RESUMEN
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.
Asunto(s)
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
OBJECTIVES: Recently, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was successfully applied for the detection of carbapenemase activity directly from Gram-negative colonies. Based on this principle, we evaluated the performance of MALDI-TOF MS for rapid detection of carbapenemase activity directly from positive blood culture vials. METHODS: A total of 100 blood culture vials were randomly selected. MALDI-TOF MS carbapenemase assay results were confirmed by the detection of carbapenemase-encoding genes. RESULTS: A total of 110 bacterial isolates were recovered. The MALDI-TOF MS carbapenemase assay identified 21 of 29 (72.4%) of the carbapenemase-producing isolates directly from the blood culture vials, especially those encoding KPC-2 (100%) and SPM-1 (100%), after a 4 h incubation period. Although the majority of OXA-23-producing Acinetobacter baumannii isolates were not identified on day 1, all isolates were identified as carbapenemase producers directly from the colony on the next day. CONCLUSIONS: The MALDI-TOF MS carbapenemase assay is a feasible and rapid test to identify carbapenemase activity directly from blood culture vials. It may contribute to faster readjustment of empirical antimicrobial therapy and implementation of infection control measures.
Asunto(s)
Proteínas Bacterianas/sangre , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , beta-Lactamasas/sangre , Acinetobacter baumannii/enzimología , Acinetobacter baumannii/aislamiento & purificación , Humanos , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/aislamiento & purificación , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/aislamiento & purificaciónRESUMEN
This study evaluates the accuracy of liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) for detecting carbapenem hydrolytic activity among SPM-1-, GIM-1-, and GES-5-producing Pseudomonas aeruginosa isolates and OXA-143-, IMP-10-, and OXA-58-producing Acinetobacter baumannii isolates. Class A and B carbapenemase activities were rapidly detected by MALDI-TOF in a 2-h assay. However, an extended incubation time was necessary for detection of carbapenem-hydrolyzing class D ß-lactamase (CHDL) activity in Acinetobacter spp.
Asunto(s)
Acinetobacter baumannii/química , Acinetobacter baumannii/enzimología , Técnicas Bacteriológicas/métodos , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/enzimología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , beta-Lactamasas/metabolismo , Acinetobacter baumannii/aislamiento & purificación , Humanos , Pseudomonas aeruginosa/aislamiento & purificaciónRESUMEN
N-glycosylation is implicated in cancers and aberrant N-glycosylation is recognized as a hallmark of cancer. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 displays hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells. This comparative site-specific N-glycoproteome analysis provides a pool of potential N-glycosylation-related cancer biomarkers, but also gives insights into the M6P pathway in cancer.
Asunto(s)
Manosafosfatos , Neoplasias , Humanos , Glicosilación , Manosafosfatos/química , Manosafosfatos/metabolismo , Neoplasias/genéticaRESUMEN
BACKGROUND: The malaria burden remains a major public health concern, especially in sub-Saharan Africa. The complex biology of Plasmodium, the apicomplexan parasite responsible for this disease, challenges efforts to develop new strategies to control the disease. Proteolysis is a fundamental process in the metabolism of malaria parasites, but roles for proteases in generating vasoactive peptides have not previously been explored. RESULTS: In the present work, it was demonstrated by mass spectrometry analysis that Plasmodium parasites (Plasmodium chabaudi and Plasmodium falciparum) internalize and process plasma kininogen, thereby releasing vasoactive kinins (Lys-BK, BK and des-Arg9-BK) that may mediate haemodynamic alterations during acute malaria. In addition, it was demonstrated that the P. falciparum cysteine proteases falcipain-2 and falcipain-3 generated kinins after incubation