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1.
Breast Cancer Res Treat ; 173(3): 533-543, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30361874

RESUMO

PURPOSE: There is an urgent need for the development of a predictor of response to chemotherapy for ER-positive breast cancer which is less chemosensitive than for ER-negative breast cancer in order to avoid unnecessary chemotherapy. In the present study, intrinsic subtyping by PAM50 was evaluated for its ability to predict a response to chemotherapy. PATIENTS AND METHODS: For this study, 124 patients with ER-positive breast cancer treated with neoadjuvant sequential paclitaxel and FEC (NAC) were evaluated. Tumor biopsy specimens obtained before NAC were subjected to intrinsic subtyping (IS) by gene expression (GE) using PAM50 (PAM50-IS) or immunohistochemistry (IHC-IS). RESULTS: Of the PAM50-ISs (Luminal A, Luminal B, HER2-enriched, and Basal-like), GE-Luminal A showed the lowest pCR rate (1.9%), and multivariate analysis revealed that GE-Luminal A was a significant (P = 0.031) predictor of non-pCR independently of other clinicopathological parameters, including Ki67, and tumor-infiltrating lymphocytes. Of the IHC-ISs, on the other hand, IHC-Luminal A was not significantly associated with pCR. We also found that breast tumors with low ER levels (1-9%), like ER-negative tumors, were mostly GE-HER2-enriched and GE-Basal-like, and more sensitive to NAC than those with high ER levels (≥ 10%). CONCLUSIONS: GE-Luminal A intrinsically subtyped by PAM50 was the least sensitive to NAC and very unlikely to attain pCR. IHC-Luminal A identified by IHC, on the other hand, was not significantly predictive of pCR. In addition, PAM50 revealed that tumors with low ER (1-9%) were more like ER-negative tumors than ER-positive tumors, and most such cases should therefore would better be treated with chemotherapy.


Assuntos
Amidina-Liases/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Oxigenases de Função Mista/genética , Receptores Estrogênicos/genética , Adulto , Idoso , Amidina-Liases/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/mortalidade , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Oxigenases de Função Mista/metabolismo , Terapia Neoadjuvante , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Receptores Estrogênicos/metabolismo , Fatores de Risco , Análise de Sobrevida , Resultado do Tratamento
2.
Diabetes Obes Metab ; 20 Suppl 2: 64-76, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30230179

RESUMO

Biosynthesis of peptide hormones by pancreatic islet endocrine cells is a tightly orchestrated process that is critical for metabolic homeostasis. Like neuroendocrine peptides, insulin and other islet hormones are first synthesized as larger precursor molecules that are processed to their mature secreted products through a series of proteolytic cleavages, mediated by the prohormone convertases Pc1/3 and Pc2, and carboxypeptidase E. Additional posttranslational modifications including C-terminal amidation of the ß-cell peptide islet amyloid polypeptide (IAPP) by peptidyl-glycine α-amidating monooxygenase (Pam) may also occur. Genome-wide association studies (GWAS) have showed genetic linkage of these processing enzymes to obesity, ß-cell dysfunction, and type 2 diabetes (T2D), pointing to their important roles in metabolism and blood glucose regulation. In both type 1 diabetes (T1D) and T2D, and in the face of metabolic or inflammatory stresses, islet prohormone processing may become impaired; indeed elevated proinsulin:insulin (PI:I) ratios are a hallmark of the ß-cell dysfunction in T2D. Recent studies suggest that genetic or acquired defects in proIAPP processing may lead to the production and secretion of incompletely processed forms of proIAPP that could contribute to T2D pathogenesis, and additionally that impaired processing of both PI and proIAPP may be characteristic of ß-cell dysfunction in T1D. In islet α-cells, the prohormone proglucagon is normally processed to bioactive glucagon by Pc2 but may express Pc1/3 under certain conditions leading to production of GLP-1(7-36NH2 ). A better understanding of how ß-cell processing of PI and proIAPP, as well as α-cell processing of proglucagon, are impacted by genetic susceptibility and in the face of diabetogenic stresses, may lead to new therapeutic approaches for improving islet function in diabetes.


