , Metástase Neoplásica , Neoplasias/fisiopatologia , Peptídeo Hidrolases/farmacologia , Biblioteca de Peptídeos , Neoplasias da Próstata/enzimologia , Proteínas/metabolismo , Endopeptidases/isolamento & purificação , Humanos , Hidrólise , Masculino , Peptídeo Hidrolases/isolamento & purificação , Células Tumorais CultivadasRESUMO
A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters. Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization. Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate. Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized. FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C. For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids. The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis. As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Ativação Enzimática , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Peptídeo Hidrolases/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/químicaRESUMO
The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The BT1/PTR1 null mutant is hypersensitive to MTX, but in a step-by-step fashion, we could induce resistance to MTX in these cells. Several resistance mechanisms were found to co-exist including a reduced folate and MTX accumulation, demonstrating that cells with no measurable biopterin uptake but also greatly reduced folate uptake are viable, despite their auxotrophy for each of these substrates. The resistant cells have also amplified the gene coding for the MTX target dihydrofolate reductase. Finally, we found a marked reduction in MTX polyglutamylation in resistant cells. These studies further highlight the formidable ability of Leishmania cells to bypass the blockage of key metabolic pathways.
Assuntos
Proteínas de Transporte/genética , Antagonistas do Ácido Fólico/farmacologia , Ácido Fólico/metabolismo , Leishmania/metabolismo , Proteínas de Membrana Transportadoras , Metotrexato/farmacologia , Oxirredutases/genética , Proteínas de Protozoários/genética , Animais , Biopterinas/metabolismo , Sobrevivência Celular , Resistência a Medicamentos , Inativação Gênica , Leishmania/citologia , Leishmania/genética , Peptídeo Sintases/metabolismoRESUMO
The reactive site loop of serpins undoubtedly defines in part their ability to inhibit a particular enzyme. Exchanges in the reactive loop of serpins might reassign the targets and modify the serpin-protease interaction kinetics. Based on this concept, we have developed a procedure to change the specificity of known serpins. First, reactive loops are very good substrates for the target enzymes. Therefore, we have used the phage-display technology to select from a pentapeptide phage library the best substrates for the human prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J. & Deperthes, D. (2002) Eur. J. Biochem. 269, 2747-2754]. Selected substrates were then transplanted into the reactive site loop of alpha1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease. Thus, we have developed some highly specific alpha1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity. These inhibitors might be useful to help elucidate the importance of hK2 in prostate cancer progression.
Assuntos
Bacteriófagos/genética , Inibidores de Serina Proteinase/farmacologia , Calicreínas Teciduais/antagonistas & inibidores , Sequência de Bases , Western Blotting , Primers do DNA , Humanos , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/farmacologiaRESUMO
Folates are polyglutamylated in most organisms, and in cancer cells the polyglutamylation of folates and of the antifolate methotrexate (MTX) is an important determinant of MTX susceptibility. The folylpolyglutamate synthetase (FPGS) responsible for polyglutamylation of folates was recently characterized in the parasite Leishmania. We show here that MTX is polyglutamylated in Leishmania tarentolae and that triglutamates are the predominant form. The glutamate chain length of MTX increases significantly in Leishmania cells transfected with the FPGS gene and decreases in cells with one FPGS allele disrupted. Modulation in the expression of the FPGS gene also has a profound effect on MTX susceptibility and this effect was found to be dependent on the folate concentration of the medium. In the folate-rich medium SDM-79, overexpression of FPGS will confer MTX resistance while in M-199 medium, which has much less folates, FPGS transfectants are more sensitive to MTX. Cells with one allele of FPGS disrupted are more resistant to MTX in low folate medium. The modulation of FPGS expression affects both the short-term and long-term accumulation of folate and MTX, showing a marked decrease in accumulation in the FPGS haploid mutant. This differential accumulation was mediated by decreased influx of the drug into the cell. Finally, the analysis of MTX-resistant Leishmania mutants indicated that the presence of shorter glutamate chains on MTX is correlated with MTX resistance.