with human kininogen, suggesting that these enzymes have an important role in this process. The biologic activity of peptides released by Plasmodium parasites was observed by measuring ileum contraction and activation of kinin receptors (B1 and B2) in HUVEC cells; the peptides elicited an increase in intracellular calcium, measured by Fluo-3 AM fluorescence. This effect was suppressed by the specific receptor antagonists Des-Arg9[Leu8]-BK and HOE-140. CONCLUSIONS: In previously undescribed means of modulating host physiology, it was demonstrated that malaria parasites can generate active kinins by proteolysis of plasma kininogen.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Quininógenos/metabolismo , Cininas/metabolismo , Plasmodium chabaudi/enzimología , Plasmodium falciparum/enzimología , Animales , Calcio/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Cobayas , Humanos , Íleon/efectos de los fármacos , Espectrometría de Masas , Contracción Muscular/efectos de los fármacos , Plasmodium chabaudi/metabolismo , Plasmodium falciparum/metabolismo , ProteolisisRESUMEN
SARS-CoV-2 infection poses a global health crisis. In parallel with the ongoing world effort to identify therapeutic solutions, there is a critical need for improvement in the prognosis of COVID-19. Here, we report plasma proteome fingerprinting that predict high (hospitalized) and low-risk (outpatients) cases of COVID-19 identified by a platform that combines machine learning with matrix-assisted laser desorption ionization mass spectrometry analysis. Sample preparation, MS, and data analysis parameters were optimized to achieve an overall accuracy of 92%, sensitivity of 93%, and specificity of 92% in dataset without feature selection. We identified two distinct regions in the MALDI-TOF profile belonging to the same proteoforms. A combination of SDS-PAGE and quantitative bottom-up proteomic analysis allowed the identification of intact and truncated forms of serum amyloid A-1 and A-2 proteins, both already described as biomarkers for viral infections in the acute phase. Unbiased discrimination of high- and low-risk COVID-19 patients using a technology that is currently in clinical use may have a prompt application in the noninvasive prognosis of COVID-19. Further validation will consolidate its clinical utility.
Asunto(s)
COVID-19/diagnóstico , Aprendizaje Automático , Proteoma/metabolismo , Proteómica/métodos , SARS-CoV-2/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Adulto , Anciano , Biomarcadores/sangre , COVID-19/epidemiología , COVID-19/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Pronóstico , Reproducibilidad de los Resultados , SARS-CoV-2/fisiología , Sensibilidad y Especificidad , Proteína Amiloide A Sérica/análisisRESUMEN
In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.
Asunto(s)
Metaloendopeptidasas/biosíntesis , Metaloendopeptidasas/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Secuencia de Aminoácidos , Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia , Humanos , Metaloendopeptidasas/genética , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Proteínas Recombinantes/genética , Especificidad por SustratoRESUMEN
Here we report a detailed analysis of magnesium (Mg²+) ion effects on furin hydrolysis of fluorescent resonance energy transfer decapeptide substrates derived from canonical R-X-K/R-R furin cleavage motifs within certain viral envelope glycoproteins and eukaryotic proproteins. Using virus-derived sequences a selective activation of furin by Mg²+) ions was observed as a result of cooperativity between furin subsites. Furin hydrolysis of the peptides Abz-SRRHKR↓FAGV-Q-EDDnp (from measles virus fusion protein F0 and Abz-RERRRKKR↓GLFG-Q-EDDnp (from Asian avian influenza A, H5N1) was activated between 60- and 80-fold by MgCl2. It appears that virus envelope glycoprotein mutations have been selected to increase their susceptibility to furin within cells, a location where Mg²+ is present in adequate concentrations for activation. Both the pH profile of furin and its intrinsic fluorescence were modified by Mg²+ ions, which bind to furin with a K(d) value of 1.1 mM.
