RESUMEN
cdk2.cyclin E and cdk5.p25 are two members of the cyclin-dependent kinase family that are potential therapeutic targets for oncology and Alzheimer's disease, respectively. In this study we have investigated the mechanism for these enzymes. Kinases catalyze the transfer of phosphate from ATP to a protein acceptor, thus utilizing two substrates, ATP and the target protein. For a two-substrate reaction, possible kinetic mechanisms include: ping-pong, sequential random, or sequential ordered. To determine the kinetic mechanism of cdk2.GST-cyclin E and cdk5.GST-p25, kinase activity was measured in experiments in which concentrations of peptide and ATP substrates were varied in the presence of dead-end inhibitors. A peptide identical to the peptide substrate, but with a substitution of valine for the phosphoacceptor threonine, competed with substrate with a K(i) value of 0.6 mm. An aminopyrimidine, PNU 112455A, was identified in a screen for inhibitors of cdk2. Nonlinear least squares and Lineweaver-Burk analyses demonstrated that the inhibitor PNU 112455A was competitive with ATP with a K(i) value of 2 microm. In addition, a co-crystal of PNU 112455A with cdk2 showed that the inhibitor binds in the ATP binding pocket of the enzyme. Analysis of the inhibitor data demonstrated that both kinases use a sequential random mechanism, in which either ATP or peptide may bind first to the enzyme active site. For both kinases, the binding of the second substrate was shown to be anticooperative, in that the binding of the first substrate decreases the affinity of the second substrate. For cdk2.GST-cyclin E the kinetic parameters were determined to be K(m, ATP) = 3.6 +/- 1.0 microm, K(m, peptide) = 4.6 +/- 1.4 microm, and the anticooperativity factor, alpha = 130 +/- 44. For cdk5.GST-p25, the K(m, ATP) = 3.2 +/- 0.7 microm, K(m, peptide) = 1.6 +/- 0.3 microm, and alpha = 7.2 +/- 1.8.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Quinasa 2 Dependiente de la Ciclina , Quinasa 5 Dependiente de la Ciclina , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Cinética , Unión Proteica , Conformación Proteica , Pirimidinas/química , Pirimidinas/metabolismo , Especificidad por Sustrato , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMEN
Protein tyrosine phosphatase 1B (PTP1B) attenuates insulin signaling by catalyzing dephosphorylation of insulin receptors (IR) and is an attractive target of potential new drugs for treating the insulin resistance that is central to type II diabetes. Several analogues of cholecystokinin(26)(-)(33) (CCK-8) were found to be surprisingly potent inhibitors of PTP1B, and a common N-terminal tripeptide, N-acetyl-Asp-Tyr(SO(3)H)-Nle-, was shown to be necessary and sufficient for inhibition. This tripeptide was modified to reduce size and peptide character, and to replace the metabolically unstable sulfotyrosyl group. This led to the discovery of a novel phosphotyrosine bioisostere, 2-carboxymethoxybenzoic acid, and to analogues that were >100-fold more potent than the CCK-8 analogues and >10-fold selective for PTP1B over two other PTP enzymes (LAR and SHP-2), a dual specificity phosphatase (cdc25b), and a serine/threonine phosphatase (calcineurin). These inhibitors disrupted the binding of PTP1B to activated IR in vitro and prevented the loss of tyrosine kinase (IRTK) activity that accompanied PTP1B-catalyzed dephosphorylation of IR. Introduction of these poorly cell permeant inhibitors into insulin-treated cells by microinjection (oocytes) or by esterification to more lipophilic proinhibitors (3T3-L1 adipocytes and L6 myocytes) resulted in increased potency, but not efficacy, of insulin. In some instances, PTP1B inhibitors were insulin-mimetic, suggesting that in unstimulated cells PTP1B may suppress basal IRTK activity. X-ray crystallography of PTP1B-inhibitor complexes revealed that binding of an inhibitor incorporating phenyl-O-malonic acid as a phosphotyrosine bioisostere occurred with the mobile WPD loop in the open conformation, while a closely related inhibitor with a 2-carboxymethoxybenzoic acid bioisostere bound with the WPD loop closed, perhaps accounting for its superior potency. These CCK-derived peptidomimetic inhibitors of PTP1B represent a novel template for further development of potent, selective inhibitors, and their cell activity further justifies the selection of PTP1B as a therapeutic target.
