RESUMO
Insulin-degrading enzyme (IDE) is a 110 kDa chambered zinc metalloendopeptidase that degrades insulin, amyloid ß, and other intermediate-sized aggregation prone peptides that adopt ß-structures. Structural studies of IDE in complex with multiple physiological substrates have suggested a role for hydrophobic and aromatic residues of the IDE active site in substrate binding and catalysis. Here, we examine functional requirements for conserved hydrophobic and aromatic IDE active site residues that are positioned within 4.5 Å of IDE-bound insulin B chain and amyloid ß peptides in the reported crystal structures for the respective enzyme-substrate complexes. Charge, size, hydrophobicity, aromaticity, and other functional group requirements for substrate binding IDE active site residues were examined through mutational analysis of the recombinant human enzyme and enzyme kinetic studies conducted using native and fluorogenic derivatives of human insulin and amyloid ß peptides. A functional requirement for IDE active site residues F115, A140, F141, Y150, W199, F202, F820, and Y831 was established, and specific contributions of residue charge, size, and hydrophobicity to substrate binding, specificity, and proteolysis were demonstrated. IDE mutant alleles that exhibited enhanced or diminished proteolytic activity toward insulin or amyloid ß peptides and derivative substrates were identified.
Assuntos
Insulisina/química , Insulisina/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Humanos , Insulina/metabolismo , Cinética , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
Here we report the enzymologic characterization of recombinant human pitrilysin metallopeptidase 1 (Pitrm1) and derivative mutants including the arginine-to-glutamine substitution mutant Pitrm1 R183Q, which has been implicated in inherited amyloidogenic neuropathy. Recombinant Pitrm1 R183Q was readily expressed in and purified from Escherichia coli, but was less active than the recombinant wild-type enzyme against recombinant amyloid beta-peptide (Aß 1-40). A novel fluorogenic substrate derived from the reported Aß 1-40 core peptide cleavage sequence, Mca-KLVFFAEDK-(Dnp)-OH, was synthesized and applied to real-time kinetic study of Pitrm1 and derivative mutants including Pitrm1 R183Q. The Pitrm1 R183Q mutant exhibited significantly decreased rate of fluorogenic peptide hydrolysis, yet retained similar binding affinity by comparison with the wild-type enzyme. Targeted mutagenic analysis revealed a functional requirement for uncharged or electropositive residues in place of Pitrm1 R183. Residue R183 is positioned within an N-terminal strand-loop-strand motif that is conserved among M16C, but not M16A or M16B family metallopeptidases. Truncation analysis revealed that this strand-loop-strand motif inclusive of residue R183 is essential Pitrm1 function. A requirement for charged residues within 4.5 Å of residue R183 was demonstrated, and Pitrm1 R183Q was found to exhibit increased sensitivity to heat inactivation. Our findings indicate that charge sharing in the vicinity of Pitrm1 R183 is critical to enzyme activity, providing potential insight into a molecular basis of Pitrm1 dysfunction.
Assuntos
Arginina/genética , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Motivos de Aminoácidos , Substituição de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Arginina/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Hidrólise , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Eletricidade EstáticaRESUMO
Histidine decarboxylase (HDC) is an enzyme that converts histidine to histamine. Inhibition of HDC has several medical applications, and HDC inhibitors are of considerable interest for the study of histidine metabolism. (S)-α-Fluoromethylhistidine di-hydrochloride (α-FMH) is a potent HDC inhibitor that is commercially available at high cost in small amounts only. Here we report a novel, inexpensive, and efficient method for synthesis of α-FMH using methyl 2-aziridinyl-3-(N-triphenylmethyl-4-imidazolyl) propionate and HF/pyridine, with experimental yield of 57%. To identify novel targets for α-FMH, we developed a three step in silico work-flow for identifying physically plausible protein targets. The work-flow resulted in 21 protein target hits, including several enzymes involved in glutathione metabolism, and notably, two isozymes of the glutathione S-transferase (GST) superfamily, which plays a central role in drug metabolism. In view of this predictive data, the efficacy of α-FMH as a GST inhibitor was investigated in vitro. α-FMH was demonstrated to be an effective inhibitor of GST at micromolar concentration, suggesting that off-target effects of α-FMH may limit physiological drug metabolism and elimination by GST-dependent mechanisms. The present study therefore provides new avenues for obtaining α-FMH and for studying its biochemical effects, with potential implications for drug development.
Assuntos
Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Colorimetria , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/síntese química , Cinética , Espectrometria de Massas , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4-mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4-mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in RNAi-deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream of Clr4 recruitment during heterochromatin assembly.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Heterocromatina/metabolismo , Metiltransferases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Montagem e Desmontagem da Cromatina , Heterocromatina/genética , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Lisina/metabolismo , Metiltransferases/genética , Mutação , Interferência de RNA , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Especificidade por Substrato , Telômero/genética , Telômero/metabolismoRESUMO
Here, we describe a capillary electrophoresis method for evaluating proteolysis of the amyloid beta peptide (Aß) 1-40. This method is suitable for kinetic studies, demands little specialized equipment, and consumes only small quantities of a commercially available substrate whose physiological accumulation is thought to underlie the development of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Proteólise , Eletroforese Capilar/métodos , Eletroforese Capilar/normas , Estudos de Avaliação como Assunto , Humanos , Insulisina/química , Cinética , Padrões de ReferênciaRESUMO
The insulin-degrading enzyme (IDE) cleaves numerous small peptides, including biologically active hormones and disease-related peptides. The propensity of IDE to degrade neurotoxic Aß peptides marks IDE as a potential therapeutic target for Alzheimer disease. Using a synthetic reporter based on the yeast a-factor mating pheromone precursor, which is cleaved by multiple IDE orthologs, we identified seven small molecules that stimulate rat IDE activity in vitro. Half-maximal activation of IDE by the compounds is observed in vitro in the range of 43 to 198 µM. All compounds decrease the K(m) of IDE. Four compounds activate IDE in the presence of the competing substrate insulin, which disproportionately inhibits IDE activity. Two compounds stimulate rat IDE activity in a cell-based assay, indicating that they are cell permeable. The compounds demonstrate specificity for rat IDE since they do not enhance the activities of IDE orthologs, including human IDE, and they appear specific for a-factor-based reporters since they do not enhance rat IDE-mediated cleavage of Aß-based reporters. Our results suggest that IDE activators function in the context of specific enzyme-substrate pairs, indicating that the choice of substrate must be considered in addition to target validation in IDE activator screens.
Assuntos
Permeabilidade da Membrana Celular , Ativadores de Enzimas/farmacologia , Insulisina/metabolismo , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/farmacocinética , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Fator de Acasalamento , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Proteólise , Ratos , Bibliotecas de Moléculas Pequenas , Especificidade por Substrato , Leveduras/efeitos dos fármacos , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismoRESUMO
Distinct regions of the eukaryotic genome are packaged into different types of chromatin, with euchromatin representing gene rich, transcriptionally active regions and heterochromatin more condensed and gene poor. The assembly and maintenance of heterochromatin is important for many aspects of genome control, including silencing of gene transcription, suppression of recombination, and to ensure proper chromosome segregation. The precise mechanisms underlying heterochromatin establishment and maintenance are still unclear, but much progress has been made towards understanding this process during the last few years, particularly from studies performed in fission yeast. In this review, we hope to provide a conceptual model of centromeric heterochromatin in fission yeast that integrates our current understanding of the competing forces of transcription, replication, and RNA decay that influence its assembly and propagation.
Assuntos
Centrômero/metabolismo , Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Interferência de RNA , Schizosaccharomyces/metabolismo , Transcrição Gênica , Animais , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Epigênese Genética , Histonas/metabolismo , Mamíferos , Metilação , Complexos Multiproteicos/metabolismo , Estabilidade de RNARESUMO
Insulin-degrading enzyme (IDE) (insulysin) is a zinc metallopeptidase that metabolizes several bioactive peptides, including insulin and the amyloid ß peptide. IDE is an unusual metallopeptidase in that it is allosterically activated by both small peptides and anions, such as ATP. Here, we report that the ATP-binding site is located on a portion of the substrate binding chamber wall arising largely from domain 4 of the four-domain IDE. Two variants having residues in this site mutated, IDEK898A,K899A,S901A and IDER429S, both show greatly decreased activation by the polyphosphate anions ATP and PPPi. IDEK898A,K899A,S901A is also deficient in activation by small peptides, suggesting a possible mechanistic link between the two types of allosteric activation. Sodium chloride at high concentrations can also activate IDE. There are no observable differences in average conformation between the IDE-ATP complex and unliganded IDE, but regions of the active site and C-terminal domain do show increased crystallographic thermal factors in the complex, suggesting an effect on dynamics. Activation by ATP is shown to be independent of the ATP hydrolysis activity reported for the enzyme. We also report that IDEK898A,K899A,S901A has reduced intracellular function relative to unmodified IDE, consistent with a possible role for anion activation of IDE activity in vivo. Together, the data suggest a model in which the binding of anions activates by reducing the electrostatic attraction between the two halves of the enzyme, shifting the partitioning between open and closed conformations of IDE toward the open form.
Assuntos
Insulisina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Ânions/metabolismo , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Ativação Enzimática , Insulisina/química , Insulisina/genética , Espaço Intracelular/metabolismo , Modelos Moleculares , Mutagênese , Mutação , Conformação Proteica , RatosRESUMO
The S. cerevisiae genome encodes two M16A enzymes: Axl1p and Ste23p. Of the two, Ste23p shares significantly higher sequence identity with M16A enzymes from other species, including mammalian insulin-degrading enzymes (IDEs). In this study, recombinant Ste23p and R. norvegicus IDE (RnIDE) were isolated from E. coli, and their enzymatic properties compared. Ste23p was found to cleave established RnIDE substrates, including the amyloid-beta peptide (Abeta1-40) and insulin B-chain. A novel internally quenched fluorogenic substrate (Abz-SEKKDNYIIKGV-nitroY-OH) based on the polypeptide sequence of the yeast P2 a-factor mating propheromone was determined to be a suitable substrate for both Ste23p and RnIDE, and was used to conduct comparative enzymological studies. Both enzymes were most active at 37 degrees C, in alkaline buffers and in high salt environments. In addition, the proteolytic activities of both enzymes towards the fluorogenic substrate were inhibited by metal chelators, thiol modifiers, inhibitors of cysteine protease activity and insulin. Characteristics of STE23 expression were also evaluated. Our analysis indicates that the 5' terminus of the STE23 gene has been mischaracterized, with the physiologically relevant initiator corresponding to residue M53 of the publicly annotated protein sequence. Finally, we demonstrate that, unlike haploid-specific Axl1p, Ste23p is expressed in both haploid and diploid cell types. Our study presents the first comprehensive biochemical analysis of a yeast M16A enzyme, and provides evidence that S. cerevisiae Ste23p has enzymatic properties that are highly consistent with mammalian IDEs and other M16A enzymes.
Assuntos
Insulisina/genética , Insulisina/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Animais , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Insulina/metabolismo , Insulisina/química , Metaloendopeptidases/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Sais/farmacologia , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
According to the amyloid hypothesis, Abeta peptides are neurotoxic and underlie development and progression of Alzheimer's disease (AD). Multiple Abeta clearance mechanisms, including destruction of the peptides by proteolytic enzymes, are hypothesized to regulate physiological Abeta peptide levels. The insulin-degrading enzyme (IDE) is considered one of the predominant enzymes having Abeta degrading activity. Despite its putative role in protecting against AD, relatively few methods exist for studying IDE activity in vitro. We report the application of capillary electrophoresis (CE) as a novel method for evaluating IDE-mediated Abeta 1-40 proteolysis. This method employs chemically unmodified substrates that are readily obtained from commercial sources. It involves minimal sample preparation, and requires no specialized equipment beyond a CE instrument equipped with a standard fused silica capillary. In the present analysis, we demonstrate that this CE-based method is amenable to kinetic analysis, and show that IDE-mediated Abeta proteolysis is significantly and disproportionately inhibited in the presence of insulin, an alternative IDE substrate.
Assuntos
Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/metabolismo , Eletroforese Capilar/métodos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Peptídeos beta-Amiloides/efeitos dos fármacos , Animais , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Eletroforese Capilar/normas , Humanos , Insulina/farmacologia , Insulisina/metabolismo , Fragmentos de Peptídeos/efeitos dos fármacos , Ratos , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Pitrilysin is a bacterial protease that is similar to the mammalian insulin-degrading enzyme, which is hypothesized to protect against the onset of Alzheimer's disease, and the yeast enzymes Axl1p and Ste23p, which are responsible for production of the a-factor mating pheromone in Saccharomyces cerevisiae. The lack of a phenotype associated with pitrilysin deficiency has hindered studies of this enzyme. Herein, we report that pitrilysin can be heterologously expressed in yeast such that it functionally substitutes for the shared roles of Axl1p and Ste23p in pheromone production, resulting in a readily observable phenotype. We have exploited this phenotype to conduct structure-function analyses of pitrilysin and report that residues within four sequence motifs that are highly conserved among M16A enzymes are essential for its activity. These motifs include the extended metalloprotease motif, a second motif that has been hypothesized to be important for the function of M16A enzymes, and two others not previously recognized as being important for pitrilysin function. We have also established that the two self-folding domains of pitrilysin are both required for its proteolytic activity. However, pitrilysin does not possess all the enzymatic properties of the yeast enzymes since it cannot substitute for the role of Axl1p in the repression of haploid invasive growth. These observations further support the utility of the yeast system for structure-function and comparative studies of M16A enzymes.