Asunto(s)
Furina/metabolismo , Magnesio/farmacología , Proteínas del Envoltorio Viral/metabolismo , Biocatálisis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/química , Subtipo H5N1 del Virus de la Influenza A/genética , Iones/química , Iones/farmacología , Magnesio/química , Oligopéptidos/química , Proproteína Convertasas/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Proteínas del Envoltorio Viral/genéticaRESUMEN
Plasminogen is a glycoprotein implicated in angiogenesis and fibrin clot degradation associated with the release of angiostatin and plasmin activation, respectively. We have recently reported that cathepsin V, but not cathepsins L, B, and K, can release angiostatin-like fragments from plasminogen. Here, we extended the investigation to cathepsin S which has been implicated in angiogenesis and tumor cell proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasminogen hydrolysis by cathepsin S revealed generation of two fragments (60 and 38 kDa). Amino-terminal sequencing indicated that cleavage occurs at the Leu469-Leu470 peptide bond. In contrast to cathepsin V, which possesses antiangiogenic activity, cathepsin S plasminogen cleavage products were not capable of inhibiting angiogenesis on endothelial cells. Moreover, we explored the different selectivities presented by cathepsins V and S towards plasminogen and synthesized fluorescence resonance energy transfer peptides encompassing the hydrolyzed peptide bonds by both enzymes. The peptide Abz-VLFEKKQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp= N-[2,4-dinitrophenyl]ethylenediamine), hydrolyzed by cath-epsin V at the Phe-Glu bond, is a selective substrate for the enzyme when compared with cathepsins B, L, and S, whereas Abz-VLFEKKVYLQ-EDDnp is an efficient cathepsin L inhibitor. The demonstrated importance of the S(3)'-P(3)' interaction indicates the significance of the extended subsites for enzyme specificity and affinity.
Asunto(s)
Catepsina L/antagonistas & inhibidores , Catepsinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Fragmentos de Péptidos/metabolismo , Plasminógeno/metabolismo , Secuencia de Aminoácidos , Humanos , Hidrólisis , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
We report the enzymatic properties and substrate specificity of human recombinant KLK3 in the presence of glycosaminoglycans (GAGs) and sodium citrate. This salt is highly concentrated in prostate and in its presence KLK3 had a similar hydrolytic efficiency as chymotrypsin. In contrast to the latter peptidase, KLK3 activated by sodium citrate efficiently hydrolyzed substrates containing R, H and P at the P1 position. Activated KLK3 also cleaved peptides derived from the bradykinin domain of human kininogen at the same sites as human kallikrein KLK1, but presented low kininogenase activity. Angiotensin I has several sites for hydrolysis by KLK3; however, it was cleaved only at the Y-I bond (DRVY downward arrowIHPFHL). Sodium citrate modulated KLK3 conformation as observed by alterations to the intrinsic fluorescence of phenylalanines and tryptophans. Activated KLK3 was reversibly inhibited by Z-Pro-Prolinal and competitively inhibited by ortho-phenantroline. Together, these are noteworthy observations for the future design of specific non-peptide inhibitors of KLK3 and to find natural substrates.
Asunto(s)
Citratos/farmacología , Glicosaminoglicanos/farmacología , Calicreínas/antagonistas & inhibidores , Calicreínas/metabolismo , Inhibidores de Proteasas/farmacología , Secuencia de Aminoácidos , Angiotensinas/metabolismo , Cloruros/química , Cloruros/farmacología , Activación Enzimática/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Calicreínas/química , Quininógeno de Bajo Peso Molecular/metabolismo , Datos de Secuencia Molecular , Antígeno Prostático Específico , Sales (Química)/química , Sales (Química)/farmacología , Citrato de Sodio , Especificidad por SustratoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Species Cissus gongylodes has been used in the traditional medicine in South America and India for the treatment of urolithiasis, biliary and inflammatory problems without any scientific evidence. AIM OF THE STUDY: This work was developed to investigate for the first time the anti-inflammatory and anti-urolithiatic activities of leaf decoction of C. gongylodes. MATERIALS AND METHODS: Decoction was subjected to anti-inflammatory evaluation by the in vivo assay of ear oedema and quantification of the main mediators of inflammation PGE2 and LTB4, and the cytokine TNF-α. The decoction's anti-urolithiatic activity was determined by different in vitro assays to evaluate the inhibition and dissolution of the most prevalent types of kidney stones: calcium oxalate (CaOx) and struvite. Diffusion in gel technique and fresh urine of a patient with renal stone were used to investigate the inhibition and dissolution of CaOx, respectively, and the single diffusion gel growth technique was used to evaluate the inhibition and dissolution of struvite crystals. The decoction was chemically characterized by UHPLC-ESI-HRMS analysis. RESULTS: Decoction showed in vivo anti-inflammatory activity by potent decreasing the level of both the main mediators of inflammation and dose-dependent in vitro anti-urolithiatic action by inhibition and dissolution of both type of crystals, CaOx and struvite. CONCLUSIONS: Results obtained corroborate the reports of the traditional use of the decoction of Cissus gongylodes. Besides, it showed multi-target mechanisms actions, inhibition of the main inflammatory pathways, and inhibition/dissolution of the most prevalent types of crystals on urolithiasis. These actions make the decoction a promissory source to the development of new and more efficient drugs.
