RESUMEN
The kallikrein-related peptidase KLK2 has restricted expression in the prostate luminal epithelium, and its protein target is unknown. The present work reports the hydrolytic activities of KLK2 on libraries of fluorescence resonance energy-transfer peptides from which the sequence SYRIF was the most susceptible substrate for KLK2. The sequence SYRIF is present at the extracellular N-terminal segment (58SYRIF63Q) of IL-10R2. KLK2 was fully active at pH 8.0-8.2, found only in prostate inflammatory conditions, and strongly activated by sodium citrate and glycosaminoglycans, the quantities and structures controlled by prostate cells. Bone-marrow-derived macrophages (BMDM) have IL-10R2 expressed on the cell surface, which is significantly reduced after KLK2 treatment, as determined by flow cytometry (FACS analysis). The IL-10 inhibition of the inflammatory response to LPS/IFN-γ in BMDM cells due to decreased nitric oxide, TNF-α, and IL-12 p40 levels is significantly reduced upon treatment of these cells with KLK2. Similar experiments with KLK3 did not show these effects. These observations indicate that KLK2 proteolytic activity plays a role in prostate inflammation and makes KLK2 a promising target for prostatitis treatment.
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Calicreínas , Humanos , Masculino , Calicreínas/metabolismo , Calicreínas/química , Arginina/metabolismo , Arginina/química , Próstata/metabolismo , Próstata/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Animales , Ratones , Péptidos/química , Péptidos/farmacología , Péptidos/metabolismo , Dominios Proteicos , Interleucina-10/metabolismo , Especificidad por SustratoRESUMEN
Acanthoscurria juruenicola is an Amazonian spider described for the first time almost a century ago. However, little is known about their venom composition. Here, we present a multiomics characterization of A. juruenicola venom by a combination of transcriptomics, proteomics, and peptidomics approaches. Transcriptomics of female venom glands resulted in 93,979 unique assembled mRNA transcript encoding proteins. A total of 92 proteins were identified in the venom by mass spectrometry, including 14 mature cysteine-rich peptides (CRPs). Quantitative analysis showed that CRPs, cysteine-rich secretory proteins, metalloproteases, carbonic anhydrases, and hyaluronidase comprise >90% of the venom proteome. Relative quantification of venom toxins was performed by DIA and DDA, revealing converging profiles of female and male specimens by both methods. Biochemical assays confirmed the presence of active hyaluronidases, phospholipases, and proteases in the venom. Moreover, the venom promoted in vivo paralytic activities in crickets, consistent with the high concentration of CRPs. Overall, we report a comprehensive analysis of the arsenal of toxins of A. juruenicola and highlight their potential biotechnological and pharmacological applications. Mass spectrometry data were deposited to the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD013149 and via the MassIVE repository with the dataset identifier MSV000087777.
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Venenos de Araña , Arañas , Animales , Masculino , Femenino , Arañas/genética , Arañas/metabolismo , Venenos de Araña/genética , Venenos de Araña/química , Venenos de Araña/metabolismo , Cisteína/metabolismo , Proteómica/métodos , Espectrometría de Masas/métodos , Proteoma/genética , Proteoma/metabolismo , Péptidos/análisisRESUMEN
Peptide-based hydrogels have attracted much attention due to their extraordinary applications in biomedicine and offer an excellent mimic for the 3D microenvironment of the extracellular matrix. These hydrated matrices comprise fibrous networks held together by a delicate balance of intermolecular forces. Here, we investigate the hydrogelation behavior of a designed decapeptide containing a tetraleucine self-assembling backbone and fibronectin-related tripeptides near both ends of the strand. We have observed that this synthetic peptide can produce hydrogel matrices entrapping >99% wt/vol % water. Ultrastructural analyses combining atomic force microscopy, small-angle neutron scattering, and X-ray diffraction revealed that amyloid-like fibrils form cross-linked networks endowed with remarkable thermal stability, the structure of which is not disrupted up to temperatures >80 °C. We also examined the interaction of peptide hydrogels with either NIH3T3 mouse fibroblasts or HeLa cells and discovered that the matrices sustain cell viability and induce morphogenesis into grape-like cell spheroids. The results presented here show that this decapeptide is a remarkable building block to prepare highly stable scaffolds simultaneously endowed with high water retention capacity and the ability to instruct cell growth into tumor-like spheroids even in noncarcinoma lineages.