Assuntos
Carboxipeptidase H/fisiologia , Ilhotas Pancreáticas/metabolismo , Pró-Proteína Convertase 1/fisiologia , Pró-Proteína Convertase 2/fisiologia , Amidina-Liases/metabolismo , Células Secretoras de Glucagon/metabolismo , Humanos , Insulina/biossíntese , Células Secretoras de Insulina/metabolismo , Oxigenases de Função Mista/metabolismo , Proinsulina/metabolismo
3.
Nat Genet ; 50(8): 1122-1131, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30054598

RESUMO

The molecular mechanisms underpinning susceptibility loci for type 2 diabetes (T2D) remain poorly understood. Coding variants in peptidylglycine α-amidating monooxygenase (PAM) are associated with both T2D risk and insulinogenic index. Here, we demonstrate that the T2D risk alleles impact negatively on overall PAM activity via defects in expression and catalytic function. PAM deficiency results in reduced insulin content and altered dynamics of insulin secretion in a human ß-cell model and primary islets from cadaveric donors. Thus, our results demonstrate a role for PAM in ß-cell function, and establish molecular mechanisms for T2D risk alleles at this locus.


Assuntos
Amidina-Liases/genética , Diabetes Mellitus Tipo 2/genética , Secreção de Insulina/genética , Células Secretoras de Insulina/patologia , Oxigenases de Função Mista/genética , Alelos , Animais , Linhagem Celular , Predisposição Genética para Doença , Células HEK293 , Humanos , Insulina/genética , Camundongos , Polimorfismo de Nucleotídeo Único
4.
J Biol Chem ; 293(36): 13897-13909, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-29997255

RESUMO

PHR (PAM/Highwire/RPM-1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F-box (SCF) complex that contains the F-box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.


Assuntos
Amidina-Liases/química , Oxigenases de Função Mista/química , Nicotinamida-Nucleotídeo Adenililtransferase/química , Proteínas Ligases SKP Culina F-Box/química , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal , Animais , Caenorhabditis elegans , Proteínas F-Box/metabolismo , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/fisiologia , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Ligação Proteica , Proteínas Quinases Associadas a Fase S , Proteínas Ligases SKP Culina F-Box/fisiologia , Ubiquitina-Proteína Ligases
5.
Cardiovasc Pathol ; 34: 50-57, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29677652

RESUMO

Skeletal myocytes have well-established fast and slow twitch fibers with unique gene and protein specific expression patterns. By immunohistochemical staining, these show a mosaic pattern across myocytes. We hypothesized cardiac myocytes may behave similarly where some proteins are differentially expressed between mature cardiomyocytes. We utilized the tool HPASubC on over 52,000 cardiac images of the Human Protein Atlas to identify differential protein expression patterns by immunohistochemistry across the cardiomyocytes. We matched identified proteins to open chromatin and gene expression data. We identified 143 putative proteins with mosaic patterns of expression across the cardiomyocytes. We validated four of these proteins (MYL3, MYL4, PAM, and MYOM1) and demonstrated unique atrial or ventricular patterns of expression for each. Acetylation of histone H3K27 at the promoters of these four genes were consistent with the atrial/ventricular expression patterns. Despite the generally accepted homogeneity of cardiomyocytes, a small subset of proteins varies between cardiomyocytes in a mosaic pattern. This fundamental process has been previously uncharacterized. These changes may inform on different functional and disease-related activities of proteins in individual cardiomyocytes.