Asunto(s)
Antiinflamatorios/uso terapéutico , Cissus , Edema/tratamiento farmacológico , Cálculos Renales/tratamiento farmacológico , Extractos Vegetales/uso terapéutico , Animales , Antiinflamatorios/química , Oxalato de Calcio/química , Aceite de Crotón , Cristalización , Dinoprostona/metabolismo , Edema/inducido químicamente , Edema/metabolismo , Humanos , Cálculos Renales/química , Leucotrieno B4/metabolismo , Masculino , Ratones , Extractos Vegetales/química , Hojas de la Planta , Estruvita/química , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We explored furin substrate requirements in addition to the motif R-X-K/R-R using synthetic fluorescent resonance energy transfer (FRET) decapeptides. These decapeptides were derived from furin cleavage sites in viral coat glycoproteins and human and bacterial protein precursors. The hydrolysis by furin of most substrate was activated by K(+) ion, whereas kosmotropic anions of the Hofmeister series were inhibitors. The analysis of furin hydrolytic activity showed that its efficiency is highly dependent on the particular combinations of amino acids at different substrate positions. There is a clear interdependence of furin subsites that must be taken in account in determining its specificity and also for the design of inhibitors. However, clear preferences were detected for substrates with S at P(1)', and V at P(2)', at P(3)' the amino acids D, S, L and A are almost equally frequent. In the non-prime subsites the best substrates presented S and H at P(6); basic amino acids at P(5); and no clear tendency at P(3). Interestingly, two amino acid substitutions on the prime side of the peptide derived from H5N1 influenza hemagglutinin furin processing site highly improved its hydrolysis. These modifications are possible by single point mutations, suggesting a potential yield of a more infectious virus.
Asunto(s)
Furina/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Cloruro de Potasio/farmacología , Secuencia de Aminoácidos , Transferencia Resonante de Energía de Fluorescencia , Furina/química , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis/efectos de los fármacos , Cinética , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Cloruro de Sodio/farmacología , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Candida haemulonii is now considered a complex of two species and one variety: C. haemulonii sensu stricto, Candida duobushaemulonii and the variety C. haemulonii var. vulnera. Identification (ID) of these species is relevant for epidemiological purposes and for therapeutic management, but the different phenotypic commercial systems are unable to provide correct species ID for these emergent pathogens. Hence, we evaluated the MALDI-TOF MS performance for the ID of C. haemulonii species, analyzing isolates/strains of C. haemulonii complex species, Candida pseudohaemulonii and Candida auris by two commercial platforms, their databases and softwares. To differentiate C. haemulonii sensu sctricto from the variety vulnera, we used the ClinProTools(TM) models and a single-peak analysis with the software FlexAnalysis(TM). The Biotyper(TM) database gave 100% correct species ID for C. haemulonii sensu stricto, C. pseudohaemulonii and C. auris, with 69% of correct species ID for C. duobushaemulonii. Vitek MS(TM) IVD database gave 100% correct species ID for C. haemulonii sensu stricto, misidentifying all C. duobushaemulonii and C. pseudohaemulonii as C. haemulonii, being unable to identify C. auris. The Vitek MS(TM) RUO database needed to be upgraded with in-house SuperSpectra to discriminate C. haemulonii sensu stricto, C. duobushaemulonii, C. pseudohaemulonii, and C. auris strains/isolates. The generic algorithm model from ClinProTools(TM) software showed recognition capability of 100% and cross validation of 98.02% for the discrimination of C. haemulonii sensu stricto from the variety vulnera. Single-peak analysis showed that the peaks 5670, 6878, or 13750 m/z can distinguish C. haemulonii sensu stricto from the variety vulnera.
