RESUMO
OBJECTIVE: This study aimed to evaluate the role of pepsin inhibitors in the inflammatory response and their effects on laryngeal mucosal integrity during gastroesophageal reflux (GERD) under in vivo conditions. METHODS: A surgical model of GERD was used, in which mice were treated with pepstatin (0.3 mg/kg) or darunavir (8.6 mg/kg) for 3 days. On the third day after the experimental protocol, the laryngeal samples were collected to assess the severity of inflammation (wet weight and myeloperoxidase activity) and mucosal integrity (transepithelial electrical resistance and paracellular epithelial permeability to fluorescein). RESULTS: The surgical GERD model was reproduced. It showed features of inflammation and loss of barrier function in the laryngeal mucosa. Pepstatin and darunavir administration suppressed laryngeal inflammation and preserved laryngeal mucosal integrity. CONCLUSION: Pepsin inhibition by the administration of pepstatin and darunavir improved inflammation and protected the laryngeal mucosa in a mouse experimental model of GERD. LEVEL OF EVIDENCE: NA Laryngoscope, 134:3080-3085, 2024.
Assuntos
Modelos Animais de Doenças , Refluxo Gastroesofágico , Pepsina A , Animais , Camundongos , Refluxo Gastroesofágico/tratamento farmacológico , Pepstatinas/farmacologia , Mucosa Laríngea/efeitos dos fármacos , Mucosa Laríngea/patologia , Masculino , Inflamação/tratamento farmacológico , Inflamação/prevenção & controleRESUMO
Cathepsin D (CD) is overexpressed in several types of cancer and constitutes an important biological target. Pepstatin A, a pentapeptide incorporating two non-proteinogenic statin residues, is among the most potent inhibitor of CD but lacks selectivity and suffers from poor bioavailability. Eight analogues of Pepstatin A, were synthesized, replacing residues in P3 or P1 position by non-canonical (S)- and (R)-α-Trifluoromethyl Alanine (TfmAla), (S)- and (R)-Trifluoromethionine (TFM) or non-natural d-Valine. The biological activities of those analogues were quantified on isolated CD and Pepsin by fluorescence-based assay (FRET) and cytotoxicity of the best fluorinated inhibitors was evaluated on SKOV3 ovarian cancer cell line. (R)-TFM based analog of Pepstatin A (compound 6) returned a sub-nanomolar IC50 against CD and an increased selectivity. Molecular Docking experiments could partially rationalize these results. Stabilized inhibitor 6 in the catalytic pocket of CD showed strong hydrophobic interactions of the long and flexible TFM side chain with lipophilic residues of S1 and S3 sub-pockets of the catalytic pocket. The newly synthesized inhibitors returned no cytotoxicity at IC50 concentrations on SKOV3 cancer cells, however the compounds derived from (S)-TfmAla and (R)-TFM led to modifications of cells morphologies, associated with altered organization of F-actin and extracellular Fibronectin.
Assuntos
Catepsina D , Metionina/análogos & derivados , Pepsina A , Pepstatinas/farmacologia , Pepstatinas/química , Simulação de Acoplamento Molecular , AlaninaRESUMO
Pepsin, because of its optimal activity at low acidic pH, has gained importance in mass spectrometric proteome research as a readily available and easy-to-handle protease. Pepsin has also been study object of protein higher-order structure analyses, but questions about how to best investigate pepsin in-solution conformers still remain. We first determined dependencies of pepsin ion charge structures on solvent pH which indicated the in-solution existence of (a) natively folded pepsin (N) which by nanoESI-MS analysis gave rise to a narrow charge state distribution with an 11-fold protonated most intense ion signal, (b) unfolded pepsin (U) with a rather broad ion charge state distribution whose highest ion signal carried 25 protons, and (c) a compactly folded pepsin conformer (C) with a narrow charge structure and a 12-fold protonated ion signal in the center of its charge state envelope. Because pepsin is a protease, unfolded pepsin became its own substrate in solution at pH 6.6 since at this pH some portion of pepsin maintained a compact/native fold which displayed enzymatic activity. Subsequent mass spectrometric ITEM-TWO analyses of pepstatin A - pepsin complex dissociation reactions in the gas phase confirmed a very strong binding of pepstatin A by natively folded pepsin (N). ITEM-TWO further revealed the existence of two compactly folded in-solution pepsin conformers (Ca and Cb) which also were able to bind pepstatin A. Binding strengths of the respective compactly folded pepsin conformer-containing complexes could be determined and apparent gas phase complex dissociation constants and reaction enthalpies differentiated these from each other and from the pepstatin A - pepsin complex which had been formed from natively folded pepsin. Thus, ITEM-TWO turned out to be well suited to pinpoint in-solution pepsin conformers by interrogating quantitative traits of pepstatin A - pepsin complexes in the gas phase.