Assuntos
Proteínas Musculares/análise , Miócitos Cardíacos/química , Acetilação , Amidina-Liases/análise , Conectina/análise , Regulação da Expressão Gênica , Histonas/química , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Oxigenases de Função Mista/análise , Mosaicismo , Proteínas Musculares/genética , Cadeias Leves de Miosina/análise , Reconhecimento Automatizado de Padrão , Fenótipo , Regiões Promotoras Genéticas , Mapas de Interação de Proteínas , Proteômica/métodos
6.
J Biol Chem ; 293(16): 6052-6063, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29487130

RESUMO

Neuropeptides constitute a vast and functionally diverse family of neurochemical signaling molecules and are widely involved in the regulation of various physiological processes. The nematode Caenorhabditis elegans is well-suited for the study of neuropeptide biochemistry and function, as neuropeptide biosynthesis enzymes are not essential for C. elegans viability. This permits the study of neuropeptide biosynthesis in mutants lacking certain neuropeptide-processing enzymes. Mass spectrometry has been used to study the effects of proprotein convertase and carboxypeptidase mutations on proteolytic processing of neuropeptide precursors and on the peptidome in C. elegans However, the enzymes required for the last step in the production of many bioactive peptides, the carboxyl-terminal amidation reaction, have not been characterized in this manner. Here, we describe three genes that encode homologs of neuropeptide amidation enzymes in C. elegans and used tandem LC-MS to compare neuropeptides in WT animals with those in newly generated mutants for these putative amidation enzymes. We report that mutants lacking both a functional peptidylglycine α-hydroxylating monooxygenase and a peptidylglycine α-amidating monooxygenase had a severely altered neuropeptide profile and also a decreased number of offspring. Interestingly, single mutants of the amidation enzymes still expressed some fully processed amidated neuropeptides, indicating the existence of a redundant amidation mechanism in C. elegans All MS data are available via ProteomeXchange with the identifier PXD008942. In summary, the key steps in neuropeptide processing in C. elegans seem to be executed by redundant enzymes, and loss of these enzymes severely affects brood size, supporting the need of amidated peptides for C. elegans reproduction.


Assuntos
Amidina-Liases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Neuropeptídeos/metabolismo , Amidina-Liases/química , Amidina-Liases/genética , Sequência de Aminoácidos , Animais , Vias Biossintéticas , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Cobre/metabolismo , Deleção de Genes , Humanos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação , Neuropeptídeos/genética , Alinhamento de Sequência , Espectrometria de Massas em Tandem
7.
Hum Genomics ; 11(1): 29, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29162152

RESUMO

BACKGROUND: Peptidylglycine-α-amidating monooxygenase (PAM) may play a role in the secretion of atrial natriuretic peptide (ANP), which is a hormone involved in the maintenance of blood pressure (BP). The objective of the present study was to determine whether PAM is a novel candidate gene for hypertension (HTN). RESULTS: A total of 2153 Korean participants with normotension and HTN were included. Genotype data were obtained using the Korean Chip. The rs13175330 polymorphism of the PAM gene was selected from the ten single nucleotide polymorphisms (SNPs) most strongly associated with BP. The presence of the G allele of the PAM rs13175330 A>G SNP was associated with a higher risk of HTN after adjustments for age, sex, BMI, smoking, and drinking [OR 1.607 (95% CI 1.220-2.116), p = 0.001]. The rs13175330 G allele carriers in the HTN group treated without antihypertensive therapy (HTN w/o therapy) had significantly higher systolic and diastolic BP than the AA carriers, whereas the G allele carriers in the HTN group treated with antihypertensive therapy (HTN w/ therapy) showed significantly higher diastolic BP. Furthermore, rs13175330 G allele carriers in the HTN w/o therapy group had significantly increased levels of insulin, insulin resistance, and oxidized low-density lipoprotein (LDL) and significantly decreased LDL-cholesterol levels and LDL particle sizes compared to the AA carriers. CONCLUSION: These results suggest that the PAM rs13175330 A>G SNP is a novel candidate gene for HTN in the Korean population. Additionally, the PAM rs13175330 G allele might be associated with insulin resistance and LDL atherogenicity in patients with HTN.


Assuntos
Amidina-Liases/genética , Hipertensão/genética , Oxigenases de Função Mista/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Asiático/genética , Pressão Sanguínea/genética , Estudos de Casos e Controles , HDL-Colesterol/sangue , HDL-Colesterol/genética , Feminino , Predisposição Genética para Doença , Humanos , Hipertensão/etiologia , Insulina/sangue , Resistência à Insulina/genética , Lipoproteínas LDL/sangue , Lipoproteínas LDL/genética , Masculino , Pessoa de Meia-Idade
8.
Anal Chem ; 89(13): 6992-6999, 2017 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-28590120

RESUMO

The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.