RESUMEN
Phosphorylated kininogen and some of its fragments containing serine phosphorylated bradykinin ([pS(6)]-Bk) were identified in human serum and plasma by a phosphoproteomic approach. We report the kininogenase ability of human tissue and plasma kallikreins and tryptase to generate [pS(6)]-Bk or Lys-[pS(6)]-Bk having as substrate the synthetic human kininogen fluorescent fragment Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2. The pharmacological assays of [pS(6)]-Bk showed it as a full B2 bradykinin receptor agonist in smooth muscle, it produces a portal liver hypertensive response in rat and mouse paw edema that lasts longer than Bk. The rat hypotensive response to infusions of Bk is greater than that of [pS(6)]Bk, both if injected through femoral vein or aorta. [pS(6)]-Bk was more resistant than Bk to kininase digestion performed with angiotensin converting enzyme, neprilysin, thimet oligopeptidase, aminopeptidase P and carboxypeptidase M. (1)H-NMR experiments indicated that [pS(6)]-Bk has lower flexibility, with the pS(6)-P(7) bond restricted to the trans conformation, and can explain [pS(6)]-Bk resistance to hydrolysis. In conclusion, [pS(6)]-Bk presenting lower activity than Bk, with longer lasting effects and being slowly released by kininogenases from synthetic Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2, suggests that phosphorylation of the kininogens can be an efficient kallikrein-kinin system regulator.
Asunto(s)
Bradiquinina/análogos & derivados , Bradiquinina/farmacología , Péptido Hidrolasas/farmacología , Secuencia de Aminoácidos , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Bradiquinina/genética , Cobayas , Humanos , Hidrólisis/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Técnicas de Cultivo de Órganos , Péptido Hidrolasas/genética , Conejos , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Transthyretin (TTR) is a plasma protein transporter of thyroxine (T(4)) and retinol and also has peptidase activity. In order to characterize TTR peptidase activity we used fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLRSSK-Q-EDDnp and from two portion-mixing libraries as substrates. Most of the susceptible FRET peptides were cleaved at more than one peptide bond, without particular substrate specificity. The more relevant observation was that the peptides containing E or D were cleaved at only one peptide bond and TTR was competitively inhibited by glutathione analog peptide γ-E-A-G-OH that contains two free carboxyl groups. The dependence on ionic interactions of TTR hydrolytic activity was confirmed by the large inhibitory effects of salt and ionic surfactants. TTR was not inhibited by any usual peptidase inhibitors, except by ortho-phenanthroline and EDTA. The mechanism of TTR catalysis was explored by the pH-profile of TTR hydrolytic activity in different temperatures and by proton inventory. The obtained pK and heat of ionization values suggest that a carboxylate and an ammonium group, possibly from a lysine side chain are involved. These results support the recently proposed inducible metalloprotease mechanism for TTR based on its 3D structure in presence of Zn(2+) and a series of point mutations [Liz et al., Biochem. J 443 (2012) 769-778].
Asunto(s)
Lisina/química , Prealbúmina/química , Protones , Secuencia de Aminoácidos , Técnicas Químicas Combinatorias , Ácido Edético/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Datos de Secuencia Molecular , Biblioteca de Péptidos , Fenantrolinas/química , Inhibidores de Proteasas/química , Técnicas de Síntesis en Fase Sólida , Especificidad por Sustrato , Tensoactivos/química , TemperaturaRESUMEN
X-linked hypophosphatemia (XLH/HYP)-with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscesses-is caused by mutations in the zinc-metallopeptidase PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization-inhibiting, acidic serine- and aspartate-rich motif (ASARM)-containing peptides, which are proteolytically derived from the mineral-binding matrix proteins of the SIBLING family (small, integrin-binding ligand N-linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motif-osteopontin (OPN) and bone sialoprotein (BSP)-as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full-length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an â¼35 kDa OPN fragment that was not present in wild-type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild-type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. In conclusion, these results identify full-length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization-inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP.