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Hidrogeles , Nanoestructuras , Amiloide , Animales , Células HeLa , Humanos , Hidrogeles/química , Ratones , Morfogénesis , Células 3T3 NIH , Nanoestructuras/toxicidad , Péptidos/química , AguaRESUMEN
The COVID-19 outbreak has rapidly spread on a global scale, affecting the economy and public health systems throughout the world. In recent years, peptide-based therapeutics have been widely studied and developed to treat infectious diseases, including viral infections. Herein, the antiviral effects of the lysine linked dimer des-Cys11, Lys12,Lys13-(pBthTX-I)2K ((pBthTX-I)2K)) and derivatives against SARS-CoV-2 are reported. The lead peptide (pBthTX-I)2K and derivatives showed attractive inhibitory activities against SARS-CoV-2 (EC50 = 28-65 µM) and mostly low cytotoxic effect (CC50 > 100 µM). To shed light on the mechanism of action underlying the peptides' antiviral activity, the Main Protease (Mpro) and Papain-Like protease (PLpro) inhibitory activities of the peptides were assessed. The synthetic peptides showed PLpro inhibition potencies (IC50s = 1.0-3.5 µM) and binding affinities (Kd = 0.9-7 µM) at the low micromolar range but poor inhibitory activity against Mpro (IC50 > 10 µM). The modeled binding mode of a representative peptide of the series indicated that the compound blocked the entry of the PLpro substrate toward the protease catalytic cleft. Our findings indicated that non-toxic dimeric peptides derived from the Bothropstoxin-I have attractive cellular and enzymatic inhibitory activities, thereby suggesting that they are promising prototypes for the discovery and development of new drugs against SARS-CoV-2 infection.
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Venenos de Crotálidos/química , Dimerización , Papaína/antagonistas & inhibidores , Péptidos/química , Péptidos/farmacología , SARS-CoV-2/enzimología , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Simulación del Acoplamiento Molecular , Papaína/química , Papaína/metabolismo , Péptidos/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Conformación Proteica , SARS-CoV-2/efectos de los fármacosRESUMEN
Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (NEL, EC 3.4.24.16) are closely related zinc-dependent metalo-oligopeptidases, which take part in the metabolism of oligopeptides (from 5 to 17 amino acid residues) inside and outside cells. Both peptidases are ubiquitously distributed in tissues. TOP is one of the main intracellular peptide-processing enzymes being important for the antigen selection in the MHC Class I presentation route, while NEL function has been more associated with the extracellular degradation of neurotensin. Despite efforts being made to develop specific inhibitors for these peptidases, the most used are: CPP-Ala-Ala-Tyr-PABA, described by Orlowski et al. in 1988, and CPP-Ala-Aib-Tyr-PABA (JA-2) that is an analog more resistant to proteolysis, which development was made by Shrimpton et al. in 2000. In the present work, we describe other analogs of these compounds but, with better discriminatory capacity to inhibit specifically NEL or TOP. The modifications introduced in these new analogs were based on a key difference existent in the extended binding sites of NEL and TOP: the negatively charged Glu469 residue of TOP corresponds to the positively charged Arg470 residue of NEL. These residues are in position to interact with the residue at the P1' and/or P2' of their substrates (mimicked by the Ala-Ala/P1'-P2' residues of the CPP-Ala-Ala-Tyr-PABA). Therefore, exploring this single difference, the following compounds were synthesized: CPP-Asp-Ala-Tyr-PABA, CPP-Arg-Ala-Tyr-PABA, CPP-Ala-Asp-Tyr-PABA, CPP-Ala-Arg-Tyr-PABA. Confirming the predictions, the replacement of each non-charged residue of the internal portion Ala-Ala by a charged residue Asp or Arg resulted in compounds with higher selectivity for NEL or TOP, especially due to the electrostatic attraction or repulsion by the NEL Arg470 or TOP Glu469 residue. The CPP-Asp-Ala-Tyr-PABA and CPP-Ala-Asp-Tyr-PABA presented higher affinities for NEL, and, the CFP-Ala-Arg-Tyr-PABA showed higher affinity for TOP.
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Metaloendopeptidasas/metabolismo , Oligopéptidos/farmacología , Cinética , Metaloendopeptidasas/antagonistas & inhibidores , Mutación/genética , Oligopéptidos/síntesis química , Oligopéptidos/química , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid ß peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to â¼95-fold, affecting primarily Vmax The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.
