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1.
Int J Cancer ; 150(7): 1123-1133, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-34817877

RESUMO

Gene variants that encode pancreatic enzymes with impaired secretion can induce pancreatic acinar endoplasmic reticulum (ER) stress, cellular injury and pancreatitis. The role of such variants in pancreatic cancer risk has received little attention. We compared the prevalence of ER stress-inducing variants in CPA1 and CPB1 in patients with pancreatic ductal adenocarcinoma (PDAC cases), enrolled in the National Familial Pancreas Tumor Registry, to their prevalence in noncancer controls in the Genome Aggregation Database (gnomAD). Variants of unknown significance were expressed and variants with reduced secretion assessed for ER stress induction. In vitro assessments were compared with software predictions of variant function. Protein variant software was used to assess variants found in only one gnomAD control ("n-of-one" variants). A meta-analysis of prior PDAC case/control studies was also performed. Of the 1385 patients with PDAC, 0.65% were found to harbor an ER stress-inducing variant in CPA1 or CPB1, compared to 0.17% of the 64 026 controls (odds ratio [OR]: 3.80 [1.92-7.51], P = .0001). ER stress-inducing variants in the CPA1 gene were identified in 4 of 1385 PDAC cases vs 77 of 64 026 gnomAD controls (OR: 2.4 [0.88-6.58], P = .087), and variants in CPB1 were detected in 5 of 1385 cases vs 33 of 64 026 controls (OR: 7.02 [2.74-18.01], P = .0001). Meta-analysis demonstrated strong associations for pancreatic cancer and ER-stress inducing variants for both CPA1 (OR: 3.65 [1.58-8.39], P < .023) and CPB1 (OR: 9.51 [3.46-26.15], P < .001). Rare variants in CPB1 and CPA1 that induce ER stress are associated with increased odds of developing pancreatic cancer.


Assuntos
Carboxipeptidase B/genética , Carboxipeptidases A/genética , Carcinoma Ductal Pancreático/etiologia , Estresse do Retículo Endoplasmático/fisiologia , Neoplasias Pancreáticas/etiologia , Carboxipeptidase B/fisiologia , Carboxipeptidases A/fisiologia , Carcinoma Ductal Pancreático/genética , Estudos de Casos e Controles , Predisposição Genética para Doença , Variação Genética , Humanos , Neoplasias Pancreáticas/genética , Risco
2.
Life Sci Alliance ; 5(1)2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34750241

RESUMO

Metallocarboxypeptidases play critical roles in the development of mosquitoes and influence pathogen/parasite infection of the mosquito midgut. Here, we report the crystal structure of Aedes aegypti procarboxypeptidase B1 (PCPBAe1), characterized its substrate specificity and mechanism of binding to and inhibiting Dengue virus (DENV). We show that the activated PCPBAe1 (CPBAe1) hydrolyzes both Arg- and Lys-substrates, which is modulated by residues Asp251 and Ser239 Notably, these residues are conserved in CPBs across mosquito species, possibly required for efficient digestion of basic dietary residues that are necessary for mosquito reproduction and development. Importantly, we characterized the interaction between PCPBAe1 and DENV envelope (E) protein, virus-like particles, and infectious virions. We identified residues Asp18A, Glu19A, Glu85, Arg87, and Arg89 of PCPBAe1 are essential for interaction with DENV. PCPBAe1 maps to the dimeric interface of the E protein domains I/II (Lys64-Glu84, Val238-Val252, and Leu278-Leu287). Overall, our studies provide general insights into how the substrate-binding property of mosquito carboxypeptidases could be targeted to potentially control mosquito populations or proposes a mechanism by which PCPBAe1 binds to and inhibits DENV.


Assuntos
Aedes/enzimologia , Aedes/virologia , Carboxipeptidase B/metabolismo , Vírus da Dengue , Dengue/transmissão , Interações entre Hospedeiro e Microrganismos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Carboxipeptidase B/química , Carboxipeptidase B/genética , Domínio Catalítico , Dengue/prevenção & controle , Dengue/virologia , Vírus da Dengue/fisiologia , Controle de Infecções , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNA , Relação Estrutura-Atividade , Especificidade por Substrato , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo
3.
Protein Sci ; 30(12): 2445-2456, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34658092

RESUMO

Metallocarboxypeptidases (MCPs) in the mosquito midgut play crucial roles in infection, as well as in mosquito dietary digestion, reproduction, and development. MCPs are also part of the digestive system of plant-feeding insects, representing key targets for inhibitor development against mosquitoes/mosquito-borne pathogens or as antifeedant molecules against plant-feeding insects. Notably, some non-mosquito insect B-type MCPs are primarily insensitive to plant protease inhibitors (PPIs) such as the potato carboxypeptidase inhibitor (PCI; MW 4 kDa), an inhibitor explored for cancer treatment and insecticide design. Here, we report the crystal structure of Aedes aegypti carboxypeptidase-B1 (CPBAe1)-PCI complex and compared the binding with that of PCI-insensitive CPBs. We show that PCI accommodation is determined by key differences in the active-site regions of MCPs. In particular, the loop regions α6-α7 (Leu242 -Ser250 ) and ß8-α8 (Pro269 -Pro280 ) of CPBAe1 are replaced by α-helices in PCI-insensitive insect Helicoverpa zea CPBHz. These α-helices protrude into the active-site pocket of CPBHz, restricting PCI insertion and rendering the enzyme insensitive. We further compared our structure with the only other PCI complex available, bovine CPA1-PCI. The potency of PCI against CPBAe1 (Ki  = 14.7 nM) is marginally less than that of bovine CPA1 (Ki  = 5 nM). Structurally, the above loop regions that accommodate PCI binding in CPBAe1 are similar to that of bovine CPA1, although observed changes in proteases residues that interact with PCI could account for the differences in affinity. Our findings suggest that PCI sensitivity is largely dictated by structural interference, which broadens our understanding of carboxypeptidase inhibition as a mosquito population/parasite control strategy.


Assuntos
Aedes/enzimologia , Carboxipeptidase B/química , Carboxipeptidases A/química , Proteínas de Insetos/química , Inibidores de Proteases/química , Sequência de Aminoácidos , Animais , Carboxipeptidase B/antagonistas & inibidores , Carboxipeptidase B/genética , Carboxipeptidase B/metabolismo , Carboxipeptidases A/antagonistas & inibidores , Carboxipeptidases A/genética , Carboxipeptidases A/metabolismo , Domínio Catalítico , Bovinos , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Cinética , Modelos Moleculares , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por Substrato
4.
Crit Care ; 25(1): 51, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33557911

RESUMO

BACKGROUND: Thrombosis and coagulopathy are highly prevalent in critically ill patients with COVID-19 and increase the risk of death. Immunothrombosis has recently been demonstrated to contribute to the thrombotic events in COVID-19 patients with coagulopathy. As the primary components of immunothrombosis, neutrophil extracellular traps (NETs) could be induced by complement cascade components and other proinflammatory mediators. We aimed to explore the clinical roles of NETs and the regulation of complement on the NET formation in COVID-19. METHODS: We recruited 135 COVID-19 patients and measured plasma levels of C5, C3, cell-free DNA and myeloperoxidase (MPO)-DNA. Besides, the formation of NETs was detected by immunofluorescent staining and the cytotoxicity to vascular endothelial HUVEC cells was evaluated by CCK-8 assay. RESULTS: We found that the plasma levels of complements C3 and MPO-DNA were positively related to coagulation indicator fibrin(-ogen) degradation products (C3: r = 0.300, p = 0.005; MPO-DNA: r = 0.316, p = 0.002) in COVID-19 patients. Besides, C3 was positively related to direct bilirubin (r = 0.303, p = 0.004) and total bilirubin (r = 0.304, p = 0.005), MPO-DNA was positively related to lactate dehydrogenase (r = 0.306, p = 0.003) and creatine kinase (r = 0.308, p = 0.004). By using anti-C3a and anti-C5a antibodies, we revealed that the complement component anaphylatoxins in the plasma of COVID-19 patients strongly induced NET formation. The pathological effect of the anaphylatoxin-NET axis on the damage of vascular endothelial cells could be relieved by recombinant carboxypeptidase B (CPB), a stable homolog of enzyme CPB2 which can degrade anaphylatoxins to inactive products. CONCLUSIONS: Over-activation in anaphylatoxin-NET axis plays a pathological role in COVID-19. Early intervention in anaphylatoxins might help prevent thrombosis and disease progression in COVID-19 patients.