Asunto(s)
Huesos/metabolismo , Raquitismo Hipofosfatémico Familiar/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X , Osteopontina/metabolismo , Endopeptidasa Neutra Reguladora de Fosfato PHEX/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Inmunohistoquímica , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Osteopontina/química , ProteolisisRESUMEN
We investigated the antimicrobial resistance profile and the occurrence of Klebsiella pneumoniae carbapenemase (KPC)-producing Gram-negative rods in sewage samples obtained from a Brazilian teaching hospital and from the wastewater treatment plant (WWTP) that receives it for treatment. We identified multidrug-resistant bacteria as well as KPC-2-producing Aeromonas spp. and several Enterobacteriaceae species, including Kluyvera spp., in the hospital effluent and in different sites of the WWTP. Most isolates showed the blaKPC-2 gene harbored on a transposon that was carried by conjugative plasmids. The presence of KPC production among Aeromonas spp., Kluyvera spp., and other Enterobacteriaceae indicates the adaptability of such isolates to aquatic environments, not only in the hospital effluent but also throughout the WWTP. Although secondary treatment seems to decrease the amount of KPC producers in sewage, multidrug-resistant isolates are continually disposed in the urban river. Thus, sewage treatment regulations are urgently needed to decelerate the evolution of antimicrobial resistance beyond hospitals.
Asunto(s)
Aeromonas/aislamiento & purificación , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple , Enterobacteriaceae/aislamiento & purificación , Kluyvera/aislamiento & purificación , Aguas del Alcantarillado/microbiología , beta-Lactamasas/genética , Acinetobacter/efectos de los fármacos , Acinetobacter/aislamiento & purificación , Aeromonas/efectos de los fármacos , Brasil , Elementos Transponibles de ADN , Enterobacteriaceae/efectos de los fármacos , Hospitales de Enseñanza , Kluyvera/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Pseudomonas/efectos de los fármacos , Pseudomonas/aislamiento & purificaciónRESUMEN
We studied the substrate specificity requirements of recombinant cysteine peptidases from Plasmodium falciparum, falcipain-2 (FP-2) and falcipain-3 (FP-3), using fluorescence resonance energy transfer (FRET) peptides as substrates. Systematic modifications were introduced in the lead sequence Abz-KLRSSKQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp=N-[2,4-dinitrophenyl]ethylenediamine) resulting in five series assayed to map S3-S'2 subsite specificity. Despite high sequence identity and structural similarity between FP-2 and FP-3, noteworthy differences in substrate specificity were observed. The S1 subsite of FP-2 preferentially accommodates peptides containing the positively charged residue Arg in P1, while FP-3 has a clear preference for the hydrophobic residue Leu in this position. The S2 subsite of FP-2 and FP-3 presents a strict specificity for hydrophobic residues, with Leu being the residue preferred by both enzymes. FP-2 did not show preference for the residues present at P3, while FP-3 hydrolysed the peptide Abz-ALRSSRQ-EDDnp, containing Ala at P3, with the highest catalytic efficiency of all series studied. FP-2 has high susceptibility for substrates containing hydrophobic residues in P'1, while FP-3 accommodates well peptides containing Arg in this position. The S'2 subsite of both enzymes demonstrated broad specificity. In addition, radioimmunoassay experiments indicated that kinins can be generated by FP-2 and FP-3 proteolysis of high molecular weight kininogen (HK). Both enzymes excised Met-Lys-bradykinin, Lys-bradykinin and bradykinin from the fluorogenic peptide Abz-MISLMKRPPGFSPFRSSRI-NH2, which corresponds to the Met(375) to Ile(393) sequence of HK. The capability of FP-2 and FP-3 to release kinins suggests the involvement of these enzymes in the modulation of malaria pathophysiology.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Calicreínas/metabolismo , Plasmodium falciparum/enzimología , Quininógenos/metabolismo , Cininas/metabolismo , Especificidad por SustratoRESUMEN
