RESUMO
A series of PdII complexes with bis-(2-pyridylmethyl)glycine as a ligand of formula [PdX(bis-(2-pyridylmethyl)glycine)] where Xâ¯=â¯Cl, Br, I were prepared and the effect of the halogen nature in the antitumor activity of eight tumorigenic and one non-tumorigenic cell line was evaluated. The chloride derivative was further functionalized with a transferrin receptor binding peptide, generating the first PdII based metallopeptide. Its antitumor activity was also evaluated. However, among all the complexes, the chloride and iodine parent compounds showed the lowest GI50 values in the panel evaluated, and lowest GI50 than cisplatin in several cell lines. In contrast, the bromine derivative showed higher values of GI50 than chloride and iodine (around 30 - 50⯵M). The same trend was observed for the bovine serum albumin binding constant with higher values for iodine, chlorine, and bromine in this order. In aqueous solution, the chloride is exchanged by water while the bromine and iodine are not. DNA was evaluated as a target and showed no significative interaction for all the compounds. The results suggest sulfur-rich proteins and not DNA as a target. This report represents the first PdII metallopeptide reported, its evaluation in solution and antitumor activity. This work opens the possibilities for further functionalization of PdII complexes and the importance of the halogen coordination in the design of novel metallodrugs.
Assuntos
Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Halogênios/química , Paládio/química , Peptídeos/química , Peptídeos/farmacologia , Proteínas de Ligação a Transferrina/química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Células HT29 , Humanos , Células MCF-7 , Espectroscopia de Ressonância Magnética , Peptídeos/síntese química , Relação Estrutura-Atividade , Difração de Raios XRESUMO
The substrate specificity of recombinant human mitochondrial intermediate peptidase (hMIP) using a synthetic support-bound FRET peptide library is presented. The collected fluorescent beads, which contained the hydrolysed peptides generated by hMIP, were sequenced by Edman degradation. The results showed that this peptidase presents a remarkable preference for polar uncharged residues at P1 and P1' substrate positions: Ser = Gln > Thr at P1 and Ser > Thr at P1'. Non-polar residues were frequent at the substrate P3, P2, P2' and P3' positions. Analysis of the predicted MIP processing sites in imported mitochondrial matrix proteins shows these cleavages indeed occur between polar uncharged residues. Previous analysis of these processing sites indicated the importance of positions far from the MIP cleavage site, namely the presence of a hydrophobic residue (Phe or Leu) at P8 and a polar uncharged residue (Ser or Thr) at P5. To evaluate this, additional kinetic analyses were carried out, using fluorogenic substrates synthesized based on the processing sites attributed to MIP. The results described here underscore the importance of the P1 and P1' substrate positions for the hydrolytic activity of hMIP. The information presented in this work will help in the design of new substrate-based inhibitors for this peptidase.
RESUMO
Trypanosoma cruzi trypomastigotes invade a great variety of mammalian cells, with several molecules being implicated in this complex event. Herein, the sequence GGIALAG present in prokineticin-2 receptor (PKR2), selected by phage display technology, is described as a new T. cruzi receptor for the Tc85 group of glycoproteins belonging to the gp85/TS superfamily and involved in cellular invasion of mammalian hosts. This finding is confirmed by the inhibitory activity of MCF10-A (human mammary) cell invasion by T. cruzi either by anti-PKR2 antibodies (77%) or GGIALAG-synthetic peptide (42%). Furthermore, interference RNA (iRNA) inhibition of PKR2 expression in MCF10-A cells reduces T. cruzi invasion by 50%. The binding site of Tc85 to PKR2 was localized at the C-terminal end of the molecule, upstream of the conserved FLY sequence, previously implicated in parasite cell invasion. PKR2, a receptor formed by seven membrane-spanning α-helical segments, is mainly present in the central nervous system, peripheral organs, and mature blood cells. Due to its wide distribution, PKR2 could be a suitable receptor for T. cruzi natural infection, contributing to the parasite dissemination throughout the mammalian organism. These findings augment the number and diversity of possible in vivo receptors for T. cruzi and reassure the multiplicity of Tc85 binding sites to mammalian hosts.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Bacteriófagos , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Glicoproteínas/genética , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genéticaRESUMO
It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10th or the 30th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10th, but not on the 30th day. Sulfatase decreased 30% on the 30th day, while glycosidases did not vary (or presented a transitory and slight decrease). There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.