Asunto(s)
Inhibidores Enzimáticos/química , Insulina/farmacología , Imitación Molecular , Péptidos/química , Fosfotirosina/química , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Células 3T3 , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células CHO , Células CACO-2 , Cricetinae , Cristalografía por Rayos X , Sinergismo Farmacológico , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Isomerismo , Ratones , Datos de Secuencia Molecular , Péptidos/metabolismo , Péptidos/farmacología , Fosfotirosina/metabolismo , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Sincalida/análogos & derivados , Sincalida/química , Sincalida/metabolismo , Sincalida/farmacología , Soluciones , Xenopus laevisRESUMEN
Glutathione S-transferase (GST)-cdc25B(31-566) induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. Purified, N-terminally truncated forms of cdc25B did not induce GVBD, even though many had phosphatase activity and activated cdc2 in vitro. N-terminally truncated forms of cdc25B inhibited induction of GVBD by longer forms of the enzyme suggesting a direct interaction in vivo. cdc25B(356-556), but not cdc25B(364-529), inhibited GVBD induction by GST-cdc25B(31-566) suggesting that a region of cdc25B near to the C-terminus was responsible for the inhibition. To determine the region of peptide sequence that was inhibitory, cdc25B(356-556) was subjected to proteolysis with endoproteinase lys-C. Following a demonstration that the resulting peptide mixture inhibited GST-cdc25B-dependent GVBD, a series of peptides spanning amino acids at the C-terminus were synthesized. The peptide TRSWAGERSR inhibited GVBD induced by GST-cdc25B. An alanine scan of the peptide revealed residues critical for GVBD inhibition, and site-directed mutagenesis of the corresponding residues in GST-cdc25B(31-566) eliminated its ability to induce GVBD. These results demonstrate that a cdc25B C-terminal domain, involved in dominant-negative inhibition of GVBD-competent cdc25B, is required for induction of GVBD following microinjection into oocytes.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/farmacología , Oocitos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Fosfatasas cdc25/química , Fosfatasas cdc25/farmacología , Secuencia de Aminoácidos , Animales , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Secuencia Conservada/genética , Activación Enzimática/efectos de los fármacos , Metaloendopeptidasas/metabolismo , Microinyecciones , Mutagénesis Sitio-Dirigida/genética , Oocitos/citología , Oocitos/efectos de los fármacos , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Alineación de Secuencia , Eliminación de Secuencia/genética , Xenopus laevis , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismoRESUMEN
Human platelet heparanase has been purified to homogeneity and shown to consist of two, non-covalently associated polypeptide chains of molecular masses 50 and 8 kDa. Protein sequencing provided the basis for determination of the full-length cDNA for this novel protein. Based upon this information and results from protein analysis and mass spectrometry, we propose a scheme to define the structural organization of heparanase in relation to its precursor forms, proheparanase and pre-proheparanase. The 8- and 50-kDa chains which make up the active enzyme reside, respectively, at the NH(2)- and COOH-terminal regions of the inactive precursor, proheparanase. The heparanase heterodimer is produced by excision and loss of an internal linking segment. This paper is the first to suggest that human heparanase is a two-chain enzyme.
Asunto(s)
Precursores Enzimáticos/metabolismo , Glucuronidasa , Glicósido Hidrolasas/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Plaquetas/enzimología , Cromatografía Líquida de Alta Presión , ADN Complementario , Dimerización , Activación Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Humanos , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
We screened a bacteriophage display library of random decapeptides to identify peptide inhibitors of cholesteryl ester transfer protein (CETP). After affinity selection against CETP, bacteriophage-infected Escherichia coli were plated at clonal density and 36 random clones were isolated. Analysis of the relevant portion of the bacteriophage DNA from a group of 12 clones that had a relatively high affinity for CETP revealed that the corresponding amino acid sequences of the displayed peptides exhibited an ... Xaa-Arg-Met-Arg-Tyr-Xaa ... composite motif. Based on those results, decapeptides from this group were synthesized and one of them, DP1 (NH2-VTWRMWYVPA-COOH), inhibited CETP-catalyzed transfer of cholesteryl esters and triglycerides. Amino- and carboxy-terminal truncations of DP1 demonstrated that the original decapeptide could be reduced to a pentapeptide without loss of either its ability to bind to CETP or its ability to inhibit CETP-mediated lipid transfer. That pentapeptide, NH2-WRMWY-COOH (WRMWY, PNU-107368E), binds directly to CETP and its inhibition is consistent with that of a competitive inhibitor of CETP with a Ki of 164 microM. WRMWY or modified versions of this peptide may be useful in studying the interactions between CETP and plasma lipoproteins.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Colifagos/genética , Glicoproteínas , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas Portadoras/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol , Ésteres del Colesterol/metabolismo , Cricetinae , Escherichia coli/virología , Macaca fascicularis , Oligopéptidos/metabolismo , Unión Proteica , Triglicéridos/metabolismoRESUMEN
Solid-phase synthesis of the autoinhibitory domain of calcineurin, CaN A467-491, also produced [aspartimide477]CaN A467-491 and [iso-Asp477]CaN467-491 when Boc-based chemistry was employed. In addition, the truncated peptide CaN A467-488 was obtained when Fmoc-based chemistry was employed. All four peptides proved to be effective inhibitors of protein phosphatase activity of calcineurin. The full-length peptide and the C-terminally truncated peptide (CaN467-488) were indistinguishable, with Ki values of 28 +/- 3 and 31 +/- 5 mu M, respectively. The internally modified peptides, [iso-Asp477]CaN A467-491 and [aspartimide477]-CaN A467-491, possessed lower inhibitory potencies (Ki values of 87 +/- 10 and 55 +/- 3 mu M, respectively).