In this study we investigated the peptidase activity in Leishmania (L.) amazonensis live amastigote by confocal microscopy using peptidyl-MCA as substrates, the hydrolysis of which releases the MCA fluorophore inside the cells. Cell pre-treatment with peptidase inhibitors indicated the presence of cysteine and serine peptidases. It was noteworthy that Leishmania amastigotes incorporate only substrates (Z-FR-MCA, Z-RR-MCA) or inhibitors (E64, TLCK) containing positively charged groups. The peptidase activities in the supernatants of amastigotes and promastigotes lysates were also evaluated with the same peptidyl-MCA substrates and inhibitors in the pH range 4.5-9.0. The effects of temperature and different salts were also included in this study. The hydrolytic activities of supernatants on Z-FR-MCA clearly indicate the presence of different cysteine peptidases that adapted to work in different environment conditions. Intact Leishmania cells incorporated Z-RR-MCA, the hydrolysis of which was inhibited only by TLCK indicating the presence of at least one serine peptidase. The pH profile of Z-RR-MCA hydrolysis by amastigotes and promastigotes lysate supernatants, and the hydrolysis time course of the FRET peptide Abz-AGRRRAQ-EDDnp at RA bond, followed by removal of the two C-termini R to yield Abz-AGR-OH that is a unique characteristic of oligopeptidase B, indicate its presence in the parasite.
Asunto(s)
Leishmania/enzimología , Péptido Hidrolasas/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Citratos/química , Cricetinae , Inhibidores de Cisteína Proteinasa/farmacología , Concentración de Iones de Hidrógeno , Leishmania/citología , Leucina/análogos & derivados , Leucina/farmacología , Mesocricetus , Oligopéptidos/química , Pepstatinas/farmacología , Péptido Hidrolasas/química , Proteolisis , Proteínas Protozoarias/química , Salinidad , Inhibidores de Serina Proteinasa/farmacología , Citrato de Sodio , Sulfatos/química , Temperatura , Clorometilcetona Tosilisina/farmacologíaRESUMEN
Foot and mouth disease virus expresses its genetic information as a single polyprotein that is translated from the single-stranded RNA genome. Proteinases contained within the polyprotein then generate the mature viral proteins. The leader protease (Lb(pro)) performs the initial cleavage by freeing itself from the growing polypeptide chain; subsequently, Lb(pro) cleaves the two homologues of the host cell protein eukaryotic initiation factor 4G (eIF4G). We showed that Lb(pro) possesses specific binding sites at the non prime side from S(1) down to S(7) [Santos et al. (2009) Biochemistry, 48, 7948-7958]. Here, we demonstrate that Lb(pro) has high prime side specificity at least down to the S'(5) site. Lb(pro) is thus not only one of the smallest papain-like cysteine peptidases but also one of the most specific. It can still however cleave between both K↓G and G↓R pairs. We further determined the two-step irreversible inhibition (E + I â EIâ E - I) kinetic parameters of two known irreversible epoxide-based inhibitors of cysteine proteinases, E64 and CA074 on Lb(pro) that show for the reversible step (E + I â EI) K(i) = 3.4 µM and 11.6 µM, and for the irreversible step (EIâE-I) k(4) = 0.16 and 0.06 min(-1), respectively. Knowledge of the Lb(pro) specificity led us to extend E64 by addition of the dipeptide R-P. This compound, termed E64-R-P-NH(2), irreversibly inhibited Lb(pro) with a K(i) = 30 nM and k(4) = 0.01 min(-1) and can serve as the basis for design of specific inhibitors of FMDV replication.