RESUMO
The understanding of the pathophysiology of a large number of cancer types provides a strategy to target cancer cells with minimal effect on normal cells. Protein phosphorylation and dephosphorylation play a pivotal role in intracellular signaling; to regulate signal transduction pathways, there are approximately 700 protein kinases and 100 protein phosphatases encoded within the human genome. In cancer, as well as in other proliferative diseases, unregulated cell proliferation, differentiation and survival frequently results from abnormal protein phosphorylation. Although it is often possible to identify a single kinase that plays a pivotal role in a given disease, the development of drugs based upon protein kinase inhibition has been hampered by unacceptable side effects resulting from a lack of target selectivity. With the growing understanding of the molecular biology of protein tyrosine kinases and the use of structural information, the design of potential drugs directed towards the bind adenosine triphosphate (ATP)-binding site of a single target has become possible. These advances have transferred emphasis away from the identification of potent kinase inhibitors and more towards issues of target selectivity, cellular efficacy, therapeutic effectiveness and tolerability. In this paper, the relationship between molecular biology and drug discovery methods, as utilized for the identification of anticancer drugs, will be illustrated.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Piperazinas/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/uso terapêutico , Trifosfato de Adenosina/metabolismo , Antineoplásicos/química , Benzamidas , Comunicação Celular , Desenho de Fármacos , Inibidores Enzimáticos/química , Proteínas de Fusão bcr-abl , Humanos , Mesilato de Imatinib , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Proteínas Oncogênicas/antagonistas & inibidores , Piperazinas/química , Ligação Proteica , Proteínas Proto-Oncogênicas c-kit , Pirimidinas/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
The production of recombinant leech-derived tryptase inhibitor (rLDTI) by two different strains of Saccharomyces cerevisiae resulted in the secretion of non-glycosylated and glycosylated rLTDI. Monosaccharide analysis and a-mannosidase treatment demonstrated that glycosylated rLDTI was exclusively alpha-mannosylated. A trypsin digest of reduced and S-carboxymethylated glycosylated rLDTI was separated on a reverse-phase HPLC column. Glycopeptides identified by a combination of matrix-assisted laser desorption mass spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed the presence of different glycoforms. It was found that Ser24, Ser33 and Ser36 were partially glycosylated with a single mannose residue, whereas Thr42 in glycosylated rLDTI from both strains was fully occupied with manno-oligosaccharides with a degree of polymerization ranging over 1-3 and 1-13 depending on the yeast strain. In phosphorylated rLDTI a single phosphate group was predominantly located at the innermost Man residue of units of mannobiose, mannotriose, mannotetraose and mannopentaose at Thr42. Oligosaccharides released by alkaline treatment were reduced by sodium borohydride and separated by high-pH anion-exchange chromatography on a CarboPac MA1 column, and analyzed by one- and two-dimensional 1H-NMR spectroscopy. Besides the major oligosaccharide Man alpha1-2Man-ol, the (for yeast protein O-glycosylation) unusual Man alpha1-3Man alpha1-2Man-ol was determined. The solution conformation of glycosylated rLDTI was investigated by two-dimensional NMR spectroscopy. Structure calculations by means of distance geometry showed that glycosylated rLDTI is compactly folded and contained small secondary structure elements. Analysis of the chemical shifts showed that amino acids Val32-Ser33, Ser36-Ser39 and Thr42 were affected by the O-mannosylation. In addition, changes in chemical shift were observed within the beta-hairpin peptide regions Val13-Ser16 and Gly18-Tyr21 attributed to direct interactions of the mannose residue at Ser36. Furthermore, the protein-linked oligosaccharides were spatially grouped in a position opposite of the canonical binding loop.