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Endosomas/metabolismo , Fosfatos de Inositol/metabolismo , Insulisina/metabolismo , Fosfatidilinositoles/metabolismo , Androstadienos/farmacología , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Endosomas/efectos de los fármacos , Activación Enzimática , Enzimas Inmovilizadas/metabolismo , Concentración de Iones de Hidrógeno , Insulisina/química , Insulisina/genética , Liposomas/química , Liposomas/metabolismo , Mutación , WortmaninaRESUMEN
Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.
Asunto(s)
Colágeno Tipo XVIII/metabolismo , Endostatinas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Colágeno Tipo XVIII/química , Espacio Extracelular/enzimología , Transferencia Resonante de Energía de Fluorescencia , Hemangioendotelioma/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Péptidos/metabolismo , Subunidades de Proteína/metabolismo , ProteolisisRESUMEN
MAIN CONCLUSION: A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M-1 cm-1. MpBBI inhibits strongly trypsin with K i in the 10-10 M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.
Asunto(s)
Maclura/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Semillas/química , Inhibidores de Tripsina/metabolismo , Clonación Molecular , Modelos Moleculares , Mapeo Peptídico , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Conformación Proteica , Homología de Secuencia de Aminoácido , Tripsina/metabolismo , Inhibidor de la Tripsina de Soja de Bowman-Birk , Inhibidores de Tripsina/química , Inhibidores de Tripsina/aislamiento & purificaciónRESUMEN
Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys55 and Lys268 increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys55 and Lys268 with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2.
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Caspasas/química , Proteínas Protozoarias/química , Trypanosoma brucei brucei/enzimología , Sustitución de Aminoácidos , Caspasas/genética , Caspasas/metabolismo , Activación Enzimática , Mutación Missense , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Especificidad por Sustrato , Trypanosoma brucei brucei/genéticaRESUMEN
Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R↓R-ACC and Z-R↓R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates.
Asunto(s)
Calicreínas/metabolismo , Cinética , Péptido Hidrolasas/metabolismo , Péptidos/química , Aminoácidos Básicos/química , Aminoácidos Básicos/genética , Encefalinas/química , Encefalinas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Furina/química , Furina/metabolismo , Humanos , Hidrólisis , Calicreínas/química , Calicreínas/genética , Metaloproteinasa 14 de la Matriz/química , Metaloproteinasa 14 de la Matriz/metabolismo , Modelos Moleculares , Factor de Crecimiento Nervioso/química , Factor de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/metabolismo , Neurotrofina 3 , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Péptidos/metabolismo , Conformación Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteolisis , Especificidad por SustratoRESUMEN
KLK7 substrate specificity was evaluated by families of fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLFSSK-Q-EDDnp (Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-[2,4-dinitrophenyl] ethylenediamine), by one bead-one peptide FRET peptide library in PEGA resin, and by the FRET peptide libraries Abz-GXX-Z-XX-Q-EDDnp (Z and X are fixed and random natural amino acids, respectively). KLK7 hydrolyzed preferentially F, Y or M, and its S1' and S2' subsites showed selectivity for hydrophilic amino acids, particularly R and K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz-MISLM(↓)KRPPGFSPF(↓)RSSRI-NH2 ((↓)indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. The peptide Abz-NLY(↓)RVE-Q-EDDnp is the best synthetic substrate so far described for KLK7 [kcat/Km=455 (mMs)(-1)] that was designed from the KLK7 substrate specificity analysis. It is noteworthy that the NLYRVE sequence is present in human semaphorin 6B. KLK7 is activated by GAGs, inhibited by neutral salts, and activated by high concentration of kosmotropic salt. Pyroglutamic acid inhibited KLK7 (Ki=33mM) and is present in skin moisturizing factor (124mM). The KLK7 specificity described here and elsewhere reflects its participation in patho-physiological events in skin, the gastrointestinal tract and central nervous system, where KLK7 is significantly expressed.