Asunto(s)
Proteínas de Unión a Calmodulina/química , Inhibidores Enzimáticos/química , Fragmentos de Péptidos/química , Fosfoproteínas Fosfatasas/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Calcineurina , Proteínas de Unión a Calmodulina/antagonistas & inhibidores , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/antagonistas & inhibidoresRESUMEN
Active, recombinant p68 reverse transcriptase (RT) from human immunodeficiency virus type 2 (HIV-2), with an NH2-terminal extension containing a hexahistidine sequence was isolated from extracts of Escherichia coli by immobilized metal affinity chromatography. Treatment of the purified p68/p68 homodimer of HIV-2 RT with recombinant HIV-2 protease generates stable, active heterodimer (p68/p58) that is resistant to further hydrolysis. Analysis of this p68/p58 HIV-2 RT heterodimer revealed that while one subunit is intact p68, the p58 subunit is COOH-terminally truncated by cleavage, not at Phe440 as is seen in processing of the p66/p66 HIV-1 RT homodimer by HIV-1 protease, but at Met484. The expected COOH-terminal p10 fragment resulting from hydrolysis of p68 at Met484 is not released intact, but undergoes further cleavage at Asn494, Met503, and Tyr532. Processing of p68/p68 HIV-2 RT with the HIV-1 protease led to cleavage of the Phe440-Tyr441 bond, exactly as is seen with p66/p66 HIV-1 RT, to give the analogous p53 subunit. Studies of a peptide substrate modeled after residues 437-444 in HIV-2 RT showed that while the HIV-1 protease was able to cleave the Phe440 bond, this bond was resistant to cleavage by the HIV-2 enzyme. Our findings provide a rationale for the previous observation that the RT heterodimer isolated from HIV-2 lysates is larger than that from HIV-1. We conclude that the p68/p58 HIV-2 RT heterodimer, containing the Met484 truncated p58 subunit, is a biologically relevant form of the enzyme in vivo.
Asunto(s)
Proteasa del VIH/metabolismo , VIH-1/enzimología , VIH-2/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Transcriptasa Inversa del VIH , Hidrólisis , Datos de Secuencia Molecular , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , ADN Polimerasa Dirigida por ARN/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two-stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag octapeptide epitope system. We have analyzed two peptides: Ac-Asp-Tyr-Lys-Leu-Gly-Asp-Asp-Leu-NH2 (peptide 1), which contains several non-core positions randomized, and Ac-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Leu-NH2 (peptide 2), which closely corresponds to the original Flag sequence. Enrichment of the peptides with 15N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab-bound peptide were found to be largest for the amide groups of Asp-1 and Asp-6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp-1 and Asp-4, with the other aspartic acid residues also showing significant but smaller changes.
Asunto(s)
Reacciones Antígeno-Anticuerpo , Bacteriófagos/genética , Biblioteca Genómica , Fragmentos Fab de Inmunoglobulinas/química , Péptidos/inmunología , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Distribución AleatoriaRESUMEN
Site-directed mutagenesis of autolysis sites in the human immunodeficiency virus type 1 (HIV-1) protease was applied in an analysis of enzyme specificity; the protease served, therefore, as both enzyme and substrate in this study. Inspection of natural substrates of all retroviral proteases revealed the absence of beta-branched amino acids at the P1 site and of Lys anywhere from P2 through P2'. Accordingly, several mutants of the HIV-1 protease were engineered in which these excluded amino acids were substituted at their respective P positions at the three major sites of autolysis in the wild-type protease (Leu5-Trp6, Leu33-Glu34, and Leu63-Ile64), and the mutant enzymes were evaluated in terms of their resistance to autodegradation. All of the mutant HIV-1 proteases, expressed as inclusion bodies in Escherichia coli, were enzymatically active after refolding, and all showed greatly diminished rates of cleavage at the altered autolysis sites. Some, however, were not viable enzymatically because of poor physical characteristics. This was the case for mutants having Lys replacements of Glu residues at P2' and for another in which all three P1 leucines were replaced by Ile. However, one of the mutant proteases, Q7K/L33I/L63I, was highly resistant to autolysis, while retaining the physical properties, specificity, and susceptibility to inhibition of the wild-type enzyme. Q7K/L33I/L63I should find useful application as a stable surrogate of the HIV-1 protease. Overall, our results can be interpreted relative to a model in which the active HIV-1 protease dimer is in equilibrium with monomeric, disordered species which serve as the substrates for autolysis.