Assuntos
Manose/análise , Oligopeptídeos/química , Conformação Proteica , Proteínas/química , Inibidores de Serina Proteinase/química , Sequência de Aminoácidos , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Glicosilação , Sanguessugas , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes/química , Saccharomyces cerevisiae , Soluções , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pattern of cysteine residues. Unlike the closely related inhibitors, antistasin/ghilanten and guamerin, which are selective for coagulation factor Xa or neutrophil elastase, hirustasin binds specifically to tissue kallikrein. The conservation of the pattern of cysteine residues and the significant sequence homology suggest that these related inhibitors possess a similar three-dimensional structure to hirustasin. RESULTS: The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 resolution. Hirustasin folds into a brick-like structure that is dominated by five disulfide bridges and is sparse in secondary structural elements. The cysteine residues are connected in an abab cdecde pattern that causes the polypeptide chain to fold into two similar motifs. As a hydrophobic core is absent from hirustasin the disulfide bridges maintain the tertiary structure and present the primary binding loop to the active site of the protease. The general structural topography and disulfide connectivity of hirustasin has not previously been described. CONCLUSIONS: The crystal structure of the kallikrein-hirustasin complex reveals that hirustasin differs from other serine protease inhibitors in its conformation and its disulfide bond connectivity, making it the prototype for a new class of inhibitor. The disulfide pattern shows that the structure consists of two domains, but only the C-terminal domain interacts with the protease. The disulfide pattern of the N-terminal domain is related to the pattern found in other proteins. Kallikrein recognizes hirustasin by the formation of an antiparallel beta sheet between the protease and the inhibitor. The P1 arginine binds in a deep negatively charged pocket of the enzyme. An additional pocket at the periphery of the active site accommodates the sidechain of the P4 valine.
Assuntos
Hormônios de Invertebrado/química , Calicreínas/química , Conformação Proteica , Serpinas/classificação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Cistina/química , Hormônios de Invertebrado/metabolismo , Calicreínas/antagonistas & inibidores , Calicreínas/metabolismo , Sanguessugas/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serpinas/químicaRESUMO
A synthetic gene coding for the 55-amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast alpha-factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N-terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N-terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 A and 2.4 A, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.
Assuntos
Quimotripsina/metabolismo , Hormônios de Invertebrado/química , Calicreínas/metabolismo , Tripsina/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Cristalização , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
An efficient expression/purification procedure has been developed which allows the production of pure, biologically active recombinant leech-derived tryptase inhibitor (rLDTI), originally found in the leech Hirudo medicinalis. The gene for LDTI was generated synthetically from three overlapping oligonucleotides by PCR synthesis. LDTI was expressed in the yeast Saccharomyces cerevisiae under the control of the copper-inducible CUP1 promoter and fused to the invertase signal sequence (SUC2). The entire expression cassette was inserted into the yeast high-copy vector pDP34. Appropriate host strains transformed with the expression plasmid secreted rLDTI into the medium upon copper addition. Proteinchemical analysis of the secreted rLDTI revealed exclusively inhibitor with the correct N-terminal sequence. Up to 60% of the rLDTI, however, appeared to be modified by glycosylation and the unglycosylated material showed heterogeneity at the C-terminus. Besides full-length rLDTI, truncated rLDTI species lacking either the terminal Asn46 or in addition the penultimate Leu45 were isolated. The C-terminally truncated variants were eliminated using a S. cerevisiae host strain disrupted in the structural genes of carboxypeptidases yscY and ysca, thus identifying these proteases as being responsible for the degradation of rLDTI. Mature rLDTI was purified in high yields from the culture supernatant of the carboxypeptidase-deficient yeast strain by cation-exchange chromatography and reverse-phase HPLC. The recombinant protein is at least 98% pure, based on HPLC and capillary electrophoresis, and is fully biologically active. Structural identity with the authentic leech protein was confirmed by sequence analysis and molecular-mass determination. The purified protein was tested for its ability to inhibit tryptase and trypsin in vitro and to interfere with the tryptase-induced proliferation of human fibroblasts and keratinocytes. Recombinant LDTI appears to be as potent as the authentic leech protein, exhibiting Ki-values of approximately 1.5 nM and approximately 1.6 nM against human tryptase and bovine trypsin, respectively. The tryptase-induced proliferation of human fibroblasts and keratinocytes was inhibited with half-maximum values of approximately 0.1 nM and approximately 1 nM, respectively. The availability of the recombinant material will allow evaluation of the concept of tryptase inhibition in various disease models and to test the therapeutic potential of LDTI in mast-cell-related disorders.