Assuntos
Calcitonina/análogos & derivados , Calcitonina/análise , Cromatografia Líquida de Alta Pressão/métodos , Glicopeptídeos/análise , Precursores de Proteínas/análise , Amidina-Liases/antagonistas & inibidores , Calcitonina/biossíntese , Calcitonina/metabolismo , Carboxipeptidase H/antagonistas & inibidores , Linhagem Celular Tumoral , Ácidos Graxos Monoinsaturados/farmacologia , Glicopeptídeos/biossíntese , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Oxigenases de Função Mista/antagonistas & inibidores , Precursores de Proteínas/biossíntese , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Extração em Fase Sólida/métodos , Succinatos/farmacologia
9.
Eur J Cell Biol ; 96(5): 407-417, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28377049

RESUMO

Peptidylglycine α-amidating monooxygenase (PAM) is highly expressed in neurons and endocrine cells, where it catalyzes one of the final steps in the biosynthesis of bioactive peptides. PAM is also expressed in unicellular organisms such as Chlamydomonas reinhardtii, which do not store peptides in secretory granules. As for other granule membrane proteins, PAM is retrieved from the cell surface and returned to the trans-Golgi network. This pathway involves regulated entry of PAM into multivesicular body intralumenal vesicles (ILVs). The aim of this study was defining the endocytic pathways utilized by PAM in cells that do not store secretory products in granules. Using stably transfected HEK293 cells, endocytic trafficking of PAM was compared to that of the mannose 6-phosphate (MPR) and EGF (EGFR) receptors, established markers for the endosome to trans-Golgi network and degradative pathways, respectively. As in neuroendocrine cells, PAM internalized by HEK293 cells accumulated in the trans-Golgi network. Based on surface biotinylation, >70% of the PAM on the cell surface was recovered intact after a 4h chase and soluble, bifunctional PAM was produced. Endosomes containing PAM generally contained both EGFR and MPR and ultrastructural analysis confirmed that all three cargos accumulated in ILVs. PAM containing multivesicular bodies made frequent dynamic tubular contacts with younger and older multivesicular bodies. Frequent dynamic contacts were observed between lysosomes and PAM containing early endosomes and multivesicular bodies. The ancient ability of PAM to localize to ciliary membranes, which release bioactive ectosomes, may be related to its ability to accumulate in ILVs and exosomes.


Assuntos
Amidina-Liases/metabolismo , Oxigenases de Função Mista/metabolismo , Corpos Multivesiculares/metabolismo , Transporte Proteico/fisiologia , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Receptor IGF Tipo 2/metabolismo , Vesículas Secretórias/metabolismo
10.
Metallomics ; 8(8): 729-33, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27242196

RESUMO

Cuproproteins such as PHM and DBM mature in late endosomal vesicles of the mammalian secretory pathway where changes in vesicle pH are employed for sorting and post-translational processing. Colocation with the P1B-type ATPase ATP7A suggests that the latter is the source of copper and supports a mechanism where selectivity in metal transfer is achieved by spatial colocation of partner proteins in their specific organelles or vesicles. In previous work we have suggested that a lumenal loop sequence located between trans-membrane helices TM1 and TM2 of the ATPase, and containing five histidines and four methionines, acts as an organelle-specific chaperone for metallation of the cuproproteins. The hypothesis posits that the pH of the vesicle regulates copper ligation and loop conformation via a mechanism which involves His to Met ligand switching induced by histidine protonation. Here we report the effect of pH on the HM loop copper coordination using X-ray absorption spectroscopy (XAS), and show via selenium substitution of the Met residues that the HM loop undergoes similar conformational switching to that found earlier for its partner PHM. We hypothesize that in the absence of specific chaperones, HM motifs provide a template for building a flexible, pH-sensitive transfer site whose structure and function can be regulated to accommodate the different active site structural elements and pH environments of its partner proteins.