Assuntos
Resistência a Medicamentos/fisiologia , Antagonistas do Ácido Fólico/metabolismo , Leishmania/metabolismo , Metotrexato/metabolismo , Peptídeo Sintases/metabolismo , Animais , Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Leishmania/efeitos dos fármacos , Metotrexato/farmacologia , Ácido Poliglutâmico/metabolismoRESUMO
Folates are polyglutamylated in most organisms by the enzyme folylpolyglutamate synthetase (FPGS). The Leishmania tarentolae FPGS gene was isolated. Its predicted product contains 538 amino acids and shows 33 and 30% identity with the human and yeast FPGS proteins, respectively. The level of folate polygtutamylation was studied in L. tarentolae promastigotes and in Leishmania infantum promastigotes and axenic amastigotes. In all species examined, folates were found predominantly as pentaglutamates, although monoglutamates were found in higher proportion in L. infantum axenic amastigote cells. Leishmania cells transfected with a FPGS containing plasmid (FPGS transfectant) exhibited a 6-fold increase in FPGS activity (32.7 pmol mg(-1) h(-1)) compared with wild-type cells (4.7 pmol mg(-1) h(-1)). HPLC analysis of the polyglutamylated forms of folates indicated a 2-fold increase of hexaglutamates in the FPGS transfectant compared with wild-type cells, while cells with one FPGS allele interrupted showed a higher proportion of short chain glutamates. The long-term accumulation of folates was greatly increased in the FPGS transfectant. Overall, this work indicates that FPGS activity is expressed in all forms of the parasite, and modulates the retention of folate, thereby possibly playing an important role in physiology.
Assuntos
Ácido Fólico/metabolismo , Leishmania/enzimologia , Peptídeo Sintases/genética , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Genes de Protozoários , Humanos , Leishmania/genética , Leishmania/crescimento & desenvolvimento , Leishmania infantum/enzimologia , Leishmania infantum/genética , Leishmania infantum/crescimento & desenvolvimento , Dados de Sequência Molecular , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , TransfecçãoRESUMO
The protozoan parasite Leishmania is a folate auxotroph and thus depends on the uptake of folate from the environment to meet its folate requirement. We show here that Leishmania contains several putative pteridine transporter genes. Some of these genes are deleted in methotrexate-resistant Leishmania cells where there is no measurable uptake of methotrexate. Transport studies suggest that Leishmania has more than one active folate transporter, and one of these, named FT5, corresponds to a very high affinity folate transporter (K(m) 84 nm). The uptake of both folate and methotrexate was impaired in an FT5 null mutant at low substrate concentrations (50 nm), although transport properties at higher concentrations (1000 nm) were not statistically different between wild-type and the FT5 null mutant. Modulation of the expression of FT5 also changes the susceptibility of Leishmania cells to methotrexate. These results have permitted the characterization of a novel class of folate transporters and suggest that the parasite Leishmania has several gene products possibly transporting folates and related molecules under varying conditions.
Assuntos
Resistência a Medicamentos , Deleção de Genes , Leishmania/metabolismo , Metotrexato/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Leishmania/genética , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The folate metabolic pathway has been exploited successfully for the development of antimicrobial and antineoplasic agents. Inhibitors of this pathway, however, are not useful against Leishmania and other trypanosomatids. Work on the mechanism of methotrexate resistance in Leishmania has dramatically increased our understanding of folate and pterin metabolism in this organism. The metabolic and cellular functions of the reduced form of folates and pterins are beginning to be established and this work has led to several unexpected findings. Moreover, the currently ongoing sequencing efforts on trypanosomatid genomes are suggesting the presence of several gene products that are likely to require folates and pterins. A number of the properties of folate and pterin metabolism are unique suggesting that these pathways are valid and worthwhile targets for drug development.
Assuntos
Ácido Fólico/metabolismo , Leishmania/metabolismo , Pterinas/metabolismo , Trypanosomatina/metabolismo , Animais , Transporte BiológicoRESUMO
Pterins are essential for the growth of Leishmania species, and recent work has led to the isolation of the biopterin transporter BT1. In this study, we inactivated the Leishmania donovani biopterin transporter BT1 by gene disruption mediated by homologous recombination. No transport of biopterin was detected in this mutant. The L. donovani BT1 null mutant showed a much lesser capacity for inducing infection in mice than wild-type parasites and could elicit protective immunity in mice susceptible to infection against a L. donovani challenge. Splenocytes isolated from mice immunized with the BT1 null mutant parasites produced significant amounts of interferon gamma following stimulation with L. donovani promastigotes as measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assays. Overall, these results show that by genetically manipulating the pterin transport in L. donovani, it is possible to generate an attenuated organism that could be part of a vaccination strategy.