Assuntos
Pepsina A , Espectrometria de Massas por Ionização por Electrospray , Pepsina A/química , Pepsina A/metabolismo , Pepstatinas/química , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Malaria is a devastating disease, and its management is only achieved through chemotherapy. However, resistance to available medication is still a challenge; therefore, there is an urgent need for the discovery and development of therapeutics with a novel mechanism of action to counter the resistance scourge consistent with the currently available antimalarials. Recently, plasmepsin V was validated as a therapeutic target for the treatment of malaria. The pepsin-like aspartic protease anchored in the endoplasmic reticulum is responsible for the trafficking of parasite-derived proteins to the erythrocytic surface of the host cells. In this study, a small library of compounds was preliminarily screened in vitro to identify novel modulators of Plasmodium falciparum plasmepsin V (PfPMV). The results obtained revealed kaempferol, quercetin, and shikonin as possible PfPMV inhibitors, and these compounds were subsequently probed for their inhibitory potentials using in vitro and in silico methods. Kaempferol and shikonin noncompetitively and competitively inhibited the specific activity of PfPMV in vitro with IC50 values of 22.4 and 43.34 µM, respectively, relative to 62.6 µM obtained for pepstatin, a known aspartic protease inhibitor. Further insight into the structure-activity relationship of the compounds through a 100 ns molecular dynamic (MD) simulation showed that all the test compounds had a significant affinity for PfPMV, with quercetin (-36.56 kcal/mol) being the most prominent metabolite displaying comparable activity to pepstatin (-35.72 kcal/mol). This observation was further supported by the compactness and flexibility of the resulting complexes where the compounds do not compromise the structural integrity of PfPMV but rather stabilized and interacted with the active site amino acid residues critical to PfPMV modulation. Considering the findings in this study, quercetin, kaempferol, and shikonin could be proposed as novel aspartic protease inhibitors worthy of further investigation in the treatment of malaria.
Assuntos
Quempferóis , Plasmodium falciparum , Quempferóis/farmacologia , Pepstatinas , Quercetina/farmacologiaRESUMO
Aspartic proteases are a small class of proteases implicated in a wide variety of human diseases. Covalent chemical probes for photoaffinity labeling (PAL) of these proteases are underdeveloped. We here report a full on-resin synthesis of clickable PAL probes based on the natural product inhibitor pepstatin incorporating a minimal diazirine reactive group. The position of this group in the inhibitor determines the labeling efficiency. The most effective probes sensitively detect cathepsin D, a biomarker for breast cancer, in cell lysates. Moreover, through chemical proteomics experiments and deep learning algorithms, we identified sequestosome-1, an important player in autophagy, as a direct interaction partner and substrate of cathepsin D.
