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1.
J Mol Biol ; 435(6): 168012, 2023 03 15.
Article in English | MEDLINE | ID: mdl-36792007

ABSTRACT

The Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is pathogenic to both humans and equines. The VEEV non-structural protein 2 (nsP2) is a cysteine protease (nsP2pro) that processes the polyprotein and thus it is a drug target for inhibitor discovery. The atomic structure of the VEEV nsP2 catalytic domain was previously characterized by both X-ray crystallography and computational studies. A modified nsP2pro harboring a N475A mutation in the N terminus was observed to exhibit an unexpected conformation: the N-terminal residues bind to the active site, mimicking binding of a substrate. The large conformational change of the N terminus was assumed to be induced by the N475A mutation, as N475 has an important role in stabilization of the N terminus and the active site. This conformation was first observed in the N475A mutant, but we also found it while determining a crystal structure of the catalytically active nsP2pro containing the wild-type N475 active site residue and K741A/K767A surface entropy reduction mutations. This suggests that the N475A mutation is not a prerequisite for self-inhibition. Here, we describe a high resolution (1.46 Å) crystal structure of a truncated nsP2pro (residues 463-785, K741A/K767A) and analyze the structure further by molecular dynamics to study the active and self-inhibited conformations of nsP2pro and its N475A mutant. A comparison of the different conformations of the N-terminal residues sheds a light on the interactions that play an important role in the stabilization of the enzyme.


Subject(s)
Catalytic Domain , Cysteine Proteases , Encephalitis Virus, Venezuelan Equine , Animals , Humans , Crystallography, X-Ray , Cysteine Proteases/chemistry , Cysteine Proteases/genetics , Encephalitis Virus, Venezuelan Equine/enzymology , Horses , Molecular Dynamics Simulation
2.
Biochimie ; 135: 72-81, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28115185

ABSTRACT

Human plasma kallikrein (huPK) potentiates platelet responses to subthreshold doses of ADP, although huPK itself, does not induce platelet aggregation. In the present investigation, we observe that huPK pretreatment of platelets potentiates ADP-induced platelet activation by prior proteolysis of the G-protein-coupled receptor PAR-1. The potentiation of ADP-induced platelet activation by huPK is mediated by the integrin αIIbß3 through interactions with the KGD/KGE sequence motif in huPK. Integrin αIIbß3 is a cofactor for huPK binding to platelets to support PAR-1 hydrolysis that contributes to activation of the ADP signaling pathway. This activation pathway leads to phosphorylation of Src, AktS473, ERK1/2, and p38 MAPK, and to Ca2+ release. The effect of huPK is blocked by specific antagonists of PAR-1 (SCH 19197) and αIIbß3 (abciximab) and by synthetic peptides comprising the KGD and KGE sequence motifs of huPK. Further, recombinant plasma kallikrein inhibitor, rBbKI, also blocks this entire mechanism. These results suggest a new function for huPK. Formation of plasma kallikrein lowers the threshold for ADP-induced platelet activation. The present observations are consistent with the notion that plasma kallikrein promotes vascular disease and thrombosis in the intravascular compartment and its inhibition may ameliorate cardiovascular disease and thrombosis.


Subject(s)
Adenosine Diphosphate/pharmacology , Plasma Kallikrein/pharmacology , Platelet Aggregation/drug effects , Humans , Phosphorylation/drug effects , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Receptor, PAR-1/metabolism , Signal Transduction/drug effects
3.
J Agric Food Chem ; 63(48): 10431-6, 2015 Dec 09.
Article in English | MEDLINE | ID: mdl-26568149

ABSTRACT

Callosobruchus maculatus is an important predator of cowpeas. Due to infestation during storage, this insect affects the quality of seed and crop yield. This study aimed to investigate the effects of CrataBL, a multifunction protein isolated from Crataeva tapia bark, on C. maculatus larva development. The protein, which is stable even in extreme pH conditions, showed toxic activity, reducing the larval mass 45 and 70% at concentrations of 0.25 and 1.0% (w/w), respectively. Acting as an inhibitor, CrataBL decreased by 39% the activity of cysteine proteinases from larval gut. Conversely, the activity of serine proteinases was increased about 8-fold. The toxic properties of CrataBL may also be attributed to its capacity of binding to glycoproteins or glycosaminoglycans. Such binding interferes with larval metabolism, because CrataBL-FITC was found in the fat body, Malpighian tubules, and feces of larvae. These results demonstrate the potential of this protein for controlling larva development.