Assuntos
Sanguessugas/química , Serina Endopeptidases , Inibidores de Serina Proteinase/isolamento & purificação , Sequência de Aminoácidos , Animais , Bovinos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Quimases , Humanos , Cinética , Sanguessugas/genética , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Saccharomyces cerevisiae/genética , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/farmacologia , TriptasesRESUMO
Human interferon-alpha 8 was expressed in Saccharomyces cerevisiae and found to accumulate intracellularly in an insoluble form. The protein could be solubilized and converted to a biologically active form with high yield by a denaturation-refolding procedure. The interferon-alpha 8 was further purified to apparent homogeneity by copper-chelate affinity chromatography and anion-exchange chromatography and fully characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, mass spectrometry, circular-dichroism (CD) spectroscopy and specific activity. Secondary-structure predictions from CD spectroscopy indicate that the molecule is correctly folded. Peptide mapping supported the correct sequence and the expected disulfide-bridge connectivity. The purified protein elutes on reversed-phase high-pressure liquid chromatography (RP-HPLC) as two peaks. Electrospray mass spectrometry and N-terminal sequence analysis of the minor component indicated the existence of an N-terminal acetyl group for the later eluting HPLC-component. In anti-viral assays, the two IFN forms were equally active. Hexagonal crystals of this interferon preparation could be obtained. On the basis of the electrophoretic mobility, HPLC profile, and biological activity assay, the crystalline material was judged to be identical to the uncrystallized interferon. Interferon in crystallized form was found to be stable for up to 24 months and, therefore, could be used for long-term storage, particularly for material intended for clinical use.
Assuntos
Interferon Tipo I/química , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Biotecnologia , Dicroísmo Circular , Cristalização , Estabilidade de Medicamentos , Humanos , Interferon Tipo I/genética , Interferon Tipo I/isolamento & purificação , Dados de Sequência Molecular , Mapeamento de Peptídeos , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes , Saccharomyces cerevisiae/genética , SolubilidadeRESUMO
Duramycins B and C, two new lanthionine containing antibiotics, have been isolated from Streptoverticillium strain R2075 and Streptomyces griseoluteus (R2107). The known antibiotics duramycin and cinnamycin were reisolated from Streptoverticillium hachijoense (DSM 40114) and Streptomyces longisporoflavus (DSM 40165). The structures of these latter two compounds should be revised by changing amino acid residue 3 to glutamine and 17 to asparagine, respectively. Cinnamycin therefore seems to be identical to Ro 09-0198. Leucopeptin has been shown to be identical to duramycin. Physico-chemical data of these compounds provide evidence for a similar structure for all duramycin antibiotics. All compounds of this group inhibit human phospholipase A2 at a concentration of 10(-6) molar.
Assuntos
Antibacterianos , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Cíclicos , Peptídeos/farmacologia , Fosfolipases A/antagonistas & inibidores , Sequência de Aminoácidos , Aminoácidos/análise , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos , Bacillus subtilis/efeitos dos fármacos , Bacteriocinas , Cromatografia Líquida de Alta Pressão , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Neutrófilos/enzimologia , Peptídeos/química , Peptídeos/isolamento & purificação , Fosfolipases A2 , Conformação Proteica , Estereoisomerismo , Streptomyces/classificação , Streptomyces/metabolismo , Streptomycetaceae/classificação , Streptomycetaceae/metabolismo , Relação Estrutura-AtividadeRESUMO
Orthorhombic crystals of the complex formed between bovine alpha-chymotrypsin and a recombinant human mucous proteinase inhibitor (SLPI) were grown. Data to 2.3 A resolution were collected on the area-detector diffractometer FAST. The crystal structure of the complex was solved by Patterson search techniques using chymotrypsin as a search model. A cyclic procedure of modeling and crystallographic refinement enabled the determination of the SLPI structure. The current crystallographic R-value is 0.19. SLPI has a boomerang-like shape with both wings comprising two well separated domains of similar architecture. In each domain the polypeptide chain is arranged like a stretched spiral. Two internal strands form a regular beta-hairpin loop which is accompanied by two external strands linked by the proteinase binding segment. The polypeptide segment of each domain is interconnected by four disulfide bridges with a connectivity pattern hitherto unobserved. The reactive site loop of the second domain has elastase and chymotrypsin binding properties. It contains the scissile peptide bond between Leu72I and Met73I and has a similar conformation to that observed in other serine proteinase protein inhibitors. Eight residues of this loop, two of the adjacent hairpin loop, the C-terminal segment and Trp30I are in direct contact with the cognate enzyme. The binding loop of the first domain (probably with anti-trypsin activity) is disordered due to proteolytic cleavage occurring in the course of crystallization.
Assuntos
Quimotripsina/metabolismo , Mucosa/metabolismo , Inibidores de Proteases/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Simulação por Computador , Dissulfetos/análise , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Difração de Raios XRESUMO
The acetylenic thio ester (3-pentynoyl)pantetheine irreversibly inactivates butyryl-CoA dehydrogenase from Megasphaera elsdenii. The inactivator becomes covalently attached to the protein (0.61 +/- 0.1 mol of 14C-labeled inactivator/mol of enzyme flavin). No modification of the flavin cofactor is seen. The covalent enzyme-inactivator adduct is labile toward base and neutral hydroxylamine. These treatments release 85 +/- 5% of the incorporated 14C label from the protein. Base-catalyzed hydrolysis of the adduct releases 3-oxopentanoic acid (0.6 mol/mol of incorporated inactivator). Treatment with hydroxylamine leads to formation of a hydroxamic acid on the protein (0.64 +/- 0.09 mol/mol of incorporated inactivator). The covalent adduct can be reduced with sodium borohydride with release of 1,3-pentanediol. Hydrolysis of the protein with 6 N HCl after sodium borohydride reduction yields 2-amino-5-hydroxyvaleric acid and proline. We conclude that the inactivator has reacted with the gamma-carboxyl group of a glutamate residue at the enzyme active site. The inactivation proceeds through enzyme-catalyzed rearrangement of the acetylene to an allene, followed by nucleophilic addition of the carboxyl group to the allene. (3-Chloro-3-butenoyl)pantetheine irreversibly inactivates the enzyme in a fashion similar to the acetylenic thio ester and also modifies a glutamate residue. Butyryl-CoA dehydrogenase catalyzes the isomerization of (3-butenoyl)pantetheine to (2-butenoyl)pantetheine. The enzyme catalyzes the elimination of HF from 3-fluoropropionyl-CoA and (3,3-difluorobutyryl)pantetheine. We suggest, that these results together support an oxidation mechanism for butyryl-CoA dehydrogenase which is initiated by alpha-proton abstraction.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Acetilação , Butiril-CoA Desidrogenase , Fenômenos Químicos , Química , Eletroforese em Papel , Fluoretos/metabolismo , Panteteína/análogos & derivados , Panteteína/metabolismoRESUMO
Glutaryl-CoA dehydrogenase, a flavoprotein, catalyzes the reaction -OOCCH3CH2--CH2COSR (FAD leads to FADH2) leads to CH3CH = CHCOSR + CO2 (SR = CoA or pantetheine). With the isolated enzyme, a dye serves as the final electron acceptor. The enzyme from Pseudomonas fluorescens (ATCC 11250) has been purified to homogeneity. It was established with appropriate isotopic substitutions that the proton which is added to the gamma position of the product, subsequent to decarboxylation, is not derived from the solvent but is derived from the alpha position of the substrate. Under conditions where no net conversion of substrate occurs, i.e., in the absence of electron acceptor, the enzyme catalyzes the exchange of the beta hydrogen of the substrate with solvent protons. Butyryl-CoA dehydrogenase (M. elsedenii), which catalyzes an analogous reaction, catalyzes the exchange of both the alpha and beta hydrogens with solvent protons in the absence of electron acceptor. Glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase are irreversibly inactivated by the substrate analogues 3-butynoylpantetheine and 3-pentynoylpantetheine. These inactivators do not form an adduct with the flavin and probably react with a nucleophile at the active site. Upon inactivation, the spectrum of the enzyme-bound flavin is essentially unchanged, and the flavin can be reduced by Na2S2O4. We suggest that inactivation involves intermediate allene formation. We proposed that these results support an oxidation mechanism for glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase which is initiated by proton abstraction. With glutaryl-CoA dehydrogenase, the base, which abstracts the substrate alpha proton, is shielded from the solvent and is then used to protonate the carbanion (CH2--CH--CHCOSCoA) formed after oxidation and decarboxylation.