Assuntos
Ácido Aspártico Endopeptidases , Catepsina D , Pepstatinas , Marcadores de Fotoafinidade , Humanos , Ácido Aspártico Endopeptidases/química , Catepsina D/química , Diazometano , Pepstatinas/química , Pepstatinas/farmacologia , Marcadores de Fotoafinidade/química , Proteína Sequestossoma-1/químicaRESUMO
A new pepstatin with a phenylacetyl group, pepstatin Pa (1), and its methyl ester (2) were isolated from Streptomyces varsoviensis DSM 40346. Their structures were determined by high-resolution mass spectrometry and nuclear magnetic resonance techniques. The absolute configuration was determined using the Marfey's method. Both pentapeptide products are inhibitors of pepsin and cathepsin D. Interestingly, the bacterial genome contains no biosynthetic gene cluster for the new pepstatin, suggesting an extrachromosomal origin of the biosynthetic genes.
Assuntos
Ácido Aspártico Proteases , Pepstatinas , Streptomyces , Ácido Aspártico Proteases/antagonistas & inibidores , Proteínas de Bactérias , Pepstatinas/farmacologia , Inibidores de Proteases , Streptomyces/químicaRESUMO
Aspergillus awamori was cultivated in a modified Breccia medium, and the extracellular fraction was obtained, which presented 260 ± 15 µg of protein/mg and specific protease activity of 3.87 ± 0.52 mM.min-1.mg of protein-1 using Nα-p-tosyl-L-arginine methyl ester hydrochloride (L-TAME) as substrate. This fraction showed major proteins about 104 and 44 kDa and maximal protease activity at pH 5.5, 6.5, and 9.0, suggesting that A. awamori secretes acidic, neutral, and alkaline proteases with expressive thermal stability, however, aspartic protease was the most important activity. When yeast extract was supplemented to a modified Breccia medium, A. awamori protein secretion and protease activity were maximal and the affinity chromatography on pepstatin-agarose was employed to isolate the aspartic protease activity, which was called ASPA, with approximately 75 kDa. ASPA maximal activity was obtained at pH 4.5 and 6.5, and 50 °C. Pepstatin inhibited about 80% of ASPA activity, with IC50 and Ki values of 0.154 and 0.072 µM, respectively. ASPA cleaved protein and peptides substrates with the highest activity against gelatin (95 U/mg) and good peptidase activity with KM 0.0589 mM and Vmax 1.909 mM.min-1.mg protein-1, using L-TAME as substrate. A. awamori extracellular fraction is a source of proteases with important activity, and the supplementation of modified Breccia medium increased the aspartic protease production. This enzyme presented different biochemical characteristics from the previously reported A. awamori aspartic proteases. Therefore, ASPA is an excellent candidate for biotechnological application due to its important activity and thermostability.
Assuntos
Ácido Aspártico Proteases , Ácido Aspártico Proteases/química , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/metabolismo , Aspergillus/metabolismo , Concentração de Íons de Hidrogênio , Pepstatinas/metabolismo , Peptídeo HidrolasesRESUMO
Aspartic proteases are the targets for structure-based drug design for their role in physiological processes and pharmaceutical applications. Structural insights into the thermal inactivation mechanism of an aspartic protease in presence and absence of bound pepstatin A have been obtained by kinetics of thermal inactivation, CD, fluorescence spectroscopy and molecular dynamic simulations. The irreversible thermal inactivation of the aspartic protease comprised of loss of tertiary and secondary structures succeeded by the loss of activity, autolysis and aggregation The enthalpy and entropy of thermal inactivation of the enzyme in presence of pepstatin A increased from 81.2 to 148.5 kcal mol-1, and from 179 to 359 kcal mol-1 K-1 respectively. Pepstatin A shifted the mid-point of thermal inactivation of the protease from 58 °C to 77 °C. The association constant (K) for pepstatin A with aspartic protease was 2.5 ± 0.3 × 10 5 M-1 and ΔGo value was -8.3 kcal mol-1. Molecular dynamic simulation studies were able to delineate the role of pepstatin A in stabilizing backbone conformation and side chain interactions. In the Cα-backbone, the short helical segments and the conserved glycines were part of the most unstable segments of the protein. Understanding the mechanism of thermal inactivation has the potential to develop re-engineered thermostable proteases.
Assuntos
Ácido Aspártico Proteases , Aspergillus niger/enzimologia , Proteínas Fúngicas , Pepstatinas/química , Ácido Aspártico Proteases/antagonistas & inibidores , Ácido Aspártico Proteases/química , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Concentração de Íons de Hidrogênio , Ligação Proteica , Espectrometria de FluorescênciaRESUMO
Mason-Pfizer monkey virus protease (PR) was crystallized in complex with two pepstatin-based inhibitors in P1 space group. In both crystal structures, the extended flap loops that lock the inhibitor/substrate over the active site, are visible in the electron density either completely or with only small gaps, providing the first observation of the conformation of the flap loops in dimeric complex form of this retropepsin. The H-bond network in the active site (with D26N mutation) differs from that reported for the P21 crystal structures and is similar to a rarely occurring system in HIV-1 PR.
Assuntos
Vírus dos Macacos de Mason-Pfizer/enzimologia , Pepstatinas/química , Peptídeo Hidrolases/química , Inibidores de Proteases/química , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Substituição de Aminoácidos , Vírus dos Macacos de Mason-Pfizer/genética , Mutação de Sentido Incorreto , Peptídeo Hidrolases/genética , Estrutura Secundária de Proteína , Proteínas Virais/genéticaRESUMO
A clinically-relevant, drug-resistant mutant of HIV-1 protease (PR), termed Flap+(I54V) and containing L10I, G48V, I54V and V82A mutations, is known to produce significant changes in the entropy and enthalpy balance of drug-PR interactions, compared to wild-type PR. A similar mutant, Flap+(I54A) , which evolves from Flap+(I54V) and contains the single change at residue 54 relative to Flap+(I54V) , does not. Yet, how Flap+(I54A) behaves in solution is not known. To understand the molecular basis of V54A evolution, we compared nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and enzymatic assay data from four PR proteins: PR (pWT), Flap+(I54V) , Flap+(I54A) , and Flap+(I54) , a control mutant that contains only L10I, G48V and V82A mutations. Our data consistently show that selection to the smaller side chain at residue 54, not only decreases inhibitor affinity, but also restores the catalytic activity.
Assuntos
Farmacorresistência Viral/genética , Inibidores da Protease de HIV/metabolismo , Protease de HIV , Calorimetria , Protease de HIV/química , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/química , Modelos Moleculares , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Pepstatinas/química , Pepstatinas/metabolismo , Ligação Proteica , TermodinâmicaRESUMO
The opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera) are notable for their intrinsic resistance to different antifungal classes. Little is known about the virulence attributes in this emerging fungal complex. However, it is well-recognized that enzymes play important roles in virulence/pathogenesis of candidiasis. Herein, we aimed to identify aspartyl-type peptidases in 12 clinical isolates belonging to the C. haemulonii complex. All isolates were able to grow in a chemically defined medium containing albumin as the sole nitrogen source, and a considerable consumption of this protein occurred after 72-96 h. C. haemulonii var. vulnera isolates showed the lowest albumin degradation capability and the poorest growth rate. The measurement of secreted aspartyl peptidase (Sap) activity, using the cathepsin D fluorogenic substrate, varied from 91.6 to 413.3 arbitrary units and the classic aspartyl peptidase inhibitor, pepstatin A, significantly blocked the Sap released by C. haemulonii complex. No differences were observed in the Sap activity among the three fungal species. Flow cytometry, using a polyclonal antibody against Sap1-3 of C. albicans, detected homologous proteins at the surface of C. haemulonii complex (anti-Sap1-3-labeled cells ranged from 24.6 to 79.1%). Additionally, the immunoblotting assay, conducted with the same Sap1-3 antibody, recognized a protein of â¼50 kDa in all fungal isolates. A glimpse in the genome of these fungi revealed several potential proteins containing Sap1-3-like conserved domain. Altogether, our results demonstrated the potential of C. haemulonii species complex to produce Saps, an important virulence factor of Candida spp.
Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candida/enzimologia , Candidíase/microbiologia , Dipeptidases/metabolismo , Candida/classificação , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Resistência a Múltiplos Medicamentos , Humanos , Pepstatinas/farmacologia , Inibidores de Proteases/farmacologia , Análise de Sequência de ProteínaRESUMO
The human retroviral-like aspartic protease 1 (ASPRV1) is a mammalian retroviral-like enzyme that catalyzes a critical proteolytic step during epidermal differentiation; therefore, it is also referred to as skin-specific aspartic protease (SASPase). Neutrophil granulocytes were also found recently to express ASPRV1 that is involved in the progression of acute chronic inflammation of the central nervous system, especially in autoimmune encephalomyelitis. Thus, investigation of ASPRV1 is important due to its therapeutic or diagnostic potential. We investigated the structural characteristics of ASPRV1 by homology modeling; analysis of the proposed structure was used for interpretation of in vitro specificity studies. For in-vitro characterization, activities of SASP28 and SASP14 enzyme forms were measured using synthetic oligopeptide substrates. We demonstrated that self-processing of SASP28 precursor causes autoactivation of the protease. The highest activity was measured for GST-SASP14 at neutral pH and at high ionic strength, and we proved that pepstatin A and acetyl-pepstatin can also inhibit the protease. In agreement with the structural characteristics, the relatively lower urea dissociation constant implied lower dimer stability of SASP14 compared to that of HIV-1 protease. The obtained structural and biochemical characteristics support better understanding of ASPRV1 function in the skin and central nervous system.
Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Mutação , Neutrófilos/metabolismo , Ácido Aspártico Endopeptidases/genética , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Pepstatinas/farmacologia , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Homologia Estrutural de ProteínaRESUMO
Serine proteases are thought to play a key role in the muscle softening of gazami crab (Portunus trituberculatus) during storage. A serine protease, Pt-sp2, was purified from the hepatopancreas of gazami crab using ammonium sulfate precipitation, anion-exchange and gel filtration chromatography, and was analyzed by mass spectrometry, transcriptome and bioinformatics. It revealed that Pt-sp2 was trypsin-like, with no 100% identical proteins in the NCBI database. The molecular weight of Pt-sp2 was approximately 37.2 kDa. Its optimum pH and temperature were 9.0 and 50 °C, respectively, using t-Butyloxycarbonyl-Phe-Ser-Arg-4-methyl-coumaryl-7-amide as a substrate. Pt-sp2 was activated in the presence of Ca2+. Both soybean trypsin inhibitor and Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride completely suppressed Pt-sp2 activity, while it was only partially inhibited by phenylmethylsulfonyl fluoride and EDTA. However, PMSF, Pepstatin A and cystatin inhibitor E-64 showed no inhibition on Pt-sp2 protease activity. The Km value of Pt-sp2 was 0.82 µM, and Pt-sp2 effectively hydrolyzed myofibrillar protein at 37 °C.
Assuntos
Braquiúros/enzimologia , Hepatopâncreas/enzimologia , Leucina/análogos & derivados , Proteínas Musculares/metabolismo , Pepstatinas/metabolismo , Serina Endopeptidases/metabolismo , Animais , Inibidores de Cisteína Proteinase/farmacologia , Leucina/farmacologia , Inibidores de Proteases/farmacologiaRESUMO
Human cathepsin D (CatD), a pepsin-family aspartic protease, plays an important role in tumor progression and metastasis. Here, we report the development of biomimetic inhibitors of CatD as novel tools for regulation of this therapeutic target. We designed a macrocyclic scaffold to mimic the spatial conformation of the minimal pseudo-dipeptide binding motif of pepstatin A, a microbial oligopeptide inhibitor, in the CatD active site. A library of more than 30 macrocyclic peptidomimetic inhibitors was employed for scaffold optimization, mapping of subsite interactions, and profiling of inhibitor selectivity. Furthermore, we solved high-resolution crystal structures of three macrocyclic inhibitors with low nanomolar or subnanomolar potency in complex with CatD and determined their binding mode using quantum chemical calculations. The study provides a new structural template and functional profile that can be exploited for design of potential chemotherapeutics that specifically inhibit CatD and related aspartic proteases.
