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1.
Anal Chem ; 96(15): 5897-5905, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38557023

RESUMO

Current diagnostic methods for thyroid diseases, including blood tests, ultrasound, and biopsy, always have difficulty diagnosing thyroiditis accurately, occasionally mistaking it for thyroid cancer. To address this clinical challenge, we developed Ox-PGP1, a novel fluorescent probe realizing rapid, noninvasive, and real-time diagnostic techniques. This is the first imaging tool capable of noninvasively distinguishing between thyroiditis and thyroid cancer. Ox-PGP1 was introduced as a fluorescent probe custom-built for the specific detection and quantification of pyroglutamate aminopeptidase 1 (PGP-1), a known pivotal biomarker of inflammation. Ox-PGP1 overcame the disadvantages of traditional enzyme-responsive fluorescent probes that relied on the intramolecular charge transfer (ICT) mechanism, including the issue of high background fluorescence, while offering exceptional photostability under laser irradiation. The spectral properties of Ox-PGP1 were meticulously optimized to enhance its biocompatibility. Furthermore, the low limit of detection (LOD) of Ox-PGP1 was determined to be 0.09 µg/mL, which demonstrated its remarkable sensitivity and precision. Both cellular and in vivo experiments validated the capacity of Ox-PGP1 for accurate differentiation between normal, inflammatory, and cancerous thyroid cells. Furthermore, Ox-PGP1 showed the potential to rapidly and sensitively differentiate between autoimmune thyroiditis and anaplastic thyroid carcinoma in a mouse model, achieving results in just 5 min. The successful design and application of Ox-PGP1 represent a substantial advancement in technology over traditional diagnostic approaches, potentially enabling earlier interventions for thyroid diseases.


Assuntos
Neoplasias da Glândula Tireoide , Tireoidite , Animais , Camundongos , Piroglutamil-Peptidase I , Corantes Fluorescentes , Tireoidite/patologia , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Imagem Óptica
3.
Acta Biochim Pol ; 70(3): 703-711, 2023 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-37722013

RESUMO

Circular RNAs (circRNAs) contribute to the malignant phenotype and progression of several types of human cancers, including renal cell carcinoma (RCC). This study probed the molecular mechanism of circPGPEP1 regulating RCC proliferation, Warburg effect, and distant metastasis by targeting the miR-378a-3p/JPT1 axis. Here identified higher circPGPEP1 expression in RCC tissues and cells by RT-qPCR, and high levels of circPGPEP1 were positively correlated with high histological grade and distant metastasis in RCC patients. Furthermore, patients with high levels of circPGPEP1 had a worse survival prognosis. Functional assays presented that knockdown of circPGPEP1 inhibited RCC proliferation, invasion, migration, EMT, and Warburg effect. Dual-luciferase reporter assay, RNA immunoprecipitation, nucleoplasmic RNA isolation, and functional rescue experiments confirmed that circPGPEP1 induced JPT1 expression by sponging miR-378a-3p, thereby promoting RCC malignant phenotype. Xenograft assays and metastasis models further demonstrated that down-regulation of circPGPEP1 effectively inhibited tumor growth and distant metastasis of RCC. Taken together, circPGPEP1, a prognostic circRNA in RCC, acts through the miR-378a-3p/JPT1 axis to regulate RCC progression.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , MicroRNAs , Humanos , Carcinoma de Células Renais/genética , Proliferação de Células/genética , Regulação para Baixo , Neoplasias Renais/genética , MicroRNAs/genética , RNA Circular/genética , Piroglutamil-Peptidase I/metabolismo
4.
Anal Chem ; 93(39): 13311-13318, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34569224

RESUMO

As a global health challenge, hepatocellular carcinoma (HCC) is strongly associated with chronic inflammation. Targeting inflammation, particularly inflammatory factors, is regarded as an important strategy for HCC diagnosis and treatment. Pyroglutamic aminopeptidase I (PGP-I), a common exopeptidase, was recently identified as a novel inflammatory cytokine in cells. However, whether PGP-I is involved in HCC development and can be regarded as a biomarker remains unclear. To address this issue, endogenous PGP-I was imaged in live cells and in vivo, and the related biochemical and pathological processes were analyzed accordingly with a newly developed fluorogenic PGP-I biosensor. Bioimaging with the specific biosensor demonstrated the aberrant expression of PGP-I in HCC cell lines and tumor-bearing nude mice. Moreover, overexpression of PGP-I in HCC cells promoted tumor progression, whereas knockdown of PGP-I significantly suppressed tumor cell growth and migration. The activity of PGP-I was further identified to be highly related to the phosphorylation of STAT3, which could be impeded by the natural product parthenolide. Collectively, these findings suggest that PGP-I, which can promote hepatocellular tumor progression through the classical inflammation-/tumor-related IL-6/STAT3 pathway, may serve as a potential HCC biomarker and therapeutic target.


Assuntos
Técnicas Biossensoriais , Carcinoma Hepatocelular , Interleucina-6/metabolismo , Neoplasias Hepáticas , Piroglutamil-Peptidase I , Fator de Transcrição STAT3/metabolismo , Animais , Camundongos , Camundongos Nus , Ácido Pirrolidonocarboxílico
5.
J Mater Chem B ; 9(22): 4546-4554, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34046652

RESUMO

Pyroglutamate aminopeptidase-1 (PGP-1) is an important enzyme that plays an indispensable role in the process of inflammation. Up to now, few reports have been reported on the detection of PGP-1 activity in vivo and in vitro, and there are no reports on ratiometric detection. Here, the first red-emitting ratiometric fluorescent sensor (DP-1) for the specific detection of PGP-1 both in vivo and in vitro was designed and synthesized by using DCD-NH2 as the luminescent parent and pyroglutamate as a recognition group. After interacting with PGP-1, the amide bond is hydrolyzed by the enzyme and the color of the solution changes from yellow (λabs = 420 nm) to red (λabs = 520 nm), accompanied by obvious fluorescence emission wavelength change (from ∼564 nm to ∼616 nm). The probe has high specificity and sensitivity towards PGP-1 in about 10 min, and the DL is as low as 0.25 ng mL-1. Interestingly, under the stimulation of Freund's incomplete adjuvant and lipopolysaccharide, the imaging of DP-1 in HepG2 and RAW264 cells shows that the expression of PGP-1 is associated with inflammation. What's more, for the first, the imaging of a mouse tumor model confirms that the enzyme is closely related to the occurrence of some inflammation and tumor diseases. These results indicate that DP-1 can be used as an effective tool for real-time monitoring of PGP-1 levels both in vivo and in vitro and the study of inflammatory tumor pathology.


Assuntos
Corantes Fluorescentes/química , Neoplasias/enzimologia , Piroglutamil-Peptidase I/metabolismo , Animais , Células Hep G2 , Humanos , Camundongos
6.
Bioorg Med Chem Lett ; 43: 128049, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33882272

RESUMO

Pyroglutamate aminopeptidase (PGP) specifically cleaves the peptide bond of pyroglutamic acid linked to the N-terminal end of a polypeptide or protein. Previous studies showed that PGP was associated with several physiological processes and diseases especially those involving inflammation. Utilizing a 'caging' strategy, we designed and synthesized a bioluminescence probe (PBL) with a limit-of-detection of 3.7 * 10-4 mU/mL. In vivo imaging in a mouse model of inflammatory liver disease revealed that the probe has excellent sensitivity and selectivity and provides a powerful tool for studying the physiological and pathological processes involving PGP.


Assuntos
Modelos Animais de Doenças , Inflamação/diagnóstico por imagem , Substâncias Luminescentes/química , Piroglutamil-Peptidase I/análise , Animais , Diagnóstico por Imagem , Inflamação/metabolismo , Substâncias Luminescentes/síntese química , Camundongos , Estrutura Molecular , Piroglutamil-Peptidase I/metabolismo
7.
Breast Cancer Res Treat ; 182(3): 751-760, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32506336

RESUMO

PURPOSE: Functional studies have demonstrated that gonadotropin-releasing hormone (GnRH) regulates cell proliferation, apoptosis, and tissue remodeling. GnRH is metabolized by the proteolytic regulatory enzyme pyrrolidone carboxypeptidase (Pcp) (E.C. 3.4.19.3), which is an omega peptidase widely distributed in fluids and tissues. We previously reported a decrease in both rat and human Pcp activity in breast cancer, suggesting that GnRH may be an important local hormonal factor in the pathogenesis of breast cancer. Recently, we have described that postmenopausal women with breast cancer show lower levels of serum Pcp activity than control postmenopausal women. To determine the effect of neoadjuvant chemotherapy (NACT) on serum Pcp specific activity and circulating levels of GnRH, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and steroid hormones 17-ß-estradiol and progesterone in pre- and postmenopausal women diagnosed with infiltrating ductal carcinoma. METHODS: Serum Pcp activity was measured fluorometrically using pyroglutamyl-ß-naphthylamide. Circulating GnRH levels were dosed using a commercial RIA kit. Circulating LH and FSH levels were measured by enzyme immunoassays. Levels of steroid hormones were measured in serum samples by dissociation-enhanced lanthanide fluorescence immunoassay. RESULTS AND CONCLUSION: Our results show the effect of NACT on the hypothalamic-pituitary axis, with the consequent alteration of circulating gonadotropins in premenopausal women with breast cancer. However, the results obtained in postmenopausal women with breast cancer treated with NACT, that is, the significant decrease in the concentration of GnRH and FSH compared to control postmenopausal women, differ from those obtained for premenopausal women. The only difference between pre- and postmenopausal women is their hormonal profile at the beginning of the study, that is, the presence of menopause and the consequent alteration of the hypothalamic-pituitary-gonadal axis.


Assuntos
Neoplasias da Mama/sangue , Carcinoma Ductal de Mama/sangue , Carcinoma Lobular/sangue , Hormônio Liberador de Gonadotropina/sangue , Gonadotropinas/sangue , Terapia Neoadjuvante/métodos , Piroglutamil-Peptidase I/sangue , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/patologia , Estudos de Casos e Controles , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Seguimentos , Humanos , Hormônio Luteinizante/sangue , Pessoa de Meia-Idade , Progesterona/sangue , Prognóstico , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo
8.
Nutrients ; 12(3)2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32121057

RESUMO

High-fat diets (HFD) have been widely associated with an increased risk of metabolic disorders and overweight. However, a high intake of sources that are rich in monounsaturated fatty acids has been suggested as a dietary agent that is able to positively influence energy metabolism and vascular function. The main objective of this study was to analyze the role of dietary fats on hepatic peptidases activities and metabolic disorders. Three diets: standard (S), HFD supplemented with virgin olive oil (VOO), and HFD supplemented with butter plus cholesterol (Bch), were administered over six months to male Wistar rats. Plasma and liver samples were collected for clinical biochemistry and aminopeptidase activities (AP) analysis. The expression of inducible nitric oxide synthase (iNOS) was also determined by Western blot in liver samples. The diet supplement with VOO did not induce obesity, in contrast to the Bch group. Though the VOO diet increased the time that was needed to return to the basal levels of plasma glucose, the fasting insulin/glucose ratio and HOMA2-%B index (a homeostasis model index of insulin secretion and valuation of ß-cell usefulness (% ß-cell secretion)) were improved. An increase of hepatic membrane-bound dipeptidyl-peptidase 4 (DPP4) activity was found only in VOO rats, even if no differences in fasting plasma glucagon-like peptide 1 (GLP-1) were obtained. Both HFDs induced changes in hepatic pyroglutamyl-AP in the soluble fraction, but only the Bch diet increased the soluble tyrosyl-AP. Angiotensinase activities that are implicated in the metabolism of angiotensin II (AngII) to AngIV increased in the VOO diet, which was in agreement with the higher activity of insulin-regulated-AP (IRAP) in this group. Otherwise, the diet that was enriched with butter increased soluble gamma-glutamyl transferase (GGT) and Leucyl-AP, iNOS expression in the liver, and plasma NO. In summary, VOO increased the hepatic activity of AP that were related to glucose metabolism (DPP4, angiotensinases, and IRAP). However, the Bch diet increased activities that are implicated in the control of food intake (Tyrosine-AP), the index of hepatic damage (Leucine-AP and GGT), and the expression of hepatic iNOS and plasma NO. Taken together, these results support that the source of fat in the diet affects several peptidases activities in the liver, which could be related to alterations in feeding behavior and glucose metabolism.


Assuntos
Dieta Hiperlipídica , Resistência à Insulina , Fígado/enzimologia , Fígado/patologia , Obesidade/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Dipeptidil Peptidase 4/metabolismo , Ingestão de Energia , Comportamento Alimentar , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Teste de Tolerância a Glucose , Masculino , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo II/metabolismo , Obesidade/sangue , Piroglutamil-Peptidase I/metabolismo , Ratos Wistar , gama-Glutamiltransferase/metabolismo
9.
Int J Med Sci ; 12(6): 458-67, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26078706

RESUMO

BACKGROUND AND OBJECTIVE: The role of peptidases in carcinogenic processes and their potential usefulness as tumor markers in colorectal cancer (CRC) have been classically attributed to cell-surface enzymes. The objective of the present study was to analyze the activity and mRNA expression of three cytosolic peptidases in the CRC and to correlate the obtained results with classic histopathological parameters for tumor prognosis and survival. METHODS: The activity and mRNA levels of puromycin-sensitive aminopeptidase (PSA), aminopeptidase B (APB) and pyroglutamyl-peptidase I (PGI) were measured by fluorimetric and quantitative RT-PCR methods in colorectal mucosa and tumor tissues and plasma samples from CRC patients (n=81). RESULTS: 1) PSA and APB activity was higher in adenomas and carcinomas than in the uninvolved mucosa. 2) mRNA levels of PSA and PGI was lower in tumors. 3) PGI activity in CRC tissue correlated negatively with histological grade, tumor size and 5-year overall survival of CRC patients. 4) Higher plasmatic APB activity was independently associated with better 5-year overall survival. CONCLUSIONS: Data suggest that cytosolic peptidases may be involved in colorectal carcinogenesis and point to the determination of this enzymes as a valuable method in the determination of CRC prognosis.


Assuntos
Aminopeptidases/biossíntese , Neoplasias Colorretais/genética , Piroglutamil-Peptidase I/biossíntese , Idoso , Aminopeptidases/genética , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Citosol/enzimologia , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Piroglutamil-Peptidase I/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
10.
Endocr Regul ; 49(1): 20-4, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25687677

RESUMO

OBJECTIVE: As a reflect of tissue damage, serum aminopeptidases have been proposed as biomarkers of various diseases. In order to search new serologic markers for liver cirrhosis we conducted a preliminary study in which we analyzed a broad range of aminopeptidase activities in serum of controls and patients diagnosed with pancreatitis, hepatitis, and liver cirrhosis without distinction among the etiological type or the degree of severity of each condition. METHODS: Alanyl-, arginyl-, glutamyl-, cystinyl- pyroglutamyl-, and aspartyl-aminopeptidase activities were analyzed fluorometrically, using aminoacyl-ß-naphthylamides as substrates. In addition, various parameters, such as alanine transaminase, aspartate transaminase, alkaline phosphatase, total bilirubin, direct bilirubin, and gamma glutamyl transpeptidase were assayed as routine laboratory test for liver function. RESULTS: Compared with control group, alanyl- and arginyl-aminopeptidase activities increased nonspecifically in pancreatitis, hepatitis and liver cirrhosis, glutamyl- and cystinyl-aminopeptidases did not differ between groups and pyroglutamyl-aminopeptidase demonstrated that while pancreatitis and hepatitis did not differ between them and with controls, this activity decreased selectively in liver cirrhosis compared with all the rest of groups (p<0.001 vs. control and p<0.01 vs. pancreatitis and hepatitis). Aspartyl-aminopeptidase also decreased significantly (p<0.05) in liver cirrhosis compared with controls. Routine parameters for liver function test increased, as expected, in the three pathologies analyzed. CONCLUSIONS: Despite the heterogeneous composition of the three patient groups, the specific reduction of the levels of pyroglutamyl-aminopeptidase activity in serum of liver cirrhosis patients might be considered as a potential candidate to be included in a combination of markers for the diagnosis of this disease.


Assuntos
Biomarcadores/sangue , Cirrose Hepática/sangue , Cirrose Hepática/diagnóstico , Piroglutamil-Peptidase I/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Diagnóstico Diferencial , Feminino , Hepatite/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Pancreatite/sangue
11.
Dis Markers ; 35(6): 825-32, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24379520

RESUMO

BACKGROUND: Papillary thyroid carcinoma (PTC), follicular thyroid adenoma (FTA), and thyroid nodular hyperplasia (TNH) are the most frequent diseases of the thyroid gland. Previous studies described the involvement of dipeptidyl-peptidase IV (DPPIV/CD26) in the development of thyroid neoplasia and proposed it as an additional tool in the diagnosis/prognosis of these diseases. However, very little is known about the involvement of other peptidases in neoplastic and hyperplastic processes of this gland. METHODS: The catalytic activity of 10 peptidases in a series of 30 PTC, 10 FTA, and 14 TNH was measured fluorimetrically in tumour and nontumour adjacent tissues. RESULTS: The activity of DPPIV/CD26 was markedly higher in PTC than in FTA, TNH, and nontumour tissues. Aspartyl aminopeptidase (AspAP), alanyl aminopeptidase (AlaAP), prolyl endopeptidase, pyroglutamyl peptidase I, and aminopeptidase B activities were significantly increased in thyroid neoplasms when compared to nontumour tissues. AspAP and AlaAP activities were also significantly higher in PTC than in FTA and TNH. CONCLUSIONS: These data suggest the involvement of DPPIV/CD26 and some cytosolic peptidases in the neoplastic development of PTC and FTA. Further studies will help to define the possible clinical usefulness of AlaAP and AspAP in the diagnosis/prognosis of thyroid neoplasms.


Assuntos
Adenocarcinoma Folicular/enzimologia , Carcinoma Papilar/enzimologia , Dipeptidil Peptidase 4/metabolismo , Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/enzimologia , Adulto , Antígenos CD13/metabolismo , Feminino , Glutamil Aminopeptidase/metabolismo , Humanos , Hiperplasia/enzimologia , Masculino , Pessoa de Meia-Idade , Prolil Oligopeptidases , Piroglutamil-Peptidase I/metabolismo , Serina Endopeptidases/metabolismo , Glândula Tireoide/patologia
12.
Biochemistry ; 51(31): 6089-96, 2012 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-22799522

RESUMO

The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114-208 residues are broadened, indicating that conformations of the 114-208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1-113 and the 114-208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114-208 segment of PCP-0SH folds into a stable compact structure with non-native helix-helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central ß-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH.


Assuntos
Temperatura Alta , Dobramento de Proteína , Pyrococcus furiosus/enzimologia , Piroglutamil-Peptidase I/química , Naftalenossulfonato de Anilina/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Estrutura Secundária de Proteína , Piroglutamil-Peptidase I/genética , Espectrometria de Fluorescência
13.
Breast ; 21(6): 751-4, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22381154

RESUMO

In breast cancer, hormonal changes are rather constant in post-menopausal women since they tend to vary only over long time spans. However, in pre-menopausal women, the development of breast cancer is associated with hormonal physiological variations. The aim of the present work was to analyse the changes in circulating levels of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in pre- and post-menopausal women that were healthy or with breast cancer, and their connection to serum pyrrolidone carboxypeptidase (Pcp) activity. We observed significant changes in the hormonal profile in post-menopausal women with breast cancer compared to the control group. In pre-menopausal women, we found significant changes in circulating GnRH levels with respect to the healthy group. Our present results support the existence of neuroendocrine misregulation that could be involved in tumour progression, with Pcp being a potentially new pharmacological target in breast cancer treatments.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/enzimologia , Carcinoma Ductal de Mama/enzimologia , Pós-Menopausa/sangue , Pré-Menopausa/sangue , Piroglutamil-Peptidase I/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/sangue , Carcinoma Ductal de Mama/sangue , Estudos de Casos e Controles , Feminino , Hormônio Foliculoestimulante Humano/sangue , Hormônio Liberador de Gonadotropina/sangue , Humanos , Hormônio Luteinizante/sangue , Pessoa de Meia-Idade
14.
Int J Pediatr Otorhinolaryngol ; 75(11): 1399-403, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21937126

RESUMO

OBJECTIVE: To analyze soluble and membrane-bound peptidase activities in the tonsils and adenoids removed from patients with adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis. METHODS: A total of 48 tissue samples from patients undergoing adenoidectomy and tonsillectomy for adenoid hyperplasia, tonsillar hyperplasia or chronic tonsillitis were analyzed. The catalytic activity of a pool of peptidases in the soluble (dipeptidyl peptidase IV, aminopeptidase A, aminopeptidase N and cystinyl aminopeptidase) and membrane-bound (prolyl endopeptidase, aspartyl aminopeptidase, aminopeptidase B and pyroglutamyl peptidase I) fractions was measured fluorometrically. RESULTS: The activity of membrane-bound aminopeptidase B was higher in cases of chronic tonsillitis and adenoid hyperplasia than in tonsillar hyperplasia, p=0.004. Soluble dipeptidyl peptidase IV and membrane-bound pyroglutamyl peptidase I were found to be more active in tissues from male chronic tonsillitis tissues, p<0.05, while membrane-bound aminopeptidase B activity was higher in tissues of females with tonsillar hyperplasia, p<0.001. In the case of chronic tonsillitis, soluble aminopeptidase A was found to have a higher level of activity in tissues from children than those from adults, p=0.005. CONCLUSIONS: Our results suggest a potential role of soluble aminopeptidase A, soluble dipeptidyl peptidase IV, membrane-bound aminopeptidase B and membrane-bound pyroglutamyl peptidase I in the pathobiology of adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis that is differently regulated as a function of gender. These finfings may modify in the future the clinical approach to these diseases.


Assuntos
Tonsila Faríngea/metabolismo , Aminopeptidases/metabolismo , Tonsila Palatina/metabolismo , Tonsila Faríngea/patologia , Aminopeptidases/análise , Análise de Variância , Biomarcadores/análise , Criança , Pré-Escolar , Doença Crônica , Estudos de Coortes , Dipeptidil Peptidase 4/análise , Dipeptidil Peptidase 4/metabolismo , Feminino , Glutamil Aminopeptidase/análise , Glutamil Aminopeptidase/metabolismo , Humanos , Hiperplasia/metabolismo , Hiperplasia/patologia , Masculino , Tonsila Palatina/patologia , Piroglutamil-Peptidase I/análise , Piroglutamil-Peptidase I/metabolismo , Estudos Retrospectivos , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Tonsilectomia/métodos , Tonsilite/metabolismo , Tonsilite/patologia , Tonsilite/cirurgia
15.
Curr Protoc Protein Sci ; Chapter 11: 11.7.1-11.7.20, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21400688

RESUMO

Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for N(α)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(α)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis.


Assuntos
Bioquímica/métodos , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Análise de Sequência/métodos , Acilação , Colorimetria , Formiatos/metabolismo , Hidrolases/metabolismo , Isotiocianatos/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Piroglutamil-Peptidase I/metabolismo , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/isolamento & purificação , Soluções , Anidridos Succínicos/metabolismo
16.
J Biol Chem ; 286(13): 11211-7, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21282104

RESUMO

Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute.


Assuntos
Anticorpos Monoclonais/química , Ácido Glutâmico/química , Imunoglobulina G/química , Ácido Pirrolidonocarboxílico/química , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Proteínas Arqueais/química , Células CHO , Cricetinae , Cricetulus , Ácido Glutâmico/metabolismo , Humanos , Imunoglobulina G/metabolismo , Imunoglobulina G/uso terapêutico , Processamento de Proteína Pós-Traducional , Pyrococcus furiosus/enzimologia , Piroglutamil-Peptidase I/química , Ácido Pirrolidonocarboxílico/metabolismo
17.
Proteins ; 71(2): 737-42, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17979195

RESUMO

In the denatured state (D(1) state) of cystein-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus, a hyperthermophile under nondenaturing conditions, a fairly stable alpha-helix (alpha6-helix) has been determined from H/D exchange-NMR experiments. On the other hand, the alpha6-helix region of the proline-mutant at position 199 (A199P) was unstructured in the D(1) state unlike that of the wild-type PCP-0SH, although the folded conformations of both proteins were almost identical to each other. This finding has been deduced from the information regarding the remaining amide hydrogens in the HSQC spectra after H/D exchanges in the D(1) state. To confirm this inference, we examined the helical propensities of two synthetic peptides from their NMR structural analysis in the presence of trifluoroethanol (TFE). One is an 18-residue peptide called the wild-type H6-peptide corresponding to the alpha6-helix (from Ser188 to Glu205) of the wild-type PCP-0SH, and the other is the mutant H6-peptide corresponding to the alpha6-helix region of A199P. The NOE-contact information obtained from the 2D-(1)H-NOESY spectra measured for both peptides in the presence of 30% TFE clearly demonstrated that the wild-type H6-peptide had a high helical propensity, but the mutant H6-peptide was almost totally unstructured. The TFE-induced helical propensities for these peptide fragments confirmed the conclusions deduced from the H/D exchange data measured in the D(1) states of two proteins.


Assuntos
Conformação Proteica , Piroglutamil-Peptidase I/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Dicroísmo Circular , Medição da Troca de Deutério , Espectroscopia de Ressonância de Spin Eletrônica , Ressonância Magnética Nuclear Biomolecular , Desnaturação Proteica , Estrutura Terciária de Proteína , Piroglutamil-Peptidase I/genética , Trifluoretanol
18.
Biochemistry ; 46(12): 3664-72, 2007 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-17309236

RESUMO

The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile, Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, corresponding to the denatured structure that exists in equilibrium with the native state under physiological conditions. The denatured state is the initial state (D1 state) in the refolding process but differs from the completely denatured state (D2 state) in the concentrated denaturant. Also, it has been found that the D1 state corresponds to the heat-denatured state. To elucidate the structural basis of the D1 state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 degrees C. The results indicated that amide protons in the C-terminal alpha6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggesting that the alpha6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order to examine the role of the alpha6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the alpha6-helix region were performed. The alpha6-helix region of A199P in the D1 state was partially unprotected, while some hydrophobic residues were protected against the H/D exchange, although these hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structure of A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (alpha6-helix) of PCP-0SH is already constructed in the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.


Assuntos
Modelos Moleculares , Dobramento de Proteína , Pyrococcus furiosus/enzimologia , Piroglutamil-Peptidase I/química , Substituição de Aminoácidos , Concentração de Íons de Hidrogênio , Mutação Puntual , Desnaturação Proteica , Estrutura Secundária de Proteína , Pyrococcus furiosus/química , Pyrococcus furiosus/genética , Piroglutamil-Peptidase I/genética , Temperatura
19.
Mol Cell Biochem ; 297(1-2): 189-97, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17119851

RESUMO

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl peptidase type-1 (PAP1). The cloned nucleotide sequence has an ORF coding for a primary sequence of 209 amino acid residues, which displays 98% identity with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary between the two sequences. The recombinant bovine PAP1 with a C-terminal His(6) tag (rBtaPAP1(6H)) was expressed in Escherichia coli XL10-Gold cells and purified by immobilised nickel ion affinity chromatography resulting in a yield of 2.6 mg of PAP1 per litre of culture. Purified rBtaPAP1(6H) had a specific activity of 3633 units mg(-1). SDS-PAGE revealed a band for bovine PAP1 with a molecular weight of approximately 24 kDa, which is in good agreement with previously reported data on PAP1. The K (m) and k (cat) values obtained for rBtaPAP1(6H) were 59 muM and 3.5 s(-1), respectively. The optimum pH for activity was 9.0-9.5 and the optimum temperature was 37 degrees C. rBtaPAP1(6H) was found to have an absolute requirement for the thiol-reducing agent DTT, consistent with the expected property of a cysteine protease. Kinetic studies using the peptides pGlu-His-Pro-NH(2) (TRH), pGlu-Ala and pGlu-Val revealed K (i) values of 44.1, 141 and 652.17 microM, respectively. The lowest K (i), observed for Thyrotropin-releasing Hormone (TRH), indicates that rBtaPAP1(6H) has a higher affinity for tripeptides over dipeptides.


Assuntos
Escherichia coli/metabolismo , Expressão Gênica , Piroglutamil-Peptidase I/isolamento & purificação , Piroglutamil-Peptidase I/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Bovinos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Cinética , Dados de Sequência Molecular , Proteínas Associadas a Pancreatite , Piroglutamil-Peptidase I/química , Piroglutamil-Peptidase I/genética , Proteínas Recombinantes/isolamento & purificação
20.
Am J Physiol Renal Physiol ; 292(2): F780-8, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16985214

RESUMO

Peptides play important roles in cell regulation and signaling in many tissues and are regulated by peptidases, most of which are highly expressed in the kidney. Several peptide convertases have a function in different tumor stages, and some have been clearly characterized as diagnostic and prognostic markers for solid tumors, including renal cancer; however, little is known about their in vivo role in kidney tumors. The present study compares the activity of a range of peptidases in human tumor samples and nontumor tissue obtained from clear cell renal cell carcinoma (CCRCC) patients. To cover the complete spectrum and subcellular distribution of peptide-converting activity, acid, neutral, basic, and omega activities were selected. CCRCC displays a selective and restricted pattern of peptidase activities. Puromycin-sensitive aminopeptidase activity in the tumor increases [tumor (t) = 10,775 vs. nontumor (n) = 7,635 units of peptidase (UP)/mg protein; P < 0.05], whereas aminopeptidase N decreases (t = 6,664 vs. n = 33,381 UP/mg protein; P < 0.001). Aminopeptidase B activity of the particulate fraction in tumors decreases (t = 2,399 vs. n = 13,536 UP/mg protein; P < 0.001) compared with nontumor tissues, and aspartyl-aminopeptidase activity decreases significantly in CCRCC (t = 137 vs. n = 223 UP/mg protein; P < 0.05). Soluble and particulate pyroglutamyl peptidase I activities, aminopeptidase A activity, and soluble aminopeptidase B activity do not vary in renal cancer. The relative expression for the aforementioned peptidases, assayed using quantitative RT-PCR, increases in CCRCC for aminopeptidases B (1.5-fold) and A (19-fold), aspartyl-aminopeptidase (3.9-fold), puromycin-sensitive aminopeptidase (2.5-fold), and pyroglutamyl peptidase I (7.6-fold). Only aminopeptidase N expression decreases in tumors (1.3-fold). This peptidase activity profile in the neoplastic kidney suggests a specific role for the studied convertases and the possible involvement of an intracrine renin-angiotensin system in the pathogenesis of CCRCC.


Assuntos
Carcinoma de Células Renais/enzimologia , Neoplasias Renais/enzimologia , Peptídeo Hidrolases/metabolismo , Adulto , Idoso , Aminopeptidases/biossíntese , Antígenos CD13/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica , Glutamil Aminopeptidase/biossíntese , Humanos , Rim/enzimologia , Masculino , Pessoa de Meia-Idade , Piroglutamil-Peptidase I/biossíntese
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