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Glicosaminoglicanos/farmacología , Calicreínas/metabolismo , Péptido Hidrolasas/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biocatálisis/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Humanos , Hidrólisis/efectos de los fármacos , Cinética , Quininógenos/metabolismo , Datos de Secuencia Molecular , Concentración Osmolar , Ácido Pirrolidona Carboxílico/farmacología , Semaforinas/metabolismo , Serpinas/metabolismo , Somatostatina/metabolismo , Sustancia P/metabolismo , Especificidad por Sustrato , Factores de TiempoRESUMEN
The human tissue kallikreins (KLK1-KLK15) comprise a family of 15 serine peptidases detected in almost every tissue of the human body and that actively participate in many physiological and pathological events. Some kallikreins are involved in diseases for which no effective therapy is available, as for example, epithelial disorders, bacterial infections and in certain cancers metastatic processes. In recent years our group have made efforts to find inhibitors for all kallikreins, based on natural products and synthetic molecules, and all the inhibitors developed by our group presented a competitive mechanism of inhibition. Here we describe fukugetin, a natural product that presents a mixed-type mechanism of inhibition against KLK1 and KLK2. This type of inhibitor is gaining importance today, especially for the development of exosite-type inhibitors, which present potential to selectively inhibit the enzyme activity only against specific substrate.
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Biflavonoides/farmacología , Productos Biológicos/farmacología , Inhibidores de Serina Proteinasa/farmacología , Calicreínas de Tejido/antagonistas & inhibidores , Biflavonoides/química , Biflavonoides/aislamiento & purificación , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Garcinia/química , Humanos , Modelos Moleculares , Conformación Molecular , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/aislamiento & purificación , Relación Estructura-Actividad , Calicreínas de Tejido/metabolismoRESUMEN
Peptidases are important because they play a central role in pharmaceutical, food, environmental, and other industrial processes. A serine peptidase from Aspergillus terreus was isolated after two chromatography steps that showed a yield of 15.5%. Its molecular mass was determined to be 43 kD, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This peptidase was active between pH 5.0 to 8.0 and had maximum activity at pH 7.0, at 45°C. When exposited with 1 M of urea, the enzyme maintained 100% activity and used azocasein as substrate. The N-terminal (first 15 residues) showed 33% identity with the serine peptidase of Aspergillus clavatus ES1. The kinetics assays showed that subsite S2 did not bind polar basic amino acids (His and Arg) nonpolar acidic amino acids (Asp and Glu). The subsite S1 showed higher catalytic efficiency than the S2 and S3 subsites.
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Aspergillus/enzimología , Serina Proteasas/aislamiento & purificación , Secuencia de Aminoácidos , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Fermentación , Concentración de Iones de Hidrógeno , Cinética , Serina Proteasas/química , Serina Proteasas/metabolismo , TemperaturaRESUMEN
Enteroaggregative and uropathogenic Escherichia coli, Shigella flexneri 2a, and the hybrid enteroaggregative/Shiga toxin-producing E. coli strain (O104:H4) are important pathogens responsible for intestinal and urinary tract infections, as well as sepsis and hemolytic uremic syndrome. They have in common the production of a serine protease called Pic. Several biological roles for Pic have been described, including protection of E. coli DH5α from complement-mediated killing. Hereby we showed that Pic significantly reduces complement activation by all 3 pathways. Pic cleaves purified C3/C3b and other proteins from the classic and lectin pathways, such as C4 and C2. Cleavage fragments of C3, C4, and C2 were also observed with HB101(pPic1) culture supernatants, and C3 cleavage sites were mapped by fluorescence resonance energy transfer peptides. Experiments using human serum as a source of complement proteins confirmed Pic proteolytic activity on these proteins. Furthermore, Pic works synergistically with the human complement regulators factor I and factor H, promoting inactivation of C3b. In the presence of both regulators, further degradation of C3 α' chain was observed. Therefore, Pic may contribute to immune evasion of E. coli and S. flexneri, favoring invasiveness and increasing the severity of the disorders caused by these pathogens.