Assuntos
Amidina-Liases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Oxigenases de Função Mista/metabolismo , Amidina-Liases/química , Sequência de Aminoácidos , Domínio Catalítico , Proteínas de Transporte de Cátions/química , Cobre/química , ATPases Transportadoras de Cobre/química , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Oxigenases de Função Mista/química , Modelos Moleculares , Chaperonas Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência , Espectroscopia por Absorção de Raios X
11.
Nat Commun ; 7: 10640, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26879543

RESUMO

Brain development requires a fine-tuned copper homoeostasis. Copper deficiency or excess results in severe neuro-pathologies. We demonstrate that upon neuronal differentiation, cellular demand for copper increases, especially within the secretory pathway. Copper flow to this compartment is facilitated through transcriptional and metabolic regulation. Quantitative real-time imaging revealed a gradual change in the oxidation state of cytosolic glutathione upon neuronal differentiation. Transition from a broad range of redox states to a uniformly reducing cytosol facilitates reduction of the copper chaperone Atox1, liberating its metal-binding site. Concomitantly, expression of Atox1 and its partner, a copper transporter ATP7A, is upregulated. These events produce a higher flux of copper through the secretory pathway that balances copper in the cytosol and increases supply of the cofactor to copper-dependent enzymes, expression of which is elevated in differentiated neurons. Direct link between glutathione oxidation and copper compartmentalization allows for rapid metabolic adjustments essential for normal neuronal function.


Assuntos
Adenosina Trifosfatases/metabolismo , Amidina-Liases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Glutationa/metabolismo , Metalochaperonas/metabolismo , Oxigenases de Função Mista/metabolismo , Neurogênese , Neurônios/metabolismo , Oxirredução , Via Secretória , Animais , Embrião de Galinha , ATPases Transportadoras de Cobre , Citosol , Eletroporação , Dissulfeto de Glutationa/metabolismo , Células HEK293 , Humanos , Immunoblotting , NADP/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Espectrofotometria Atômica , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo
12.
J Mol Endocrinol ; 56(4): T63-76, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26667899

RESUMO

A critical role for peptide C-terminal amidation was apparent when the first bioactive peptides were identified. The conversion of POMC into adrenocorticotropic hormone and then into α-melanocyte-stimulating hormone, an amidated peptide, provided a model system for identifying the amidating enzyme. Peptidylglycine α-amidating monooxygenase (PAM), the only enzyme that catalyzes this modification, is essential; mice lacking PAM survive only until mid-gestation. Purification and cloning led to the discovery that the amidation of peptidylglycine substrates proceeds in two steps: peptidylglycine α-hydroxylating monooxygenase catalyzes the copper- and ascorbate-dependent α-hydroxylation of the peptidylglycine substrate; peptidyl-α-hydroxyglycine α-amidating lyase cleaves the N-C bond, producing amidated product and glyoxylate. Both enzymes are contained in the luminal domain of PAM, a type 1 integral membrane protein. The structures of both catalytic cores have been determined, revealing how they interact with metals, molecular oxygen, and substrate to catalyze both reactions. Although not essential for activity, the intrinsically disordered cytosolic domain is essential for PAM trafficking. A phylogenetic survey led to the identification of bifunctional membrane PAM in Chlamydomonas, a unicellular eukaryote. Accumulating evidence points to a role for PAM in copper homeostasis and in retrograde signaling from the lumen of the secretory pathway to the nucleus. The discovery of PAM in cilia, cellular antennae that sense and respond to environmental stimuli, suggests that much remains to be learned about this ancient protein.


Assuntos
Amidina-Liases/metabolismo , Ácido Ascórbico/metabolismo , Cobre/metabolismo , Oxigênio/metabolismo , Pró-Opiomelanocortina/metabolismo , alfa-MSH/metabolismo , Processamento Alternativo , Amidina-Liases/química , Amidina-Liases/genética , Animais , Cílios/metabolismo , Evolução Molecular , Técnicas de Inativação de Genes , Genótipo , Humanos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Pró-Opiomelanocortina/química , Domínios e Motivos de Interação entre Proteínas , Proteólise , Relação Estrutura-Atividade
13.
Protein Expr Purif ; 119: 102-9, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26614892

RESUMO

The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)-B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5-7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5-2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.