Asunto(s)
Proteasa del VIH/metabolismo , VIH-1/enzimología , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Proteasa del VIH/genética , Inhibidores de la Proteasa del VIH/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligopéptidos/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The protease from simian immunodeficiency virus (SIV) was chemically synthesized by automated solid-phase technology as an NH2-terminally extended derivative, capped with biotin. Biotin-linker-(SIV protease (1-99)): the linker segment, Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala, corresponds to the amino acid sequence preceding that of the protease in the SIV gag/pol precursor polyprotein. Accordingly, the Ala-Pro bond joining the octapeptide linker to the protease constitutes a site naturally cleaved by the protease during viral maturation. This strategy for synthesis was designed to facilitate purification of the biotinylated protein derivative from a complex mixture of reaction products by avidin/agarose-affinity chromatography and to provide the means for autocatalytic removal of the biotin-linker segment. As anticipated, folding of the full-length construct leads to activation of the enzyme and excision of the desired 99-residue SIV protease (overall yield, approximately). The specificity of the synthetic SIV protease toward a number of well characterized protein substrates was the same as observed for the nearly identical enzyme from human immunodeficiency virus type 2 (HIV-2 protease) and distinct from that of the more disparate HIV-1 protease. The same functional ordering with respect to the human retroviral proteases was reflected in Ki values observed with a number of protease inhibitors. Thus, the folded synthetic SIV protease shows patterns of specificity and susceptibility to inhibition that are in accord with what would be expected based upon its degree of structural similarity to proteases from HIV-1 and HIV-2.
Asunto(s)
Biotina , Endopeptidasas/síntesis química , Virus de la Inmunodeficiencia de los Simios/enzimología , Secuencia de Aminoácidos , Avidina , Catálisis , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/aislamiento & purificación , Endopeptidasas/metabolismo , Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH , VIH-1/enzimología , VIH-2/enzimología , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , Especificidad por SustratoRESUMEN
The lymphocyte proliferative responses to respiratory syncytial virus (RSV) were evaluated for 10 healthy adult donors and compared with proliferative responses to a chimeric glycoprotein (FG glycoprotein) which consists of the extracellular domains of both the F and G proteins of RSV and which is produced from a recombinant baculovirus. The lymphocytes of all 10 donors responded to RSV, and the proliferative responses to the whole virus were highly correlated with the responses to the FG glycoprotein. These data suggested that one or both of these glycoproteins of RSV were major target structures for stimulation of the human lymphocyte proliferative response among virus-specific memory T cells. The lymphocytes of four donors were evaluated further for their proliferative responses to a nested set of overlapping peptides modeled on the extracellular and cytoplasmic domains of the F protein of RSV. Strikingly, the lymphocytes of all 4 donors responded primarily to a region defined by a single peptide spanning residues 338 to 355, and the lymphocytes of 2 donors responded to an overlapping peptide spanning residues 328 to 342 also, thus defining a region of the F1 subunit within residues 328 to 355 that may circumscribe an immunodominant site for stimulation of human T cells from a variety of individuals. This region of the F protein is highly conserved among A and B subgroup viruses. As revealed by monoclonal antibody blocking studies, the lymphocytes responding to this antigenic site had characteristics consistent with T helper cells. Similar epitope mapping studies were performed with BALB/c mice immunized with the FG protein in which a relatively hydrophobic peptide spanning residues 51 to 65 within the F2 subunit appeared to be the major T cell recognition determinant. The data are discussed with respect to an antigenic map of the F protein and the potential construction of a synthetic vaccine for RSV.
Asunto(s)
Antígenos Virales/inmunología , Proteína HN , Virus Sincitiales Respiratorios/inmunología , Linfocitos T/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales , Animales , Anticuerpos Monoclonales , Células Presentadoras de Antígenos/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos CD4/inmunología , Epítopos , Antígenos H-2/inmunología , Antígenos HLA-D/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Activación de Linfocitos , Ratones , Péptidos/inmunologíaRESUMEN
The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.