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Proteínas del Sistema Complemento/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Escherichia coli/fisiología , Evasión Inmune , Serina Endopeptidasas/metabolismo , Factores de Virulencia/metabolismo , Humanos , HidrólisisRESUMEN
The substrate specificity of TcoCBc1 was evaluated using two internally quenched fluorescent peptide libraries with randomized sequences designed to detect carboxydipeptidase (Abz-GXXZXK(Dnp)-OH) and endopeptidase (Abz-GXXZXXQ-EDDnp) activities at acidic and neutral pHs, respectively. All the data obtained with TcoCBc1 were compared with those of human cathepsin B, including the pH profiles of the hydrolytic reactions. The most relevant observation is the preference of TcoCBc1 for substrates with a pair of acidic amino acids at positions P(2) and P(1) for its carboxydipeptidase activity and the well acceptance for E and D at P(1) position for endopeptidase activity. These peculiar preferences for negatively charged groups of TcoCBc1 and its requirements for carboxydipeptidase activity were also observed on Abz labeled analogues of bradykinin (Abz-RPPG(↓)FSAFR-OH, Abz-RPPG(↓)FS(↓)AF-OH, Abz-RPPG(↓)DE(↓)AF-OH) and angiotensin I (Abz-DR(↓)VYIHAFHL-OH), where (↓) indicates the cleavage site. TcoCBc1 was modeled based on the atomic coordinates of the cathepsin B from Trypanosoma brucei and the positively charged environment in TcoCBc1 catalytic site contrasts with the negatively charged environment in human cathepsin B. The preferences of S1 and S2 subsites of TcoCBc1 for acidic amino acids have to be taken into consideration for future studies of physiological roles of TcoCBc1 as for instance in apoptotic processes of Trypanosoma congolense.
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Angiotensina I/metabolismo , Bradiquinina/metabolismo , Catepsina B/metabolismo , Fragmentos de Péptidos/metabolismo , Trypanosoma congolense/enzimología , Dominio Catalítico , Catepsina B/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Modelos Moleculares , Biblioteca de Péptidos , Conformación Proteica , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.
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Venenos de Crotálidos/enzimología , Calicreínas/metabolismo , Metaloproteasas/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Bothrops , Hidrólisis , Cinética , Simulación de Dinámica Molecular , Péptidos/química , Péptidos/metabolismo , Prolil Oligopeptidasas , Radioinmunoensayo , Especificidad por SustratoRESUMEN
Current therapies against malignant melanoma generally fail to increase survival in most patients, and immunotherapy is a promising approach as it could reduce the dosage of toxic therapeutic drugs. In the present study, we show that an immunotherapeutic approach based on the use of the Toll-like receptor (TLR)-5 ligand flagellin (Salmonella Typhimurium FliCi) combined with the major histocompatibility complex class II-restricted P10 peptide, derived from the Paracoccidioides brasiliensis gp43 major surface protein, reduced the number of lung metastasis in a murine melanoma model. Compounds were administered intranasally into C57Bl/6 mice intravenously challenged with syngeneic B16F10-Nex2 melanoma cells, aiming at the local (pulmonary) immune response modulation. Along with a marked reduction in the number of lung nodules, a significant increase in survival was observed. The immunization regimen induced both local and systemic proinflammatory responses. Lung macrophages were polarized towards a M1 phenotype, lymph node cells, and splenocytes secreted higher interleukin-12p40 and interferon (IFN)-γ levels when re-stimulated with tumor antigens. The protective effect of the FliCi+P10 formulation required TLR-5, myeloid differentiation primary response gene 88 and IFN-γ expression, but caspase-1 knockout mice were only partially protected, suggesting that intracellular flagellin receptors are not involved with the anti-tumor effect. The immune therapy resulted in the activation of tumor-specific CD4(+) T lymphocytes, which conferred protection to metastatic melanoma growth after adoptive transfer. Taken together, our results report a new immunotherapeutic approach based on TLR-5 activation and IFN-γ production capable to control the metastatic growth of B16F10-Nex2 melanoma, being a promising alternative to be associated with chemotherapeutic drugs for an effective anti-tumor responses.