Assuntos
Amidina-Liases/biossíntese , Hipoglicemiantes/química , Insulina/análogos & derivados , Insulina/química , Oxigenases de Função Mista/biossíntese , Proteínas Recombinantes/biossíntese , Amidina-Liases/química , Amidina-Liases/isolamento & purificação , Animais , Glicemia , Células CHO , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Diabetes Mellitus Experimental/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Feminino , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Masculino , Oxigenases de Função Mista/química , Oxigenases de Função Mista/isolamento & purificação , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
14.
J Mol Biol ; 426(17): 3028-40, 2014 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-25020232

RESUMO

In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes.


Assuntos
Amidina-Liases/química , Arabidopsis/enzimologia , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Ureo-Hidrolases/química , Domínio Catalítico , Complexos de Coordenação/química , Cristalografia por Raios X , Glioxilatos/química , Hidrólise , Cinética , Modelos Moleculares , Ligação Proteica , Homologia Estrutural de Proteína , Especificidade por Substrato , Ureia/análogos & derivados , Ureia/química
15.
Nat Genet ; 46(3): 294-8, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24464100

RESUMO

Through whole-genome sequencing of 2,630 Icelanders and imputation into 11,114 Icelandic cases and 267,140 controls followed by testing in Danish and Iranian samples, we discovered 4 previously unreported variants affecting risk of type 2 diabetes (T2D). A low-frequency (1.47%) variant in intron 1 of CCND2, rs76895963[G], reduces risk of T2D by half (odds ratio (OR) = 0.53, P = 5.0 × 10(-21)) and is correlated with increased CCND2 expression. Notably, this variant is also associated with both greater height and higher body mass index (1.17 cm per allele, P = 5.5 × 10(-12) and 0.56 kg/m(2) per allele, P = 6.5 × 10(-7), respectively). In addition, two missense variants in PAM, encoding p.Asp563Gly (frequency of 4.98%) and p.Ser539Trp (frequency of 0.65%), confer moderately higher risk of T2D (OR = 1.23, P = 3.9 × 10(-10) and OR = 1.47, P = 1.7 × 10(-5), respectively), and a rare (0.20%) frameshift variant in PDX1, encoding p.Gly218Alafs*12, associates with high risk of T2D (OR = 2.27, P = 7.3 × 10(-7)).


Assuntos
Amidina-Liases/genética , Ciclina D2/genética , Diabetes Mellitus Tipo 2/genética , Variação Genética , Proteínas de Homeodomínio/genética , Oxigenases de Função Mista/genética , Transativadores/genética , Estatura/genética , Peso Corporal/genética , Estudos de Casos e Controles , Dinamarca , Diabetes Mellitus Tipo 2/patologia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Islândia , Irã (Geográfico) , Masculino , Polimorfismo de Nucleotídeo Único , Fatores de Risco
16.
Database (Oxford) ; 2013: bat071, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24107613

RESUMO

An opaque biochemical definition, an insufficient functional characterization, an interpolated database description, and a beautiful 3D structure with a wrong reaction. All these are elements of an exemplar case of misannotation in biological databases and confusion in the scientific literature concerning genes and enzymes acting on ureidoglycolate, an intermediate of purine catabolism. Here we show biochemical evidence for the relocation of genes assigned to EC 3.5.3.19 (ureidoglycolate hydrolase, releasing ammonia), such as allA of Escherichia coli or DAL3 of Saccharomyces cerevisiae, to EC 4.3.2.3 (ureidoglycolate lyase, releasing urea). The EC 3.5.3.19 should be more appropriately named ureidoglycolate amidohydrolase and include genes equivalent to UAH of Arabidopsis thaliana. The distinction between ammonia- or urea-releasing activities from ureidoglycolate is relevant for the understanding of nitrogen metabolism in various organisms and of virulence factors in certain pathogens rather than a nomenclature problem. We trace the original fault in database annotation and provide a rationale for its incorporation and persistence in the scientific literature. Notwithstanding the technological distance, yet not surprising for the constancy of human nature, error categories and mechanisms established in the study of the work of amanuensis monks still apply to the modern curation of biological databases.