Subject(s)
Capparaceae/chemistry , Coleoptera/drug effects , Larva/growth & development , Lectins/pharmacology , Plant Bark/chemistry , Plant Extracts/pharmacology , Animals , Coleoptera/enzymology , Coleoptera/growth & development , Cysteine Proteases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Insect Proteins/metabolism , Larva/drug effects , Larva/enzymology
4.
PLoS One ; 8(6): e64426, 2013.
Article in English | MEDLINE | ID: mdl-23823708

ABSTRACT

A protein isolated from the bark of Crataeva tapia (CrataBL) is both a Kunitz-type plant protease inhibitor and a lectin. We have determined the amino acid sequence and three-dimensional structure of CrataBL, as well as characterized its selected biochemical and biological properties. We found two different isoforms of CrataBL isolated from the original source, differing in positions 31 (Pro/Leu); 92 (Ser/Leu); 93 (Ile/Thr); 95 (Arg/Gly) and 97 (Leu/Ser). CrataBL showed relatively weak inhibitory activity against trypsin (Kiapp = 43 µM) and was more potent against Factor Xa (Kiapp = 8.6 µM), but was not active against a number of other proteases. We have confirmed that CrataBL contains two glycosylation sites and forms a dimer at high concentration. The high-resolution crystal structures of two different crystal forms of isoform II verified the ß-trefoil fold of CrataBL and have shown the presence of dimers consisting of two almost identical molecules making extensive contacts (∼645 Å(2)). The structure differs from those of the most closely related proteins by the lack of the N-terminal ß-hairpin. In experiments aimed at investigating the biological properties of CrataBL, we have shown that addition of 40 µM of the protein for 48 h caused maximum growth inhibition in MTT assay (47% of DU145 cells and 43% of PC3 cells). The apoptosis of DU145 and PC3 cell lines was confirmed by flow cytometry using Annexin V/FITC and propidium iodide staining. Treatment with CrataBL resulted in the release of mitochondrial cytochrome c and in the activation of caspase-3 in DU145 and PC3 cells.


Subject(s)
Capparaceae/chemistry , Plant Lectins/pharmacology , Amino Acid Sequence , Cell Death/drug effects , Cell Line, Tumor , Crystallography, X-Ray , Dimerization , Drug Screening Assays, Antitumor , Glycosylation , Humans , Male , Molecular Structure , Plant Lectins/chemistry , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
5.
J Mol Biol ; 382(3): 763-78, 2008 Oct 10.
Article in English | MEDLINE | ID: mdl-18675276

ABSTRACT

The crystal structures of an aspartic proteinase from Trichoderma reesei (TrAsP) and of its complex with a competitive inhibitor, pepstatin A, were solved and refined to crystallographic R-factors of 17.9% (R(free)=21.2%) at 1.70 A resolution and 15.8% (R(free)=19.2%) at 1.85 A resolution, respectively. The three-dimensional structure of TrAsP is similar to structures of other members of the pepsin-like family of aspartic proteinases. Each molecule is folded in a predominantly beta-sheet bilobal structure with the N-terminal and C-terminal domains of about the same size. Structural comparison of the native structure and the TrAsP-pepstatin complex reveals that the enzyme undergoes an induced-fit, rigid-body movement upon inhibitor binding, with the N-terminal and C-terminal lobes tightly enclosing the inhibitor. Upon recognition and binding of pepstatin A, amino acid residues of the enzyme active site form a number of short hydrogen bonds to the inhibitor that may play an important role in the mechanism of catalysis and inhibition. The structures of TrAsP were used as a template for performing statistical coupling analysis of the aspartic protease family. This approach permitted, for the first time, the identification of a network of structurally linked residues putatively mediating conformational changes relevant to the function of this family of enzymes. Statistical coupling analysis reveals coevolved continuous clusters of amino acid residues that extend from the active site into the hydrophobic cores of each of the two domains and include amino acid residues from the flap regions, highlighting the importance of these parts of the protein for its enzymatic activity.