Assuntos
Catepsina D/antagonistas & inibidores , Catepsina D/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Sítios de Ligação , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Materiais Biomiméticos/toxicidade , Células CACO-2 , Catepsina D/química , Ensaios Enzimáticos , Humanos , Cinética , Estrutura Molecular , Pepstatinas/química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/toxicidade , Inibidores de Proteases/síntese química , Inibidores de Proteases/toxicidade , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
While exploring new angiogenesis inhibitors from microbial metabolites, we recently isolated ahpatinins C, E, and G from a soilderived Streptomyces sp. 15JA150. Ahpatinins C, E and G are known to have pepsin and renin inhibitory activities; however, their antiangiogenic activities and underlying molecular mechanisms have not been fully elucidated. In the present study, the antiangiogenic properties of ahpatinins C, E and G were investigated. The results revealed that the natural compounds significantly inhibited the vascular endothelial growth factor (VEGF)induced proliferation, invasion, adhesion, and tube formation of human umbilical vein endothelial cells (HUVECs) without exhibiting any cytotoxicity. It was also revealed that ahpatinin E effectively suppressed the neovascularization of the chorioallantoic membranes in growing chick embryos. Notably, ahpatinins C, E, and G led to the downregulation of VEGFinduced activation of VEGF receptor 2 (VEGFR2) and its downstream signaling mediators, including AKT, ERK1/2, JNK, p38, and NFκB, in HUVECs. Moreover, they reduced the expression of matrix metalloproteinase (MMP)2 and MMP9 in the HUVECs following stimulation with VEGF. Furthermore, ahpatinins C, E, and G reduced the tumor cellinduced invasion and tube forming abilities of HUVECs, as well as the expression of VEGF, by suppressing hypoxiainducible factor1α (HIF1α) activity in U87MG glioblastoma cells. Collectively, the present findings indicated that ahpatinins C, E, and G may be used in anticancer therapy by targeting tumor angiogenesis through the inhibition of both VEGFR2 and HIF1α pathways.
Assuntos
Inibidores da Angiogênese/farmacologia , Fatores Biológicos/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Streptomyces/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Adesão Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pepstatinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Surgical resection of the epileptogenic region is typically regarded to be practical and efficient for complete elimination of intractable seizures, which cannot be simply controlled by anti-epileptic drugs alone. To achieve a precision removal of the epileptogenic region and even a surgical cure, molecular imaging of epilepsy markers is highly essential for non-invasive accurate detection of the epileptogenic region. In this work, a peptide-targeted nanoprobe, based on ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs), PA-USPIONs, was elaborately constructed to enable highly selective delivery and sensitive T1-weighted positive magnetic resonance (MR) imaging of the epileptogenic region. Especially, Pepstatin A (PA), a small peptide which can specifically target to P-glycoprotein (P-gp) overexpressed at the epileptogenic region in a kainic acid (KA)-induced mice model of seizures, was conjugated onto the surface of PEGylated USPIONs. It has been demonstrated that the as-constructed PA-USPIONs nanoprobes have favorable T1 contrast enhancement and high r1 relaxivity compared with the clinically used T1-MR contrast agent (Gd-DTPA) by systematic in vitro and vivo assessments. Importantly, the toxicity evaluation, especially to brains, was assessed by the histological as well as hematological examinations, demonstrating that the fabricated PA-USPIONs nanoprobes are featured with excellent biocompatibility, guaranteeing the further potential clinical application. This first report on the development of USPIONs as T1-weighted MR contrast agents for active targeting of the epileptogenic region holds the high potential for precise resection of the according lesion in order to achieve therapeutic, often curative purposes.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Epilepsia , Nanopartículas Magnéticas de Óxido de Ferro , Nanopartículas de Magnetita , Pepstatinas , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Meios de Contraste , Epilepsia/diagnóstico por imagem , Epilepsia/genética , Imageamento por Ressonância Magnética , CamundongosRESUMO
In the pursuit of industrial aspartic proteases, aspergillopepsin A-like endopeptidase from the fungi Aspergillus niger, was identified and cultured by solid state fermentation. Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50⯱â¯0.5â¯kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60⯰C respectively. The enzyme was stable for 60â¯min at 50⯰C. The purified enzyme had specific activity of 40,000⯱â¯1800â¯U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045⯵M. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in ß-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin > defatted soya flour > gluten > gelatin > skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. This aspartic protease was found to be an excellent candidate for genetic manipulation for biotechnological application in food and feed industries, due to its high catalytic turn over number and thermostability.