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Antígenos Bacterianos/inmunología , Vacunas contra el Cáncer/inmunología , Flagelina/inmunología , Glicoproteínas/inmunología , Inmunoterapia/métodos , Neoplasias Pulmonares/terapia , Melanoma Experimental/terapia , Fragmentos de Péptidos/inmunología , Administración Intranasal , Administración a través de la Mucosa , Animales , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/genética , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , Caspasa 1/deficiencia , Caspasa 1/genética , Flagelina/administración & dosificación , Flagelina/genética , Expresión Génica , Glicoproteínas/administración & dosificación , Glicoproteínas/genética , Inyecciones Intravenosas , Interferón gamma/agonistas , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Subunidad p40 de la Interleucina-12/biosíntesis , Subunidad p40 de la Interleucina-12/inmunología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Masculino , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Metástasis de la Neoplasia , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/genética , Receptor Toll-Like 5/agonistas , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/inmunologíaRESUMEN
The objective of the present study was to investigate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in diabetic rat kidney. Cathepsins, glycosidases and sulfatases were studied on the 10th (DM-10) and on the 30th (DM-30) day of streptozotocin-induced diabetes mellitus (DM). The activity of cathepsin B, the main kidney cysteine protease, was decreased both in DM-10 and DM-30. Gel filtration chromatography of urinary proteins has shown the prevalence of low molecular weight peptides in normal and DM-10 urine, in contrast to the prevalence of high molecular weight peptides and intact proteins in DM-30. These results show that the decrease in lysosomal proteases could explain, at least in part, the increased albuminuria detected by radial immunodiffusion (RID), due to the excretion of less degraded or intact albumin. Concerning sulfated polysaccharides, the activities of ß-glucuronidase, N-acetyl-ß-d-glucosaminidase, and N-acetyl-ß-d-galactosaminidase were also decreased in DM-30, while aryl sulfatases did not vary. Increased toluidine blue metachromatic staining of the tissue suggests that the lower activities of glycosidases could lead to intracellular deposition of partially digested molecules, and this could explain the decreased urinary excretion and increased tissue buildup of these molecules. The main morphological changes observed in kidney were proximal convoluted tubules with thinner walls and thinner brush border. Immunohistochemistry revealed that most of cathepsin B was located in the brush border of proximal tubular cells, highlighting the involvement of proximal convoluted tubules in diabetic nephropathy.
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Diabetes Mellitus Experimental/enzimología , Riñón/enzimología , Lisosomas/enzimología , Animales , Catepsinas/genética , Catepsinas/metabolismo , Diabetes Mellitus Experimental/genética , Regulación hacia Abajo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Humanos , Masculino , Ratas , Ratas Wistar , Sulfatasas/genética , Sulfatasas/metabolismoRESUMEN
OBJECTIVE: Polyphosphate and heparin are anionic polymers released by activated mast cells and platelets that are known to stimulate the contact pathway of coagulation. These polymers promote both the autoactivation of factor XII and the assembly of complexes containing factor XI, prekallikrein, and high-molecular-weight kininogen. We are searching for salivary proteins from blood-feeding insects that counteract the effect of procoagulant and proinflammatory factors in the host, including elements of the contact pathway. APPROACH AND RESULTS: Here, we evaluate the ability of the sand fly salivary proteins, PdSP15a and PdSP15b, to inhibit the contact pathway by disrupting binding of its components to anionic polymers. We attempt to demonstrate binding of the proteins to polyphosphate, heparin, and dextran sulfate. We also evaluate the effect of this binding on contact pathway reactions. We also set out to determine the x-ray crystal structure of PdSP15b and examine the determinants of relevant molecular interactions. Both proteins bind polyphosphate, heparin, and dextran sulfate with high affinity. Through this mechanism they inhibit the autoactivation of factor XII and factor XI, the reciprocal activation of factor XII and prekallikrein, the activation of factor XI by thrombin and factor XIIa, the cleavage of high-molecular-weight kininogen in plasma, and plasma extravasation induced by polyphosphate. The crystal structure of PdSP15b contains an amphipathic helix studded with basic side chains that forms the likely interaction surface. CONCLUSIONS: The results of these studies indicate that the binding of anionic polymers by salivary proteins is used by blood feeders as an antihemostatic/anti-inflammatory mechanism.
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Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Sulfato de Dextran/metabolismo , Heparina/metabolismo , Proteínas de Insectos/farmacología , Polifosfatos/metabolismo , Psychodidae/química , Saliva/química , Animales , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Anticoagulantes/metabolismo , Pruebas de Coagulación Sanguínea , Permeabilidad Capilar/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Factor XIIa/antagonistas & inhibidores , Factor XIIa/metabolismo , Factor XIa/antagonistas & inhibidores , Factor XIa/metabolismo , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/aislamiento & purificación , Proteínas de Insectos/metabolismo , Quininógeno de Alto Peso Molecular/antagonistas & inhibidores , Quininógeno de Alto Peso Molecular/metabolismo , Ratones , Modelos Moleculares , Precalicreína/antagonistas & inhibidores , Precalicreína/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Trombina/metabolismo , Factores de TiempoRESUMEN
Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that ß-actin is the receptor of C7H2 in the tumor cells. C7H2 induces ß-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug.