RESUMO
Inhibition of Insulin-Regulated Aminopeptidase is being actively explored for the treatment of several human diseases and several classes of inhibitors have been developed although no clinical applications have been reported yet. Here, we combine enzymological analysis with x-ray crystallography to investigate the mechanism employed by two of the most studied inhibitors of IRAP, an aryl sulfonamide and a 2-amino-4H-benzopyran named HFI-419. Although both compounds have been hypothesized to target the enzyme's active site by competitive mechanisms, we discovered that they instead target previously unidentified proximal allosteric sites and utilize non-competitive inhibition mechanisms. X-ray crystallographic analysis demonstrated that the aryl sulfonamide stabilizes the closed, more active, conformation of the enzyme whereas HFI-419 locks the enzyme in a semi-open, and likely less active, conformation. HFI-419 potency is substrate-dependent and fails to effectively block the degradation of the physiological substrate cyclic peptide oxytocin. Our findings demonstrate alternative mechanisms for inhibiting IRAP through allosteric sites and conformational restricting and suggest that the pharmacology of HFI-419 may be more complicated than initially considered. Such conformation-specific interactions between IRAP and small molecules can be exploited for the design of more effective second-generation allosteric inhibitors.
Assuntos
Sítio Alostérico , Inibidores Enzimáticos , Insulina , Sulfonamidas , Humanos , Domínio Catalítico/efeitos dos fármacos , Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Insulina/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacologia , Cristalografia por Raios X , Regulação Alostérica , Sítio Alostérico/efeitos dos fármacos , Células HEK293 , Células CHO , Animais , CricetulusRESUMO
In the present study, several new analogues of hemorphin-4, modified with unnatural conformationally restricted amino acids followed the structure Aaa-Tyr-Xxx-Trp-Thr-NH2, where Aaa is the low-molecular-weight lipophilic adamantyl building block, and Xxx is Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) was synthesized, characterized and investigated for anticonvulsant activity in three seizure tests, the maximal electroshock test (MES), 6-Hz psychomotor seizure test and timed intravenous pentylenetetrazole infusion (ivPTZ) test. The acute neurological toxicity was determined using the rota-rod test. The new synthetic neuropeptide analogues were prepared by solid-phase peptide synthesis-Fmoc chemistry and were evaluated in three doses of 1, 3 and 5 µg, respectively, administered intracerebroventricularly in male ICR mice. The physicochemical properties of these peptide analogues were evaluated as pKa and pI values were calculated using potentiometry. The IR spectrum of the compounds was recorded and the characteristic lines of both adamantane moiety and the peptide backbone were registered in the wavelength range from 4000 to 400 cm-1. The hexapeptide Ang IV was used as a positive control. From the six synthesized peptide analogues, the P4-5 was the most active at doses of 1 and 3 µg in the three seizure tests. The order of potency of other peptides was as follows: P4 > P4-3 = P4-4 > P4-2 > Ang IV in MES, P4-4 ≥ P4-1 > P4-3 > P4-2 > P4 > Ang IV in 6-Hz test and P4-4 = P4-3 > P4-2 = P4 > Ang IV in ivPTZ test. None of the peptides displayed neurotoxicity in the rota-rod test. Docking study results suggest that direct H-bonding and ionic interactions between our synthetic ligands and residues, responsible for coordination of Zn2+ along with hydrophobic interactions between our ligands and IRAP active site are the most important for the ligand binding. The results propose that incorporation of adamantane and cycloalkane building blocks in the peptide chain of the hemorphin-4 scaffold is important for the potential high biological activity.
Assuntos
Anticonvulsivantes/química , Hemoglobinas/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Animais , Anticonvulsivantes/administração & dosagem , Anticonvulsivantes/síntese química , Sítios de Ligação , Cistinil Aminopeptidase/química , Hemoglobinas/administração & dosagem , Hemoglobinas/síntese química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Infusões Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos ICR , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/síntese química , Convulsões/prevenção & controle , Relação Estrutura-AtividadeRESUMO
The vasotocin/vasopressin and isotocin/mesotocin/oxytocin family of nonapeptides regulate social behaviors and physiological functions associated with reproductive physiology and osmotic balance. While experimental and correlative studies provide evidence for these nonapeptides as modulators of behavior across all classes of vertebrates, mechanisms for nonapeptide inactivation in regulating these functions have been largely overlooked. Leucyl-cystinyl aminopeptidase (LNPEP) - also known as vasopressinase, oxytocinase, placental leucine aminopeptidase (P-LAP), and insulin-regulated aminopeptidase (IRAP) - is a membrane-bound zinc-dependent metalloexopeptidase enzyme that inactivates vasopressin, oxytocin, and select other cyclic polypeptides. In humans, LNPEP plays a key role in the clearance of oxytocin during pregnancy. However, the evolutionary diversity, expression distribution, and functional roles of LNPEP remain unresolved for other vertebrates. Here, we isolated and sequenced a full-length cDNA encoding a LNPEP-like polypeptide of 1033 amino acids from the ovarian tissue of Amargosa pupfish, Cyprinodon nevadensis. This deduced polypeptide exhibited high amino acid identity to human LNPEP both in the protein's active domain that includes the peptide binding site and zinc cofactor binding motif (53.1% identity), and in an intracellular region that distinguishes LNPEP from other aminopeptidases (70.3% identity). Transcripts encoding this LNPEP enzyme (lnpep) were detected at highest relative abundance in the gonads, hypothalamus, forebrain, optic tectum, gill and skeletal muscle of adult pupfish. Further evaluation of lnpep transcript abundance in the brain of sexually-mature pupfish revealed that lnpep mRNAs were elevated in the hypothalamus of socially subordinate females and males, and at lower abundance in the telencephalon of socially dominant males compared to dominant females. These findings provide evidence of an association between behavioral social status and hypothalamic lnpep transcript abundance and suggest that variation in the rate of VT/IT peptide inactivation by LNPEP may be a contributing component in the mechanism whereby nonapeptides regulate social behavior.
Assuntos
Comportamento Animal , Cistinil Aminopeptidase/metabolismo , Peixes/genética , Peixes/metabolismo , Hipotálamo/metabolismo , Comportamento Social , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Cistinil Aminopeptidase/química , DNA Complementar/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Filogenia , Gravidez , Análise de Componente Principal , Prosencéfalo/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Insulin-regulated aminopeptidase (IRAP) is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates, although the mechanism behind this wide specificity is not clearly understood. We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at 2.53 Å resolution. In the presence of this inhibitor, the enzyme adopts a novel conformation in which domains II and IV are juxtaposed, forming a hollow structure that excludes external solvent access to the catalytic center. A loop adjacent to the enzyme's GAMEN motif undergoes structural reconfiguration, allowing the accommodation of bulky inhibitor side chains. Atomic interactions between the inhibitor and IRAP that are unique to this conformation can explain the strong selectivity compared to homologous aminopeptidases ERAP1 and ERAP2. This conformation provides insight on IRAP's catalytic cycle and reveals significant active-site plasticity that may underlie its substrate permissiveness.
Assuntos
Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/metabolismo , Inibidores Enzimáticos/farmacologia , Conformação Proteica/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Cistinil Aminopeptidase/química , Inibidores Enzimáticos/química , Humanos , Ligantes , Modelos Moleculares , Peptídeos/metabolismo , Especificidade por SubstratoRESUMO
Transition state and high energy intermediate mimetics have the potential to be very potent enzyme inhibitors. In this study, a model of peptide hydrolysis in the active site of insulin-regulated aminopeptidase (IRAP) was developed using density functional theory calculations and the cluster approach. The 3D structure models of the reaction coordinates were used for virtual screening to obtain new chemical starting points for IRAP inhibitors. This mechanism-based virtual screening process managed to identify several known peptidase inhibitors from a library of over 5 million compounds, and biological testing identified one compound not previously reported as an IRAP inhibitor. This novel methodology for virtual screening is a promising approach to identify new inhibitors mimicking key transition states or intermediates of an enzymatic reaction.
Assuntos
Cistinil Aminopeptidase/antagonistas & inibidores , Inibidores Enzimáticos/química , Teoria Quântica , Termodinâmica , Animais , Sítios de Ligação , Células CHO , Cricetulus , Cistinil Aminopeptidase/química , Avaliação Pré-Clínica de Medicamentos , Modelos Moleculares , Estrutura MolecularRESUMO
Insulin-regulated aminopeptidase (IRAP or oxytocinase) is a membrane-bound zinc-metallopeptidase that cleaves neuroactive peptides in the brain and produces memory enhancing effects when inhibited. We have determined the crystal structure of human IRAP revealing a closed, four domain arrangement with a large, mostly buried cavity abutting the active site. The structure reveals that the GAMEN exopeptidase loop adopts a very different conformation from other aminopeptidases, thus explaining IRAP's unique specificity for cyclic peptides such as oxytocin and vasopressin. Computational docking of a series of IRAP-specific cognitive enhancers into the crystal structure provides a molecular basis for their structure-activity relationships and demonstrates that the structure will be a powerful tool in the development of new classes of cognitive enhancers for treating a variety of memory disorders such as Alzheimer's disease.
Assuntos
Cistinil Aminopeptidase/química , Cistinil Aminopeptidase/metabolismo , Peptídeos Cíclicos/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Humanos , Insulina/metabolismo , Modelos Moleculares , Conformação Proteica , Especificidade por SubstratoRESUMO
Uptake of circulating glucose into the cells happens via the insulin- mediated signalling pathway, which translocates the glucose transporter 4 (GLUT4) vesicles from the intracellular compartment to the plasma membrane. RabãGTPases are involved in this vesicle trafficking, where RabãGTPase-activating proteins (RabGAP) enhance the GTP to GDP hydrolysis. TBC1D4 (AS160) and TBC1D1 are functional RabGAPs in the adipocytes and the skeletonal myocytes, respectively. These proteins contain two phosphotyrosine-binding domains (PTBs) at the amino-terminus of the catalytic RabGAP domain. The second PTB has been shown to interact with the cytoplasmic region of the insulin-regulated aminopeptidase (IRAP) of the GLUT4 vesicle. In this study, we quantitatively measured the â¼µM affinity (KD) between TBC1D4 PTB and IRAP using isothermal titration calorimetry, and further showed that IRAP residues 1-49 are the major region mediating this interaction. We also demonstrated that the IRAP residues 1-15 are necessary but not sufficient for the PTB interaction.
Assuntos
Calorimetria , Proteínas Ativadoras de GTPase/metabolismo , Cistinil Aminopeptidase/química , Cistinil Aminopeptidase/metabolismo , Proteínas Ativadoras de GTPase/química , Transportador de Glucose Tipo 4/metabolismo , Humanos , Insulina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
The development of inhibitors of insulin-regulated aminopeptidase (IRAP), a membrane-bound zinc metallopeptidase, is a promising approach for the discovery of drugs for the treatment of memory loss such as that associated with Alzheimer's disease. There is, however, no consensus in the literature about the mechanism by which inhibition occurs. Sequence alignments, secondary structure predictions, and homology models based on the structures of recently determined related metallopeptidases suggest that the extracellular region consists of four domains. Partial proteolysis and mass spectrometry reported here confirm some of the domain boundaries. We have produced purified recombinant fragments of human IRAP on the basis of these data and examined their kinetic and biochemical properties. Full-length extracellular constructs assemble as dimers with different nonoverlapping fragments dimerizing as well, suggesting an extended dimer interface. Only recombinant fragments containing domains 1 and 2 possess aminopeptidase activity and bind the radiolabeled hexapeptide inhibitor, angiotensin IV (Ang IV). However, fragments lacking domains 3 and 4 possess reduced activity, although they still bind a range of inhibitors with the same affinity as longer fragments. In the presence of Ang IV, IRAP is resistant to proteolysis, suggesting significant conformational changes occur upon binding of the inhibitor. We show that IRAP has a second Zn(2+) binding site, not associated with the catalytic region, which is lost upon binding Ang IV. Modulation of activity caused by domains 3 and 4 is consistent with a conformational change regulating access to the active site of IRAP.
Assuntos
Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Doença de Alzheimer/tratamento farmacológico , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Sítios de Ligação , Domínio Catalítico , Cistinil Aminopeptidase/genética , Cistinil Aminopeptidase/metabolismo , Bases de Dados de Proteínas , Humanos , Hidrólise , Cinética , Modelos Moleculares , Terapia de Alvo Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Zinco/metabolismoRESUMO
Insulin stimulates glucose uptake by regulating translocation of the GLUT4 glucose transporter from intracellular compartments to the plasma membrane. In the absence of insulin GLUT4 is actively sequestered away from the general endosomes into GLUT4-specialized compartments, thereby controlling the amount of GLUT4 at the plasma membrane. Here, we investigated the role of the aminopeptidase IRAP in GLUT4 trafficking. In unstimulated IRAP knockdown adipocytes, plasma membrane GLUT4 levels are elevated because of increased exocytosis, demonstrating an essential role of IRAP in GLUT4 retention. Current evidence supports the model that AS160 RabGAP, which is required for basal GLUT4 retention, is recruited to GLUT4 compartments via an interaction with IRAP. However, here we show that AS160 recruitment to GLUT4 compartments and AS160 regulation of GLUT4 trafficking were unaffected by IRAP knockdown. These results demonstrate that AS160 is recruited to membranes by an IRAP-independent mechanism. Consistent with a role independent of AS160, we showed that IRAP functions in GLUT4 sorting from endosomes to GLUT4-specialized compartments. This is revealed by the relocalization of GLUT4 to endosomes in IRAP knockdown cells. Although IRAP knockdown has profound effects on GLUT4 traffic, GLUT4 knockdown does not affect IRAP trafficking, demonstrating that IRAP traffics independent of GLUT4. In sum, we show that IRAP is both cargo and a key regulator of the insulin-regulated pathway.
Assuntos
Cistinil Aminopeptidase/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Cistinil Aminopeptidase/química , Endocitose/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Proteínas Ativadoras de GTPase/metabolismo , Técnicas de Silenciamento de Genes , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacosRESUMO
Inhibition of insulin-regulated aminopeptidase (IRAP) has been demonstrated to facilitate memory in rodents, making IRAP a potential target for the development of cognitive enhancing therapies. In this study, we generated a 3-D model of the catalytic domain of IRAP based on the crystal structure of leukotriene A4 hydrolase (LTA4H). This model identified two key residues at the 'entrance' of the catalytic cleft of IRAP, Ala427 and Leu483, which present a more open arrangement of the S1 subsite compared with LTA4H. These residues may define the size and 3-D structure of the catalytic pocket, thereby conferring substrate and inhibitor specificity. Alteration of the S1 subsite by the mutation A427Y in IRAP markedly increased the rate of substrate cleavage V of the enzyme for a synthetic substrate, although a corresponding increase in the rate of cleavage of peptide substrates Leu-enkephalin and vasopressin was was not apparent. In contrast, [L483F]IRAP demonstrated a 30-fold decrease in activity due to changes in both substrate affinity and rate of substrate cleavage. [L483F]IRAP, although capable of efficiently cleaving the N-terminal cysteine from vasopressin, was unable to cleave the tyrosine residue from either Leu-enkephalin or Cyt6-desCys1-vasopressin (2-9), both substrates of IRAP. An 11-fold reduction in the affinity of the peptide inhibitor norleucine1-angiotensin IV was observed, whereas the affinity of angiotensin IV remained unaltered. In additionm we predict that the peptide inhibitors bind to the catalytic site, with the NH2-terminal P1 residue occupying the catalytic cleft (S1 subsite) in a manner similar to that proposed for peptide substrates.
Assuntos
Cistinil Aminopeptidase/química , Sequência de Aminoácidos , Domínio Catalítico , Linhagem Celular , Cistinil Aminopeptidase/genética , Cistinil Aminopeptidase/metabolismo , Encefalina Leucina/metabolismo , Humanos , Cinética , Leucina/análogos & derivados , Leucina/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/química , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Vasopressinas/metabolismoRESUMO
It is becoming evident that several aminopeptidases belonging to the M1 family such as aminopeptidase A (APA), placental leucine aminopeptidase (P-LAP), and adipocyte-derived leucine aminopeptidase (A-LAP) play important roles in the regulation of blood pressure under both the physiological and pathological conditions. They share HEXXH(X)(18)E zinc-binding and GAMEN motifs essential for enzymatic activities. In this review, the current situation regarding the biochemical characteristics of these enzymes including enzymatic properties and modes of action is summarized.
Assuntos
Aminopeptidases/metabolismo , Pressão Sanguínea/fisiologia , Hipertensão/enzimologia , Aminopeptidases/química , Antígenos CD13/química , Antígenos CD13/metabolismo , Catálise , Cistinil Aminopeptidase/química , Cistinil Aminopeptidase/metabolismo , Glutamil Aminopeptidase/química , Glutamil Aminopeptidase/metabolismo , Humanos , Hipertensão/fisiopatologia , Modelos BiológicosRESUMO
Insulin-regulated aminopeptidase, IRAP, is an abundant protein that was initially cloned from a rat epididymal fat pad cDNA library as a marker protein for specialized vesicles containing the insulin-responsive glucose transporter GLUT4, wherein it is thought to participate in the tethering and trafficking of GLUT4 vesicles. The same protein was independently cloned from human placental cDNA library as oxytocinase and is proposed to have a primary role in the regulation of circulating oxytocin (OXY) during the later stages of pregnancy. More recently, IRAP was identified as the specific binding site for angiotensin IV, and we propose that it mediates the memory-enhancing effects of the peptide. This protein appears to have multiple physiological roles that are tissue- and domain-specific; thus the protein can be specifically targeted for treating different clinical conditions.
Assuntos
Cistinil Aminopeptidase/antagonistas & inibidores , Inibidores de Proteases/uso terapêutico , Acil-CoA Desidrogenase/metabolismo , Animais , Cognição/efeitos dos fármacos , Cistinil Aminopeptidase/química , Cistinil Aminopeptidase/fisiologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Proteínas Ativadoras de GTPase/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Humanos , Memória/efeitos dos fármacos , Camundongos , Parto , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tanquirases/metabolismoRESUMO
In muscle and adipose cells, the insulin-responsive aminopeptidase (IRAP) is localized to intracellular storage sites and undergoes insulin-dependent redistribution to the cell surface. Following expression, the newly synthesized IRAP protein traffics to the perinuclear insulin-sensitive compartment and acquires insulin sensitivity 6-9 h following biosynthesis. Knockdown of GGA1 by RNA interference prevented IRAP from entering, but not exiting, the insulin-responsive compartment. Mutation of the dileucine motif at positions 76 and 77 (EGFP-IRAP/AA(76,77)), but not the dileucine motif at positions 53 and 54, resulted in the rapid default of the reporter to the cell surface beginning at 3 h following biosynthesis. Alanine substitution of 9 residues amino- or carboxyl-terminal to LL(76,77) did not perturb basal intracellular sequestration or abrogate insulin-stimulated IRAP translocation. Moreover, a dominant interfering GGA mutant (VHS-GAT) potently inhibited insulin-stimulated translocation of EGFP-IRAP/WT but did not block the constitutive exocytotic trafficking of EGFP-IRAP/AA(76,77). In addition, the EGFP-IRAP/WT and EGFP-IRAP/AA(76,77) constructs occupied morphologically distinct tubulovesicular compartments in the perinuclear region. Taken together, these data indicate that LL(76,77) functions during the GGA-dependent sorting of newly made IRAP into the insulin-responsive storage compartment.
Assuntos
Cistinil Aminopeptidase/metabolismo , Insulina/metabolismo , Leucina/metabolismo , Células 3T3-L1 , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Forma Celular , Cistinil Aminopeptidase/química , Cistinil Aminopeptidase/genética , Citosol/metabolismo , Genes Reporter/genética , Leucina/genética , Camundongos , Dados de Sequência Molecular , Mutação/genética , Transporte ProteicoRESUMO
The placental leucine aminopeptidase (P-LAP), adipocyte-derived leucine aminopeptidase (A-LAP) and leukocyte-derived aminopeptidase (L-RAP) belong to one distinct group of the M1 family of amimopeptidases, which we term the "Oxytocinase subfamily". They share HEXXH(X)18E Zn-binding and GAMEN motifs essential for the enzymatic activities. Intracellular localization is the characteristic feature of the subfamily members. While P-LAP is translocated from intracellular vesicles to plasma membrane in a stimulus-dependent manner, both A-LAP and L-RAP are retained in the endoplasmic reticulum. They contain sequences necessary for the specific localization in the cell. It is getting evident that the subfamily members play important roles in the maintenance of homeostasis including maintenance of normal pregnancy, memory retention, blood pressure regulation and antigen presentation. In this review, current situation of this newly identified subfamily is summarized.
Assuntos
Aminopeptidases/metabolismo , Cistinil Aminopeptidase/metabolismo , Sequência de Aminoácidos , Aminopeptidases/química , Animais , Células COS , Cistinil Aminopeptidase/química , Citosol/enzimologia , Feminino , Regulação da Expressão Gênica , Transportador de Glucose Tipo 4 , Humanos , Camundongos , Antígenos de Histocompatibilidade Menor , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares/metabolismo , Ocitocina/metabolismo , Células PC12 , Placenta/enzimologia , Gravidez , Ratos , Especificidade por SubstratoRESUMO
Cystinyl aminopeptidase has one Zn2+-binding motif and is a member of the M1 aminopeptidase family. Ion modulation of its catalytic activity was studied in membranes of CHO-K1 cells (Chinese-hamster ovary K1 cells) using L-leucine-p-nitroanilide as substrate. The planar bidentate chelators 1,10-phenanthroline and 2,2'-bipyridine inhibited the activity in a concentration-dependent manner with Hill slopes of 3.32+/-1.78 and 2.10+/-0.26 respectively. The acetic acid-containing chelators EDTA, EGTA and DTPA (diethylenetriamine-N,N,N',N'',N''-penta-acetic acid) weakly affected the activity, but they increased the potency of the planar chelators up to a limit, at which Hill slopes became close to unity. Moreover, competition between 1,10-phenanthroline and the substrate only took place in the presence of EDTA. These findings are compatible with a model in which the bidentate chelators inhibit enzyme activity by decreasing the free Zn2+ concentration. By removing a modulatory ion from an allosteric site at the enzyme, the acetic acid-containing chelators facilitate the direct interaction between the bidentate chelators and the catalytic Zn2+. The inhibitory effect of EDTA plus 1,10-phenanthroline could be completely reversed by Zn2+. Ca2+ and Mg2+ increased the potency of Zn2+ for this process. This is expected if they interact with the modulatory site to decrease the sensitivity of the enzyme towards 1,10-phenanthroline. Conversely, the bidendate chelators increased the high-affinity [125I]angiotensin IV binding to the membranes and this was potentiated by the acetic acid-containing chelators. These findings support the concept that high-affinity [125I]angiotensin IV binding, previously referred to as 'AT4 receptor binding', only occurs for the cystinyl aminopeptidase apoenzyme.
Assuntos
Cálcio/química , Cistinil Aminopeptidase/química , Magnésio/química , Metais/química , Zinco/química , 2,2'-Dipiridil/química , Animais , Sítios de Ligação , Células CHO , Cátions Bivalentes/química , Membrana Celular/química , Quelantes/química , Cricetinae , Cistinil Aminopeptidase/metabolismo , Ácido Edético/química , Ácido Egtázico/química , Ácido Pentético/química , Fenantrolinas/química , Ligação ProteicaRESUMO
In this study we report the cloning and characterization of a novel human aminopeptidase, which we designate leukocyte-derived arginine aminopeptidase (L-RAP). The sequence encodes a 960-amino acid protein with significant homology to placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase. The predicted L-RAP contains the HEXXH(X)18E zinc-binding motif, which is characteristic of the M1 family of zinc metallopeptidases. Phylogenetic analysis indicates that L-RAP forms a distinct subfamily with placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in the M1 family. Immunocytochemical analysis indicates that L-RAP is located in the lumenal side of the endoplasmic reticulum. Among various synthetic substrates tested, L-RAP revealed a preference for arginine, establishing that the enzyme is a novel arginine aminopeptidase with restricted substrate specificity. In addition to natural hormones such as angiotensin III and kallidin, L-RAP cleaved various N-terminal extended precursors to major histocompatibility complex class I-presented antigenic peptides. Like other proteins involved in antigen presentation, L-RAP is induced by interferon-gamma. These results indicate that L-RAP is a novel aminopeptidase that can trim the N-terminal extended precursors to antigenic peptides in the endoplasmic reticulum.
Assuntos
Aminopeptidases/metabolismo , Cistinil Aminopeptidase/metabolismo , Leucócitos/enzimologia , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/genética , Sequência de Bases , Clonagem Molecular , Cistinil Aminopeptidase/química , Cistinil Aminopeptidase/genética , DNA Complementar , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de AminoácidosRESUMO
The human oxytocinase/insulin-regulated aminopeptidase (OTase/IRAP) is a 1024 amino acid type II integral membrane protein that is expressed mainly in fat, muscle and placenta tissues. It has been thought to be involved mainly in the control of onset of labour but recently rat OTase/IRAP was shown to participate in the regulation of glucose transporter isoform 4 vesicle trafficking in adipocytes as well. To approach an understanding of OTase/IRAP gene regulation the organization of the human gene was determined. Accordingly, three overlapping genomic clones were isolated and characterized. The human OTase/IRAP gene (OTASE) was found to span approximately 75 kb containing 18 exons and 17 introns. The gluzincin aminopeptidase motif: GAMEN-(31 amino acids)-HELAH-(18 amino acids)-E associated with Zn2+-binding, substrate binding and catalysis is encoded by exons 6 and 7. A major and a minor transcriptional initiation site in OTASE were identified by primer extension 514 bp and 551 bp, respectively, upstream of the translation start codon. Chloroamphenicol acetyltransferase-reporter assays revealed a functional CpG-rich promoter/enhancer region located between nucleotide -621 and the major transcriptional initiation site. Human OTASE was assigned to chromosome 5 by hybridization to genomic DNA from characterized somatic cell hybrids. Finally, the OTASE and the human aminopeptidase A gene were subchromosomally localized to 5q21 and 4q25, respectively, by in situ hybridization.
Assuntos
Aminopeptidases/genética , Cromossomos Humanos Par 5/genética , Cistinil Aminopeptidase/genética , Sequência de Aminoácidos , Aminopeptidases/química , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Cistinil Aminopeptidase/química , Éxons , Genes Reporter , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Íntrons , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Alinhamento de Sequência , TransfecçãoRESUMO
The complete amino acid sequence of human placental oxytocinase (placental leucine aminopeptidase) has been determined by cDNA cloning and sequencing. Oxytocinase is a type II integral membrane protein of 1025 amino acid residues, consisting of an acidic intracellular region of 110 amino acids followed by a hydrophobic transmembrane segment of 22 residues and 893 extracellular residues containing the characteristic Zn2+ coordination sequence element His-Glu-Xaa-Xaa-His-(18 residues)-Glu found in gluzincins. Two sets of cDNA clones with different 5'-ends were isolated and suggested to represent different spliced products of 3.6 kb (mature mRNA) and 12 kb, respectively. Oxytocinase mRNA is present in large amounts in placenta, heart and skeletal muscle and in small amounts in brain, kidney, liver and pancreas. A conserved sequence element, the GAMEN motif, which distinguishes the aminopeptidase family among gluzincins from other gluzincins, has been identified.
Assuntos
Cistinil Aminopeptidase/química , Placenta/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cistinil Aminopeptidase/genética , DNA Complementar/química , Feminino , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Two different forms of oxytocinase (L-cystine aminopeptidase, CAP; EC 3.4.11.3) were purified from the 9000 g and 105000 g precipitate fractions of human placenta homogenate by sequential chromatography on columns of hydroxyapatite, DE-32, nickel ion affinity, and Sephadex G-200. One species (CAP-I) purifed from the mitochondrial/lysosomal fraction migrated on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with an apparent molecular mass of 61 kDa; the other (CAP-II) from the microsomal fraction was composed of two subunits with molecular masses of 56 and 40 kDa. The molecular masses of CAP-I and CAP-II estimated by gel filtration were 64 and 97 kDa, respectively. The specific activities of the two species for S-benzyl-L-cysteine p-nitroanilide increased by 357- (for CAP-I) and 139-fold (for CAP-II) compared with the starting preparations. The optimal pH values toward the artificial substrate were approx. 7.4-8.0 for CAP-I and 6.8-8.0 for CAP-II. The Km and Vmax values toward oxytocin were 5.6 microM and 23.4 micromol/h/mg protein for CAP-I, and 38 microM and 15.6 micromol/h/mg protein for CAP-II. Both enzymes were inhibited by the metal-chelating agents, EDTA and o-phenanthroline, whereas they were specifically activated by addition of Co2+: CAP-I was more sensitive to these reagents than CAP-II. L-Methionine strongly inhibited CAP-I, while CAP-II activity was only slightly affected. CAP-II was more sensitive to amastatin than CAP-I. Thus, the two enzymes are quite distinct in their molecular nature and biochemical properties. They may play a regulatory role in the metabolism of oxytocin and other biologically active peptides in intact placenta.
Assuntos
Cistinil Aminopeptidase/isolamento & purificação , Placenta/enzimologia , Cistinil Aminopeptidase/química , Cistinil Aminopeptidase/fisiologia , Feminino , Humanos , Peso Molecular , Gravidez , Especificidade por SubstratoRESUMO
Gel filtration was performed for cystine aminopeptidase (CAP) [EC 3.4.11.3] on full term placental extracts from human, cynomolgus monkey, dog, goat, pig and horse. The enzymatic profiles of CAP were examined and compared with that of CAP in pregnancy plasma on the basis of inhibitory effects. Elution profiles of placental extracts exhibited 2 CAP activity peaks in human and pig, 3 peaks in cynomolgus monkey, dog and goat, and 4 peaks in horse. The molecular weights of placental CAP that showed identical inhibitory effects to that of pregnancy plasma CAP were estimated to be approximately 325,000 in human, 350,000 in the cynomolgus monkey, 140,000 in the dog, 140,000 in the goat, 128,000 in the pig, and 115,000 in the horse. These molecular weights tended to decrease in accordance with the increase of barrier layers present between maternal blood and placental syncytiotrophoblasts in which CAP is synthesized.