Subject(s)
Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/chemistry , Fungal Proteins/chemistry , Pepstatins/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Trichoderma/enzymology , Animals , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Binding Sites , Cluster Analysis , Crystallography, X-Ray , Fungal Proteins/genetics , Fungal Proteins/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Pepstatins/genetics , Pepstatins/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protein Binding
6.
J Mol Biol ; 369(4): 1029-40, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17467738

ABSTRACT

Although a majority of HIV-1 infections in Brazil are caused by the subtype B virus (also prevalent in the United States and Western Europe), viral subtypes F and C are also found very frequently. Genomic differences between the subtypes give rise to sequence variations in the encoded proteins, including the HIV-1 protease. The current anti-HIV drugs have been developed primarily against subtype B and the effects arising from the combination of drug-resistance mutations with the naturally existing polymorphisms in non-B HIV-1 subtypes are only beginning to be elucidated. To gain more insights into the structure and function of different variants of HIV proteases, we have determined a 2.1 A structure of the native subtype F HIV-1 protease (PR) in complex with the protease inhibitor TL-3. We have also solved crystal structures of two multi-drug resistant mutant HIV PRs in complex with TL-3, from subtype B (Bmut) carrying the primary mutations V82A and L90M, and from subtype F (Fmut) carrying the primary mutation V82A plus the secondary mutation M36I, at 1.75 A and 2.8 A resolution, respectively. The proteases Bmut, Fwt and Fmut exhibit sevenfold, threefold, and 54-fold resistance to TL-3, respectively. In addition, the structure of subtype B wild type HIV-PR in complex with TL-3 has been redetermined in space group P6(1), consistent with the other three structures. Our results show that the primary mutation V82A causes the known effect of collapsing the S1/S1' pockets that ultimately lead to the reduced inhibitory effect of TL-3. Our results further indicate that two naturally occurring polymorphic substitutions in subtype F and other non-B HIV proteases, M36I and L89M, may lead to early development of drug resistance in patients infected with non-B HIV subtypes.


Subject(s)
Drug Resistance, Viral , HIV Protease/chemistry , Isoenzymes/chemistry , Amino Acid Sequence , Crystallography, X-Ray , HIV Protease/genetics , HIV Protease/metabolism , HIV Protease Inhibitors/metabolism , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Models, Molecular , Molecular Sequence Data , Mutation , Polymorphism, Genetic , Sequence Alignment
7.
J Exp Bot ; 58(3): 521-32, 2007.
Article in English | MEDLINE | ID: mdl-17158110

ABSTRACT

An alkaline proteinase activity is present in quiescent seeds and up to the 24th day of development of Canavalia ensiformis DC (L.) plants. By a simple protocol consisting of cation exchange chromatography, followed by an anion exchange column, a serine proteinase (Q-SP) was purified to homogeneity from quiescent seeds. Q-SP consists of a 33 kDa chain with an optimum pH between 8.0 and 9.0. Arginine residues at P1 and P2 subsites favour binding to the substrate, as shown by the KM assay with N-alpha-benzoyl-DL-arginine-4-nitroanilide-hydrochloride and N-benzoylcarboxyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin. The same protocol was used for partial purification of benzamidine-sensitive enzymes from the developing plant. On the 7th day, a new benzamidine-sensitive enzyme is synthesized in the seedling, seen as the second active peak appearing in anion exchange chromatography. A benzamidine-sensitive enzyme purified from cotyledons presented a similar gel filtration profile as Q-SP, although it was eluted at different salt concentrations in the anion exchange chromatography. None of the enzymes was inhibited by PMSF, APMSF, or SBTI, but they were inactivated by benzamidine, TLCK, and leupeptin. Q-SP did not cleave in vitro C. ensiformis urease, concanavalin A, or its main storage protein, canavalin. In conclusion, a ubiquitous benzamidine-sensitive proteolytic activity was found in C. ensiformis from quiescent seeds up to 24 d of growth, which apparently is not involved in the hydrolysis of storage proteins and might participate in an as yet unidentified limited proteolysis event.


Subject(s)
Canavalia/enzymology , Serine Endopeptidases/metabolism , Benzamidines/pharmacology , Canavalia/drug effects , Canavalia/growth & development , Chromatography , Cotyledon/drug effects , Cotyledon/enzymology , Cotyledon/growth & development , Hydrogen-Ion Concentration , Plant Leaves/drug effects , Plant Leaves/enzymology , Plant Leaves/growth & development , Plant Roots/drug effects , Plant Roots/enzymology , Plant Roots/growth & development , Seeds/drug effects , Seeds/enzymology , Serine Endopeptidases/chemistry , Serine Endopeptidases/isolation & purification , Serine Proteinase Inhibitors/pharmacology
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