Assuntos
Ácido Aspártico Proteases/química , Aspergillus niger/enzimologia , Pepstatinas/química , Inibidores de Proteases/química , Ácido Aspártico Proteases/antagonistas & inibidores , Ácido Aspártico Proteases/isolamento & purificação , Ácido Aspártico Proteases/metabolismo , Aspergillus niger/classificação , Aspergillus niger/genética , Catálise , Cromatografia Líquida , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Pepstatinas/farmacologia , Filogenia , Inibidores de Proteases/farmacologia , Ligação Proteica , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem , TemperaturaRESUMO
Napsin A is an intracellular aspartic protease and biomarker of various malignancies like lung adenocarcinoma and ovarian clear cell carcinoma, but its detection is usually limited to immunohistochemical techniques gaining excellent information on its distribution but missing information about posttranslational modifications (e.g. maturation state) of the protein. We present a protocol for specific enrichment of napsin A from clinical or biological specimens, that facilitates detailed analysis of the protein. By using the exceptionally broad pH range under which napsin A binds to its inhibitor pepstatin A we achieve highly selective binding of napsin A while other aspartic proteases have negligible affinity. Using this method we demonstrate that lung napsin A in many mammals is a heterogeneous enzyme with a characteristic ladder-like appearance in SDS-PAGE that might be caused by proteolytically processed N- and/or C-termini, in contrast to the more homogeneous form found in kidneys and primary lung adenocarcinoma.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Pulmão/metabolismo , Pepstatinas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Ácido Aspártico Endopeptidases/análise , Ácido Aspártico Endopeptidases/genética , Western Blotting , Bovinos , Cobaias , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Pepstatinas/genética , Ligação Proteica , Coelhos , Ratos , Ovinos , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Triple-negative breast cancers (TNBCs) are more aggressive than other breast cancer (BC) subtypes and lack effective therapeutic options. Unraveling marker events of TNBCs may provide new directions for development of strategies for targeted TNBC therapy. Herein, we reported that Annexin A1 (AnxA1) and Cathepsin D (CatD) are highly expressed in MDA-MB-231 (TNBC lineage), compared to MCF-10A and MCF-7. Since the proposed concept was that CatD has protumorigenic activity associated with its ability to cleave AnxA1 (generating a 35.5 KDa fragment), we investigated this mechanism more deeply using the inhibitor of CatD, Pepstatin A (PepA). Fourier Transform Infrared (FTIR) spectroscopy demonstrated that PepA inhibits CatD activity by occupying its active site; the OH bond from PepA interacts with a CO bond from carboxylic acids of CatD catalytic aspartate dyad, favoring the deprotonation of Asp33 and consequently inhibiting CatD. Treatment of MDA-MB-231 cells with PepA induced apoptosis and autophagy processes while reducing the proliferation, invasion, and migration. Finally, in silico molecular docking demonstrated that the catalytic inhibition comprises Asp231 protonated and Asp33 deprotonated, proving all functional results obtained. Our findings elucidated critical CatD activity in TNBC cell trough AnxA1 cleavage, indicating the inhibition of CatD as a possible strategy for TNBC treatment.
Assuntos
Anexina A1/genética , Catepsina D/genética , Simulação de Acoplamento Molecular , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Catepsina D/antagonistas & inibidores , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Pepstatinas/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin. These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation.