RESUMO
Peptides play a key role in controlling many physiological and neurobiological pathways. Many bioactive peptides require a C-terminal α-amide for full activity. The bifunctional enzyme catalysing α-amidation, peptidylglycine α-amidating monooxygenase (PAM), is the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine functions are dependent upon amidated peptides; additional amidated peptides are growth promoters in tumours. The amidation reaction occurs in two steps, glycine α-hydroxylation followed by dealkylation to generate the α-amide product. Currently, most potentially useful inhibitors target the first reaction, which is rate-limiting. PAM is a membrane-bound enzyme that visits the cell surface during peptide secretion. PAM is then used again in the biosynthetic pathway, meaning that cell-impermeable inhibitors or inactivators could have therapeutic value for the treatment of cancer or psychiatric abnormalities. To date, inhibitor design has not fully exploited the structures and mechanistic details of PAM.
Assuntos
Oxigenases de Função Mista , Amidas , Animais , Biomarcadores/química , Biomarcadores/metabolismo , Humanos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Terapia de Alvo Molecular , Complexos Multienzimáticos , Peptídeos/químicaRESUMO
The growing issue of insecticide resistance has meant the identification of novel insecticide targets has never been more important. Arylalkylamine N-acyltransferases (AANATs) have been suggested as a potential new target. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. In insects, this process is a key step in melanism, hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each AANAT isoform characterized indicates each organism accommodates an assembly of discrete AANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology. Presented here is a thorough kinetic and structural analysis of AANAT found in one of the most common secondary pests of all plant commodities in the world, Tribolium castaneum. The enzyme, named TcAANAT0, catalyzes the formation of short-chain N-acylarylalkylamines, with short-chain acyl-CoAs (C2-C10), benzoyl-CoA, and succinyl-CoA functioning in the role of acyl donor. Recombinant TcAANAT0 was expressed and purified from E. coli and was used to investigate the kinetic and chemical mechanism of catalysis. The kinetic mechanism is an ordered sequential mechanism with the acyl-CoA binding first. pH-rate profiles and site-directed mutagenesis studies identified amino acids critical to catalysis, providing insights about the chemical mechanism of TcAANAT0. A crystal structure was obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. This combination of kinetic analysis and crystallography alongside mutagenesis and sequence analysis shines light on some approaches possible for targeting TcAANAT0 and other AANATs for novel insecticide design.
Assuntos
Arilalquilamina N-Acetiltransferase/química , Proteínas de Insetos/química , Tribolium/enzimologia , Acetilcoenzima A/metabolismo , Animais , Arilalquilamina N-Acetiltransferase/genética , Arilalquilamina N-Acetiltransferase/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Mutação , Fenetilaminas/metabolismo , Ligação Proteica , Triptaminas/metabolismoRESUMO
INTRODUCTION: A critical and as-yet unmet need in Alzheimer's disease (AD) is the discovery of peripheral small molecule biomarkers. Given that brain pathology precedes clinical symptom onset, we set out to test whether metabolites in blood associated with pathology as indexed by cerebrospinal fluid (CSF) AD biomarkers. METHODS: This study analyzed 593 plasma samples selected from the European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery study, of individuals who were cognitively healthy (n = 242), had mild cognitive impairment (n = 236), or had AD-type dementia (n = 115). Logistic regressions were carried out between plasma metabolites (n = 883) and CSF markers, magnetic resonance imaging, cognition, and clinical diagnosis. RESULTS: Eight metabolites were associated with amyloid ß and one with t-tau in CSF, these were primary fatty acid amides (PFAMs), lipokines, and amino acids. From these, PFAMs, glutamate, and aspartate also associated with hippocampal volume and memory. DISCUSSION: PFAMs have been found increased and associated with amyloid ß burden in CSF and clinical measures.
Assuntos
Peptídeos beta-Amiloides , Amiloidose/sangue , Biomarcadores , Hipocampo , Memória/fisiologia , Metabolômica , Idoso , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Amiloidose/líquido cefalorraquidiano , Amiloidose/metabolismo , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Encéfalo/patologia , Disfunção Cognitiva/diagnóstico , Estudos de Coortes , Feminino , Hipocampo/metabolismo , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Proteínas tau/sangue , Proteínas tau/líquido cefalorraquidianoRESUMO
Long-chain fatty acid amides are signaling lipids found in mammals and other organisms; however, details of the metabolic pathways for the N-acylglycines and primary fatty acid amides (PFAMs) have remained elusive. Heavy-labeled precursor and subtraction lipidomic experiments in mouse neuroblastoma N18TG2 cells, a model cell line for the study of fatty acid amide metabolism, establish the biosynthetic pathways for the N-acylglycines and the PFAMs. We provide evidence that the N-acylglycines are formed by a long-chain specific glycine-conjugating enzyme, glycine N-acyltransferase-like 3 (GLYATL3). siRNA knockdown of GLYATL3 in the N18TG2 cells resulted in a decrease in the levels of the N-acylglycines and the PFAMs. This is the first report of an enzyme responsible for long-chain N-acylglycine production in cellula. The production of the PFAMs in N18TG2 cells was reported to occur by the oxidative cleavage of the N-acylglycines, as catalyzed by peptidylglycine α-amidating monooxygenase (PAM). siRNA knockdown of PAM resulted in an accumulation of [(13)C18]N-oleoylglycine and decreased levels of [(13)C18]oleamide when the N18TG2 cells were grown in the presence of [(13)C18]oleic acid. The addition of [1-(13)C]palmitate to the N18TG2 cell growth media led to the production of a family of [1-(13)C]palmitoylated fatty acid amides, consistent with the biosynthetic pathways detailed herein.
Assuntos
Aciltransferases/fisiologia , Ácidos Graxos/biossíntese , Amidas/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Lipogênese , CamundongosRESUMO
Proteomics is a powerful approach used for investigating the complex molecular mechanisms of disease pathogenesis and progression. An important challenge in modern protein profiling approaches involves targeting of specific protein activities in order to identify altered molecular processes associated with disease pathophysiology. Adenosine-binding proteins represent an important subset of the proteome where aberrant expression or activity changes of these proteins have been implicated in numerous human diseases. Herein, we describe an affinity-based approach for the enrichment of adenosine-binding proteins from a complex cell proteome. A novel N6-biotinylated-8-azido-adenosine probe (AdoR probe) was synthesized, which contains a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Probe specificity was confirmed with protein standards prior to further evaluation in a complex protein mixture consisting of a lysate derived from mouse neuroblastoma N18TG2 cells. Protein identification and relative quantitation using mass spectrometry allowed for the identification of small variations in abundance of nucleoside- and nucleotide-binding proteins in these samples where a significant enrichment of AdoR-binding proteins in the labeled proteome from the neuroblastoma cells was observed. The results from this study demonstrate the utility of this method to enrich for nucleoside- and nucleotide-binding proteins in a complex protein mixture, pointing towards a unique set of proteins that can be examined in the context of further understanding mechanisms of disease, or fundamental biological processes in general.
Assuntos
Adenosina , Proteínas de Transporte/metabolismo , Nucleotídeos/metabolismo , Proteômica/métodos , Sondas RNA/genética , Sondas RNA/metabolismo , Ontologia Genética , Humanos , Proteoma/metabolismo , Sondas RNA/síntese química , Reprodutibilidade dos Testes , Coloração e RotulagemRESUMO
Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N(18)TG(2) and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N(18)TG(2) and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N(18)TG(2) and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation.
Assuntos
Amidas/metabolismo , Etanolamina/metabolismo , Ácidos Graxos/metabolismo , Animais , Células Cultivadas , Plexo Corióideo/citologia , Plexo Corióideo/metabolismo , Etanolaminas/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Linoleicos/metabolismo , Camundongos , Neuroblastoma/metabolismo , Ácidos Oleicos/metabolismo , Ácidos Palmíticos/metabolismo , Ovinos/metabolismo , Carneiro Doméstico , Células Tumorais CultivadasRESUMO
Amidation is a post-translational modification found at the C-terminus of ~50% of all neuropeptide hormones. Cleavage of the C(α)-N bond of a C-terminal glycine yields the α-amidated peptide in a reaction catalyzed by peptidylglycine α-amidating monooxygenase (PAM). The mass of an α-amidated peptide decreases by 58 Da relative to its precursor. The amino acid sequences of an α-amidated peptide and its precursor differ only by the C-terminal glycine meaning that the peptides exhibit similar RP-HPLC properties and tandem mass spectral (MS/MS) fragmentation patterns. Growth of cultured cells in the presence of a PAM inhibitor ensured the coexistence of α-amidated peptides and their precursors. A strategy was developed for precursor and α-amidated peptide pairing (PAPP): LC-MS/MS data of peptide extracts were scanned for peptide pairs that differed by 58 Da in mass, but had similar RP-HPLC retention times. The resulting peptide pairs were validated by checking for similar fragmentation patterns in their MS/MS data prior to identification by database searching or manual interpretation. This approach significantly reduced the number of spectra requiring interpretation, decreasing the computing time required for database searching and enabling manual interpretation of unidentified spectra. Reported here are the α-amidated peptides identified from AtT-20 cells using the PAPP method.
Assuntos
Amidas/química , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , alfa-MSH/química , Animais , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/métodos , Bases de Dados de Proteínas , Dissulfiram/farmacologia , Glicina/metabolismo , Camundongos , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Peptídeos/química , Reprodutibilidade dos Testes , Ferramenta de Busca , Fatores de TempoRESUMO
Peptidyl alpha-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of alpha-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C(6)H(5)-CO-NH-CH(2)-COOH) inhibit the enzyme with K(i) values as low as 0.5microM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, -CO-NH-CH(2)-COOH with an S-(thioacetyl)thioglycolic acid moiety, -CS-S-CH(2)-COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations >100-fold excess of their respective K(i) values. Comparison of K(i) values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed.
Assuntos
Inibidores Enzimáticos/química , Hipuratos/química , Hipuratos/farmacologia , Inseticidas/química , Oxigenases de Função Mista/antagonistas & inibidores , Complexos Multienzimáticos/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Hipuratos/síntese química , Humanos , Concentração Inibidora 50 , Inseticidas/metabolismo , Inseticidas/farmacologia , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Complexos Multienzimáticos/metabolismo , Ratos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Ubiquitin (Ub) and the ubiquitin-like proteins (UBLs) mediate an array of cellular functions. These proteins contain a C-terminal glycine residue that is key to their function. Oxidative conversion of C-terminal glycine-extended prohormones to the corresponding alpha-amidated peptide is one step in the biosynthesis of bioactive peptide hormones. The enzyme catalyzing this reaction is peptidylglycine alpha-amidating monooxygenase (PAM). We report herein that Ub is a PAM substrate with a (V/K)(amidation) that is similar to other known peptide substrates. This work is significant because PAM and the UBLs co-localize to the hypothalamus and the adrenal medulla and are both over-expressed in glioblastomas.
Assuntos
Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Peptídeos/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Glicina/metabolismo , Glioxilatos/metabolismo , Estrutura Molecular , Oxirredução , Oxigênio/metabolismo , Peptídeos/genética , Ratos , Ubiquitina/genéticaRESUMO
Oleamide is an endogenous sleep-inducing lipid that has been isolated from the cerebrospinal fluid of sleep-deprived mammals. Oleamide is the best-understood member of the primary fatty acid amide family. One key unanswered question regarding oleamide and all other primary acid amides is the pathway by which these molecules are produced. One proposed pathway involves oleoyl-CoA and N-oleoylglycine as intermediates: oleic acid --> oleoyl-CoA --> N-oleoylglycine --> oleamide. The first and third reactions are known reactions, catalyzed by acyl-CoA synthetase and peptidylglycine alpha-amidating monooxygenase (PAM). Oleoyl-CoA formation from oleic acid has been demonstrated in vitro and in vivo while, to date, N-oleoylglycine cleavage to oleamide has been established only in vitro. PAM catalyzes the final step in alpha-amidated peptide biosynthesis, and its proposed role in primary fatty acid amide biosynthesis has been controversial. Mouse neuroblastoma N(18)TG(2) cells are an excellent model system for the study of oleamide biosynthesis because these cells convert [(14)C]-oleic acid to [(14)C]-oleamide and express PAM in a regulated fashion. We report herein that growth of the N(18)TG(2) cells in the presence of [(14)C]-oleic acid under conditions known to stimulate PAM expression generates an increase in [(14)C]-oleamide or in the presence of a PAM inhibitor generates [(14)C]-N-oleoylglycine. This represents the first identification of N-oleoylglycine from a biological source. In addition, N(18)TG(2) cell growth in the presence of N-oleoylglycine yields oleamide. These results strongly indicate that N-oleoylglycine is an intermediate in oleamide biosynthesis and provide further evidence that PAM does have a role in primary fatty acid amide production in vivo.
Assuntos
Neuroblastoma/metabolismo , Ácido Oleico/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Meios de Cultura/química , Inibidores Enzimáticos/química , Ácidos Graxos Monoinsaturados/química , Glicina/análogos & derivados , Glicina/metabolismo , Camundongos , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/biossíntese , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/biossíntese , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Ácidos Oleicos/biossíntese , Ácidos Oleicos/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
The C-terminal alpha-amide moiety of most peptide hormones arises by the posttranslational cleavage of a glycine-extended precursor in a reaction catalyzed by bifunctional peptidylglycine alpha-amidating monooxygenase (PAM). Glutathione and the S-alkylated glutathiones have a C-terminal glycine and are, thus, potential substrates for PAM. The addition of PAM to glutathione, a series of S-alkylated glutathiones, and leukotriene C(4) results in the consumption of O(2) and the production of the corresponding amidated peptide and glyoxylate. This reaction proceeds in two steps with the intermediate formation of a C-terminal alpha-hydroxyglycine-extended peptide. Amidated glutathione (gammaGlu-Cys-amide) is a relatively poor substrate for glutathione S-transferase with a V/K value that is 1.3% of that for glutathione. Peptide substrates containing a penultimate hydrophobic or sulfur-containing amino acid exhibit the highest (V/K)(app) values for PAM-catalyzed amidation. The S-alkylated glutathiones incorporate both features in the penultimate position with S-decylglutathione having the highest (V/K)(app) of the substrates described in this report.