Assuntos
Amidina-Liases/metabolismo , Aminoidrolases/metabolismo , Bases de Dados como Assunto , Publicações , Arabidopsis/enzimologia , Biocatálise , Escherichia coli/enzimologia , Glicolatos/química , Glicolatos/metabolismo , Humanos , Nitrogênio/metabolismo , Saccharomyces cerevisiae/enzimologia , Terminologia como Assunto
17.
Microb Pathog ; 59-60: 19-28, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23583291

RESUMO

Coccidioides is the causative agent of a potentially life-threatening respiratory disease of humans. A feature of this mycosis is that pH measurements of the microenvironment of pulmonary abscesses are consistently alkaline due to ammonia production during the parasitic cycle. We previously showed that enzymatically active urease is partly responsible for elevated concentrations of extracellular ammonia at sites of lung infection and contributes to both localized host tissue damage and exacerbation of the respiratory disease in BALB/c mice. Disruption of the urease gene (URE) of Coccidioides posadasii only partially reduced the amount of ammonia detected during in vitro growth of the parasitic phase, suggesting that other ammonia-producing pathways exist that may also contribute to the virulence of this pathogen. Ureidoglycolate hydrolase (Ugh) expressed by bacteria, fungi and higher plants catalyzes the hydrolysis of ureidoglycolate to yield glyoxylate and the release CO2 and ammonia. This enzymatic pathway is absent in mice and humans. Ureidoglycolate hydrolase gene deletions were conducted in a wild type (WT) isolate of C. posadasii as well as the previously generated Δure knock-out strain. Restorations of UGH in the mutant stains were performed to generate and evaluate the respective revertants. The double mutant revealed a marked decrease in the amount of extracellular ammonia without loss of reproductive competence in vitro compared to both the WT and Δure parental strains. BALB/c mice challenged intranasally with the Δugh/Δure mutant showed 90% survival after 30 days, decreased fungal burden, and well-organized pulmonary granulomas. We conclude that loss of both Ugh and Ure activity significantly reduced the virulence of this fungal pathogen.


Assuntos
Amidina-Liases/metabolismo , Amônia/metabolismo , Coccidioides/metabolismo , Coccidioides/patogenicidade , Coccidioidomicose/patologia , Pneumopatias Fúngicas/patologia , Urease/metabolismo , Amidina-Liases/genética , Animais , Coccidioides/enzimologia , Coccidioides/genética , Coccidioidomicose/microbiologia , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Humanos , Pneumopatias Fúngicas/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sobrevida , Urease/genética , Virulência
18.
Nat Genet ; 45(2): 197-201, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23263489

RESUMO

Insulin secretion has a crucial role in glucose homeostasis, and failure to secrete sufficient insulin is a hallmark of type 2 diabetes. Genome-wide association studies (GWAS) have identified loci contributing to insulin processing and secretion; however, a substantial fraction of the genetic contribution remains undefined. To examine low-frequency (minor allele frequency (MAF) 0.5-5%) and rare (MAF < 0.5%) nonsynonymous variants, we analyzed exome array data in 8,229 nondiabetic Finnish males using the Illumina HumanExome Beadchip. We identified low-frequency coding variants associated with fasting proinsulin concentrations at the SGSM2 and MADD GWAS loci and three new genes with low-frequency variants associated with fasting proinsulin or insulinogenic index: TBC1D30, KANK1 and PAM. We also show that the interpretation of single-variant and gene-based tests needs to consider the effects of noncoding SNPs both nearby and megabases away. This study demonstrates that exome array genotyping is a valuable approach to identify low-frequency variants that contribute to complex traits.


Assuntos
Exoma/genética , Variação Genética , Insulina/genética , Insulina/metabolismo , Amidina-Liases/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Jejum/sangue , Finlândia , Frequência do Gene , Genética Populacional , Genótipo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Secreção de Insulina , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Oxigenases de Função Mista/genética , Anotação de Sequência Molecular , Proinsulina/sangue , Proteínas Supressoras de Tumor/genética
19.
Mol Biol Evol ; 29(10): 3095-109, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22496439

RESUMO

Secreted peptides, produced by enzymatic processing of larger precursor molecules, are found throughout the animal kingdom and play important regulatory roles as neurotransmitters and hormones. Many require a carboxy-terminal modification, involving the conversion of a glycine residue into an α-amide, for their biological activity. Two sequential enzymatic activities catalyze this conversion: a monooxygenase (peptidylglycine α-hydroxylating monooxygenase or PHM) and an amidating lyase (peptidyl-α-hydroxyglycine α-amidating lyase or PAL). In vertebrates, these activities reside in a single polypeptide known as peptidylglycine α-amidating monooxygenase (PAM), which has been extensively studied in the context of neuropeptide modification. Bifunctional PAMs have been reported from some invertebrates, but the phylogenetic distribution of PAMs and their evolutionary relationship to PALs and PHMs is unclear. Here, we report sequence and expression data for two PAMs from the coral Acropora millepora (Anthozoa, Cnidaria), as well as providing a comprehensive survey of the available sequence data from other organisms. These analyses indicate that bifunctional PAMs predate the origins of the nervous and endocrine systems, consistent with the idea that within the Metazoa their ancestral function may have been to amidate epitheliopeptides. More surprisingly, the phylogenomic survey also revealed the presence of PAMs in green algae (but not in higher plants or fungi), implying that the bifunctional enzyme either predates the plant/animal divergence and has subsequently been lost in a number of lineages or perhaps that convergent evolution or lateral gene transfer has occurred. This finding is consistent with recent discoveries that other molecules once thought of as "neural" predate nervous systems.


Assuntos
Antozoários/enzimologia , Clorófitas/enzimologia , Oxigenases de Função Mista/genética , Complexos Multienzimáticos/genética , Neurônios/enzimologia , Processamento Alternativo/genética , Amidina-Liases/química , Amidina-Liases/metabolismo , Sequência de Aminoácidos , Animais , Antozoários/genética , Biocatálise , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fatores de Tempo
20.
J Exp Bot ; 62(1): 307-18, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20813786

RESUMO

Ureidoglycolate is an intermediate in the degradation of the ureides, allantoin and allantoate, found in many organisms. In some leguminous plant species these compounds are used to transport recently fixed nitrogen in the root nodules to the aerial parts of the plant. In the present study, it was demonstrated that purified ureidoglycolases from chickpea (Cicer arietinum) and French bean (Phaseolus vulgaris) do not produce glyoxylate, and can use phenylhydrazine as a substrate with K(m) values of 4.0 mM and 8.5 mM, respectively. Furthermore, these enzymes catalyse the transfer of the ureidoglycolyl group to phenylhydrazine to produce ureidoglycolyl phenylhydrazide, which degrades non-enzymatically to glyoxylate phenylhydrazone and urea. This supports their former classification as ureidoglycolate urea-lyases. The enzymatic reaction catalysed by the characterized ureidoglycolases uncovered here can be viewed as a novel type of phenylhydrazine ureidoglycolyl transferase. The implications of these findings for ureide metabolism in legume nitrogen metabolism are discussed.


Assuntos
Amidina-Liases/metabolismo , Cicer/metabolismo , Glicolatos/metabolismo , Phaseolus/metabolismo , Proteínas de Plantas/metabolismo , Ureia/metabolismo , Amidina-Liases/genética , Cicer/enzimologia , Cicer/genética , Redes e Vias Metabólicas , Nitrogênio/metabolismo , Phaseolus/enzimologia , Phaseolus/genética , Proteínas de Plantas/genética
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