Assuntos
Animais , Masculino , Catepsina B/metabolismo , Diabetes Mellitus Experimental/enzimologia , Fígado/enzimologia , Lisossomos/enzimologia , Albuminas/análise , Western Blotting , Glicemia/efeitos dos fármacos , Catepsina L/metabolismo , Creatinina/urina , Cisteína Proteases/metabolismo , Sulfato de Dextrana/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Expressão Gênica/efeitos dos fármacos , Glucuronidase/metabolismo , Hexosaminidases/metabolismo , Imuno-Histoquímica , Rim/metabolismo , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , RNA , Sulfatases/metabolismoRESUMO
It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10th or the 30th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10th, but not on the 30th day. Sulfatase decreased 30% on the 30th day, while glycosidases did not vary (or presented a transitory and slight decrease). There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.
Assuntos
Catepsina B/metabolismo , Diabetes Mellitus Experimental/enzimologia , Fígado/enzimologia , Lisossomos/enzimologia , Albuminas/análise , Animais , Glicemia/efeitos dos fármacos , Western Blotting , Catepsina L/metabolismo , Creatinina/urina , Cisteína Proteases/metabolismo , Sulfato de Dextrana/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Expressão Gênica/efeitos dos fármacos , Glucuronidase/metabolismo , Hexosaminidases/metabolismo , Imuno-Histoquímica , Rim/metabolismo , Masculino , RNA/isolamento & purificação , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Sulfatases/metabolismoRESUMO
An antigenic conserved B domain was previously identified within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, the r-potDomain B, a 6× His-tag polypeptide belonging to the conserved B domain from the potato apyrase, and synthetic peptides LbB1LJ and LbB2LJ derived from the B domain from Leishmania NTPDase 1 were used as molecular tools for studies of the Leishmania amazonensis NTPDase 1. Widespread subcellular location of the specific NTPDase 1 was detected by Western blots of promastigote fractions and ultrastructural immunocytochemical microscopy using immune sera raised against these biomolecules. In addition, the L. amazonensis-infected BALB/c mice were evaluated at 12 to 120 days after infection, which progresses showing typical nodular lesion. High antibody reactivity with either r-potDomain B, LbB1LJ, or LbB2LJ was found in L. amazonensis-infected BALB/c mice indicating the antigenicity of the B domain from NTPDase 1 isoform. The IgG1 antibody reactivity significantly increased at 90-120 days postinfection, 18- to 24-fold when compared to the 12th day, and remained elevated even at 120th after infection, coinciding with the most active stage of the disease. In contrast, significantly higher IgG2a antibody reactivity with each biomolecule was observed at 40th day, about two- to fourfold higher than those found at 12th or 20th day, and decreased along 120-day period. Apparently, the conserved B domain is capable to induce IgG2a production in early disease stages. All together, these results suggest that r-potDomain B or synthetic peptides could be molecular starting points in experimental protocols of immunotherapy and/or vaccination for leishmaniasis.
Assuntos
Antígenos CD/metabolismo , Apirase/metabolismo , Leishmania/enzimologia , Leishmaniose/parasitologia , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Antígenos de Protozoários , Apirase/genética , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Anotação de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
Angiotensins (Angs) modulate blood pressure, hydro-electrolyte composition, and antinociception. Although Ang (5-8) has generally been considered to be inactive, we show here that Ang (5-8) was the smallest Ang to elicit dose-dependent responses and receptor-mediated antinociception in the rat ventrolateral periaqueductal gray matter (vlPAG). Ang (5-8) antinociception seems to be selective, because it did not alter blood pressure or act on vascular or intestinal smooth muscle cells. The non-selective Ang-receptor (Ang-R) antagonist saralasin blocked Ang (5-8) antinociception, but selective antagonists of Ang-R types I, II, IV, and Mas did not, suggesting that Ang (5-8) may act via an unknown receptor. Endopeptidase EP 24.11 and amastatin-sensitive aminopeptidase from the vlPAG catalyzed the synthesis (from Ang II or Ang III) and inactivation of Ang (5-8), respectively. Selective inhibitors of neuronal-nitric oxide (NO) synthase, soluble guanylyl cyclase (sGC) and a non-selective opioid receptor (opioid-R) inhibitor blocked Ang (5-8)-induced antinociception. In conclusion, Ang (5-8) is a new member of the Ang family that selectively and strongly modulates antinociception via NO-sGC and endogenous opioid in the vlPAG.
Assuntos
Angiotensina I/farmacologia , Guanilato Ciclase/metabolismo , Óxido Nítrico/metabolismo , Nociceptividade/efeitos dos fármacos , Peptídeos Opioides/metabolismo , Fragmentos de Peptídeos/farmacologia , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Antagonistas de Receptores de Angiotensina/farmacologia , Animais , Aorta/efeitos dos fármacos , Relação Dose-Resposta a Droga , Frequência Cardíaca/fisiologia , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Peptídeos Opioides/antagonistas & inibidores , Ratos , Ratos Wistar , Saralasina/farmacologia , Guanilil Ciclase Solúvel , Teprotida/farmacologiaRESUMO
Bauninia forficata is trivially known as cow paw, and popularly used in Brazil for treatment of diabetes mellitus. Denominated baupain a cysteine proteinase was purified from B. forficata leaves. In this study, we investigated the baupain effect on aggregation of isolated human platelets in vitro and the results show that baupain hinders thrombin - but not ADP- and collagen- induced platelet aggregation. With synthetic quenched-fluorescent peptides, the kinetics of the cleavage site of human proteinase-activated receptor 1 / 2 / 3 and 4 [PAR-1 / 2 / 3 and 4] by baupain was determined. In conclusion, similar to bromelain and papain, baupain hinders human platelets aggregation, probably through an unspecific cleavage in the Phe-Leu bond of PAR1.
Assuntos
Cisteína Proteases/química , Cisteína Proteases/metabolismo , Fibrinolíticos/química , Folhas de Planta/enzimologia , Agregação Plaquetária/efeitos dos fármacos , Contagem de Células , Cisteína Proteases/farmacologia , Dipeptídeos/química , Fibrinolíticos/metabolismo , Fibrinolíticos/farmacologia , Corantes Fluorescentes , Humanos , Cinética , Receptores Ativados por Proteinase/química , Receptores Ativados por Proteinase/metabolismo , Trombina/metabolismoRESUMO
Trypanosoma cruzi is a protozoan parasite that infects vertebrates, causing in humans a pathological condition known as Chagas' disease. The infection of host cells by T. cruzi involves a vast collection of molecules, including a family of 85 kDa GPI-anchored glycoproteins belonging to the gp85/trans-sialidase superfamily, which contains a conserved cell-binding sequence (VTVXNVFLYNR) known as FLY, for short. Herein, it is shown that BALB/c mice administered with a single dose (1 µg/animal, intraperitoneally) of FLY-synthetic peptide are more susceptible to infection by T. cruzi, with increased systemic parasitaemia (2-fold) and mortality. Higher tissue parasitism was observed in bladder (7·6-fold), heart (3-fold) and small intestine (3·6-fold). Moreover, an intense inflammatory response and increment of CD4+ T cells (1·7-fold) were detected in the heart of FLY-primed and infected animals, with a 5-fold relative increase of CD4+CD25+FoxP3+ T (Treg) cells. Mice treated with anti-CD25 antibodies prior to infection, showed a decrease in parasitaemia in the FLY model employed. In conclusion, the results suggest that FLY facilitates in vivo infection by T. cruzi and concurs with other factors to improve parasite survival to such an extent that might influence the progression of pathology in Chagas' disease.
Assuntos
Doença de Chagas/imunologia , Glicoproteínas/química , Neuraminidase/química , Peptídeos/administração & dosagem , Trypanosoma cruzi/patogenicidade , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/imunologia , Doença de Chagas/parasitologia , Sequência Conservada , Feminino , Fatores de Transcrição Forkhead/imunologia , Glicoproteínas/imunologia , Humanos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/imunologia , Parasitemia/imunologia , Parasitemia/parasitologia , Peptídeos/síntese química , Peptídeos/química , Peritônio/citologia , Linfócitos T Reguladores/imunologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/imunologia , VirulênciaRESUMO
An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response.