Assuntos
Anafilatoxinas/metabolismo , COVID-19/tratamento farmacológico , COVID-19/imunologia , Carboxipeptidase B/metabolismo , Carboxipeptidase B/uso terapêutico , Armadilhas Extracelulares/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Trombose/prevenção & controle , Adulto , Idoso , COVID-19/fisiopatologia , Armadilhas Extracelulares/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Trombose/imunologia
5.
Biotechnol Bioeng ; 118(9): 3334-3347, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33624836

RESUMO

The goal of cell culture process intensification is to improve productivity while maintaining acceptable quality attributes. In this report, four processes, namely a conventional manufacturing Process A, and processes intensified by enriched N-1 seed (Process B), by perfusion N-1 seed (Process C), and by perfusion production (Process D) were developed for the production of a monoclonal antibody. The three intensified processes substantially improved productivity, however, the product either failed to meet the specification for charge variant species (main peak) for Process D or the production process required early harvest to meet the specification for charge variant species, Day 10 or Day 6 for Processes B and C, respectively. The lower main peak for the intensified processes was due to higher basic species resulting from higher C-terminal lysine. To resolve this product quality issue, we developed an enzyme treatment method by introducing carboxypeptidase B (CpB) to clip the C-terminal lysine, leading to significantly increased main peak and an acceptable and more homogenous product quality for all the intensified processes. Additionally, Processes B and C with CpB treatment extended bioreactor durations to Day 14 increasing titer by 38% and 108%, respectively. This simple yet effective enzyme treatment strategy could be applicable to other processes that have similar product quality issues.


Assuntos
Anticorpos Monoclonais/biossíntese , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Carboxipeptidase B/farmacologia , Animais , Células CHO , Cricetulus
6.
Proteomics Clin Appl ; 14(6): e2000053, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33007151

RESUMO

PURPOSE: Type 1 diabetes (T1D) is characterized by autoimmune mediated self-destruction of the pancreatic islet beta cells and the resultant insulin deficiency. However, little is known about the underlying molecular pathogenesis at the pancreatic tissue level given the limited availability of clinical specimens. EXPERIMENTAL DESIGN: Quantitative proteomic studies is performed on age-matched T1D and healthy cadaveric pancreatic tissues (n = 18 each) using TMT 10plex-based isobaric labeling and BoxCar-based label-free LC-MS/MS approaches. ELISA is used to validate the differentially expressed proteins (DEPs). RESULTS: Overall, the two quantitative proteomics approaches identified 8824 proteins, of which 261 are DEPs. KEGG pathway and functional network analyses of the DEPs reveal dysregulations to pancreatic exocrine function, complement coagulation cascades, and extracellular matrix receptor interaction pathways in T1D. A selected list of the DEPs associated with pathways, subnetworks, and plasma proteome of T1D are validated using ELISA. CONCLUSIONS AND CLINICAL RELEVANCE: Integrating labeling and label-free approaches improve the confidence in quantitative profiling of pancreatic tissue proteome, which furthers the understanding of the dysregulated pathways and functional subnetworks associated with T1D pathogenesis and may aid to develop diagnostic and therapeutic strategies for T1D.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Pâncreas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Adolescente , Adulto , Biomarcadores/metabolismo , Carboxipeptidase B/metabolismo , Estudos de Casos e Controles , Colágeno Tipo VI/metabolismo , Diabetes Mellitus Tipo 1/patologia , Feminino , Humanos , Insulina/metabolismo , Masculino , Pâncreas/patologia , Adulto Jovem
7.
J Am Soc Mass Spectrom ; 31(7): 1587-1592, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32515589

RESUMO

Unprocessed C-terminal lysine (C-term Lys) is one of the most common causes for the formation of basic variants in therapeutic monoclonal antibodies (mAbs). Although the C-term Lys variants are routinely quantified by a LC-MS-based peptide mapping method using the relative MS responses from both C-terminal peptides (with and without Lys), this approach often leads to overestimation of Lys-containing peptide due to the intrinsic difference in ionization efficiency. Herein, we report an 18O-labeling assisted LC-MS method, which takes advantage of the carboxypeptidase B-catalyzed Lys removal and 18O-labeling to achieve improved accuracy of C-term Lys quantitation. The fidelity of this method was first demonstrated using synthetic peptide mixture standards that mimic a wide range of C-term Lys levels. Finally, the newly developed method was applied in a case study where C-term Lys variants in mAb samples manufactured from different processes were accurately quantified and compared. This new method provides a valuable solution for studies where accurate C-term Lys levels are needed to assist decision-making and root-cause investigation.


Assuntos
Anticorpos Monoclonais/análise , Cromatografia Líquida/métodos , Lisina/química , Espectrometria de Massas/métodos , Isótopos de Oxigênio/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Carboxipeptidase B/metabolismo , Lisina/análise , Lisina/metabolismo , Isótopos de Oxigênio/análise , Isótopos de Oxigênio/metabolismo
8.
Anal Chem ; 92(12): 8005-8009, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32441514

RESUMO

The determination of protein C-termini is of great significance for protein function annotation and proteolysis research. However, the progress of C-terminomics is still far behind its counterpart, N-terminomics, because of the low reactivity of the carboxyl group. Herein, we developed a negative selection strategy, termed carboxypeptidase B-assisted charge-based fractional diagonal chromatography (CPB-ChaFRADIC), to achieve a global C-terminome analysis. The highly reactive carboxypeptidase B cleavage was utilized to reduce the charge state of non-C-terminal peptides. Together with high-performance charge-based fractional diagonal chromatography, the C-terminal peptides could be isolated. Such a strategy was applied for profiling C-termini from Escherichia coli cell lysates and 441 canonical C-termini and 510 neo-C-termini originating from proteolytic processing were identified. These findings represent 2-fold and 5.8-fold that of identified C-termini via direct analysis, respectively. Using parallel digestion with trypsin and LysC, such a strategy enabled the identification of 604 canonical C-termini and 818 neo-C-termini, representing the largest C-terminome data set of E. coli, and no deficiency in His/Lys/Arg-containing C-terminal peptides was observed. The presented CPB-ChaFRADIC strategy is therefore a highly efficient and unbiased strategy for large-scale C-terminome analysis. Furthermore, using the CPB-ChaFRADIC strategy, we identified 107 cleavage sites and 102 substrates of caspase-3 in Jurkat cells, demonstrating that the CPB-ChaFRADIC strategy shows great promise in promoting proteolysis research. Data are available via ProteomeXchange with identifier PXD018520.


Assuntos
Carboxipeptidase B/metabolismo , Proteína C/análise , Cromatografia Líquida , Escherichia coli/enzimologia , Humanos , Peptídeos/química , Peptídeos/metabolismo , Proteína C/metabolismo , Espectrometria de Massas em Tandem
9.
Electrophoresis ; 41(12): 1109-1117, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32250465

RESUMO

A two-step CIEF with chemical mobilization was developed for charge profiling of the therapeutic mAb rituximab under non-denaturing separation conditions. CIEF of the intact mAb was combined with a middle-down approach analyzing Fc/2 and F(ab´)2 fragments after digest with a commercial cysteine protease (IdeS). CIEF methods were optimized separately for the intact mAb and its fragments due to their divergent pIs. Best resolution was achieved by combining Pharmalyte (PL) 8-10.5 with PL 3-10 for variants of intact rituximab and of F(ab´)2 fragments, respectively, whereas PL 6.7-7.7 in combination with PL 3-10 was used for Fc/2 variants. Charge heterogeneity in Fc/2 dominates over F(ab´)2 . In addition, a copy product of rituximab, and adalimumab were analyzed. Both mAbs contain additional alkaline C-terminal lysine variants as confirmed by digest with carboxypeptidase B. The optimized CIEF methods for intact mAb and Fc/2 were tested for their potential as platform approaches for these mAbs. The CIEF method for Fc/2 was slightly adapted in this process. The pI values for major intact mAb variants were determined by adjacent pI markers resulting in 9.29 (rituximab) and 8.42 (adalimumab). In total, seven to eight charge variants could be distinguished for intact adalimumab and rituximab, respectively.


Assuntos
Anticorpos Monoclonais , Eletroforese Capilar/métodos , Focalização Isoelétrica/métodos , Adalimumab/análise , Adalimumab/química , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Carboxipeptidase B/metabolismo , Cisteína Proteases/metabolismo , Lisina/metabolismo , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Rituximab/análise , Rituximab/química
10.
Anal Cell Pathol (Amst) ; 2019: 5189165, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31737467

RESUMO

Recently, there has been an increasing interest in the potential clinical use of several inflammatory indexes, namely, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and systemic-immune-inflammation index (SII). This study aimed at assessing whether these markers could be early indicators of pulmonary hypertension (PH) in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). A total of 185 patients were enrolled in our retrospective study from January 2017 to January 2019. Receiver operating characteristic curve (ROC) and area under the curve (AUC) were used to evaluate the clinical significance of these biomarkers to predict PH in patients with AECOPD. According to the diagnostic criterion for PH by Doppler echocardiography, the patients were stratified into two groups. The study group consisted of 101 patients complicated with PH, and the control group had 84 patients. The NLR, PLR, and SII values of the PH group were significantly higher than those of the AECOPD one (p < 0.05). The blood biomarker levels were positively correlated with NT-proBNP levels, while they had no significant correlation with the estimated pulmonary arterial systolic pressure (PASP) other than PLR. NLR, PLR, and SII values were all associated with PH (p < 0.05) in the univariate analysis, but not in the multivariate analysis. The AUC of NLR used for predicting PH was 0.701 and was higher than PLR and SII. Using 4.659 as the cut-off value of NLR, the sensitivity was 81.2%, and the specificity was 59.5%. In conclusion, these simple markers may be useful in the prediction of PH in patients with AECOPD.


Assuntos
Biomarcadores/metabolismo , Progressão da Doença , Hipertensão Pulmonar/complicações , Hipertensão Pulmonar/patologia , Inflamação/patologia , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/patologia , Idoso , Plaquetas/patologia , Carboxipeptidase B/metabolismo , Eletrocardiografia , Feminino , Humanos , Hipertensão Pulmonar/diagnóstico por imagem , Inflamação/imunologia , Linfócitos/patologia , Masculino , Análise Multivariada , Peptídeo Natriurético Encefálico/metabolismo , Neutrófilos/patologia , Fragmentos de Peptídeos/metabolismo , Valor Preditivo dos Testes , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Curva ROC
11.
Fish Shellfish Immunol ; 94: 434-446, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31536767

RESUMO

Carboxypeptidase plays an important physiological role in the tissues and organs of animals. In this study, we cloned an entire 2316 bp carboxypeptidase B-like (CPB) sequence with a 1302 bp open reading frame encoding a 434 amino acid peptide from Scylla paramamosain. The CPB gene was expressed highly in hepatopancreas and decreased in crab hemocytes after challenges with white spot syndrome virus (WSSV) or Vibrio alginolyticus. After CPB gene knockdown using double-stranded RNA (CPB-dsRNA), the expression of JAK, STAT, C-type lectin, crustin antimicrobial peptide, Toll-like receptors, prophenoloxidase, and myosin II essential light chain-like protein were down-regulated in hemocytes at 24 h post dsRNA treatment. CPB knockdown decreases total hemocyte count in crabs indicated that CPB may negatively regulate crab hemocyte proliferation in crabs. CPB showed an inhibitory effect on hemocyte apoptosis in crabs infected with WSSV or V. alginolyticus. The phagocytosis rate of WSSV by hemocytes was increased after CPB-dsRNA treatment. After WSSV challenge, the mortality and WSSV copy number were both decreased but the rate of hemocyte apoptosis was increased in CPB-dsRNA-treated crabs. The results indicate that the antiviral activity of the crabs was enhanced when CPB was knocked down, indicating WSSV may take advantage of CPB to benefit its replication. In contrast, the absence of CPB in crabs increased mortality following the V. alginolyticus challenge. The phagocytosis rate of V. alginolyticus by hemocytes was increased after CPB-dsRNA treatment. It was revealed that CPB may play a positive role in the immune response to V. alginolyticus through increasing the phagocytosis rate of V. alginolyticus. This research further adds to our understanding of the CPB and identifies its potential role in the innate immunity of crabs.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Carboxipeptidase B/genética , Carboxipeptidase B/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Carboxipeptidase B/química , Perfilação da Expressão Gênica , Hemócitos/imunologia , Fagocitose , Filogenia , Distribuição Aleatória , Alinhamento de Sequência , Vibrio alginolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
12.
Anal Chim Acta ; 1057: 36-43, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30832916

RESUMO

Carboxypeptidase B (CPB) is a protease that specifically hydrolyzes C-terminal alkaline amino acid of a peptide, which plays an important role in biological analysis. The activity and inhibition of CPB are significant for peptide sequencing and protein engineering. In this paper, a sensitive and easily-prepared nanochannel system was used to detect the activity of CPB. We assembled the peptides composing of alkaline amino acids into the nanochannels to detect the activity of CPB based on its hydrolysis characteristic. With CPB, the peptides would be cleaved, causing less blocking-effect on the ionic current through nanochannels. This system exhibited high sensitivity (detection limit of 0.01 U mL-1), wide linear range (0.01-10 U mL-1), and fast response (less than 10 s) for specific CPB detection. We also used the system to detect the effect of CPB inhibitors and detect in complex actual samples. The strategy exhibits effective analytical characteristics and it can be regarded as a hopeful prospect for the rapid diagnosis of patients with pancreatitis.


Assuntos
Carboxipeptidase B/metabolismo , Ensaios Enzimáticos/métodos , Nanotecnologia/métodos , Peptídeos/metabolismo , Carboxipeptidase B/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Humanos , Hidrólise , Porosidade
13.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 10): 638-643, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30279315

RESUMO

A site-directed mutagenesis method has been used to obtain the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT), in which the amino-acid residues of the S1' subsite are substituted by the corresponding residues from pancreatic carboxypeptidase B (CPB). It was shown that the mutant enzyme retained the broad, mainly hydrophobic selectivity of wild-type CPT. The mutant containing the implanted CPB S1' subsite was crystallized and its three-dimensional structure was determined at 1.29 Šresolution by X-ray crystallography. A comparison of the three-dimensional structures of CPT, the G215S/A251G/T257A/D260G/T262D CPT mutant and CPB showed that the S1' subsite of CPT has not been distorted by the mutagenesis and adequately reproduces the structure of the CPB S1' subsite. The CPB-like mutant differs from CPB in substrate selectivity owing to differences between the two enzymes outside the S1' subsite. Moreover, the difference in substrate specificity between the enzymes was shown to be affected by residues other than those that directly contact the substrate.


Assuntos
Proteínas de Bactérias/química , Carboxipeptidase B/química , Carboxipeptidases/química , Mutação , Thermoactinomyces/química , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboxipeptidase B/genética , Carboxipeptidase B/metabolismo , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Pâncreas/química , Pâncreas/enzimologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Suínos , Thermoactinomyces/enzimologia , Termodinâmica
14.
Bioorg Med Chem Lett ; 28(13): 2256-2260, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29859906

RESUMO

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) is a target molecule for treating thromboembolic disorders. We previously reported that design and synthesis of compound 1 containing a selenol group and chloloaminopyridine. Compound 1 showed high inhibitory activity towards TAFIa, with a high degree of selectivity for TAFIa over carboxypeptidase N (CPN). Here we report investigation of this selectivity. To obtain co-crystal of 1/pp-CPB (a surrogate of TAFIa), we synthesized protected compound 5 as a stabilized precursor of 1. The X-ray crystal structure and docking study indicated that the Cl substituent is accommodated in the pp-CPB specific pocket whereas CPN has no identical pocket. This is important information for the design of drugs targeting TAFIa with high selectivity.


Assuntos
Aminopiridinas/química , Carboxipeptidase B2/antagonistas & inibidores , Compostos Organosselênicos/química , Inibidores de Proteases/química , Aminopiridinas/síntese química , Animais , Sítios de Ligação , Carboxipeptidase B/antagonistas & inibidores , Carboxipeptidase B/química , Humanos , Ligação de Hidrogênio , Lisina Carboxipeptidase/antagonistas & inibidores , Simulação de Acoplamento Molecular , Compostos Organosselênicos/síntese química , Inibidores de Proteases/síntese química , Suínos
15.
Proc Natl Acad Sci U S A ; 115(18): 4767-4772, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29669919

RESUMO

To evaluate whether germline variants in genes encoding pancreatic secretory enzymes contribute to pancreatic cancer susceptibility, we sequenced the coding regions of CPB1 and other genes encoding pancreatic secretory enzymes and known pancreatitis susceptibility genes (PRSS1, CPA1, CTRC, and SPINK1) in a hospital series of pancreatic cancer cases and controls. Variants in CPB1, CPA1 (encoding carboxypeptidase B1 and A1), and CTRC were evaluated in a second set of cases with familial pancreatic cancer and controls. More deleterious CPB1 variants, defined as having impaired protein secretion and induction of endoplasmic reticulum (ER) stress in transfected HEK 293T cells, were found in the hospital series of pancreatic cancer cases (5/986, 0.5%) than in controls (0/1,045, P = 0.027). Among familial pancreatic cancer cases, ER stress-inducing CPB1 variants were found in 4 of 593 (0.67%) vs. 0 of 967 additional controls (P = 0.020), with a combined prevalence in pancreatic cancer cases of 9/1,579 vs. 0/2,012 controls (P < 0.01). More ER stress-inducing CPA1 variants were also found in the combined set of hospital and familial cases with pancreatic cancer than in controls [7/1,546 vs. 1/2,012; P = 0.025; odds ratio, 9.36 (95% CI, 1.15-76.02)]. Overall, 16 (1%) of 1,579 pancreatic cancer cases had an ER stress-inducing CPA1 or CPB1 variant, compared with 1 of 2,068 controls (P < 0.00001). No other candidate genes had statistically significant differences in variant prevalence between cases and controls. Our study indicates ER stress-inducing variants in CPB1 and CPA1 are associated with pancreatic cancer susceptibility and implicate ER stress in pancreatic acinar cells in pancreatic cancer development.


Assuntos
Carboxipeptidase B , Carboxipeptidases A , Estresse do Retículo Endoplasmático/genética , Predisposição Genética para Doença , Mutação , Proteínas de Neoplasias , Neoplasias Pancreáticas , Idoso , Idoso de 80 Anos ou mais , Carboxipeptidase B/genética , Carboxipeptidase B/metabolismo , Carboxipeptidases A/genética , Carboxipeptidases A/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia
16.
J Biomol Struct Dyn ; 36(4): 956-965, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28274181

RESUMO

Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1'-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1'-subsite.


Assuntos
Carboxipeptidase B/química , Modelos Moleculares , Fenilalanina/química , Conformação Proteica , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica , Especificidade por Substrato
17.
Biochemistry (Mosc) ; 83(12): 1594-1602, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30878033

RESUMO

It is generally accepted that the primary specificity of metallocarboxypeptidases is mainly determined by the structure of the so-called primary specificity pocket. However, the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT) with the primary specificity pocket fully reproducing the one in pancreatic carboxypeptidase B (CPB) retained the broad, mainly hydrophobic substrate specificity of the wild-type enzyme. In order to elucidate factors affecting substrate specificity of metallocarboxypeptidases and the reasons for the discrepancy with the established views, we have solved the structure of the complex of the CPT G215S/A251G/T257A/D260G/T262D mutant with the transition state analogue N-sulfamoyl-L-phenylalanine at a resolution of 1.35 Å and compared it with the structure of similar complex formed by CPB. The comparative study revealed a previously underestimated structural determinant of the substrate specificity of metallocarboxypeptidases and showed that even if substitution of five amino acid residues in the primary specificity pocket results in its almost complete structural correspondence to the analogous pocket in CPB, this does not lead to fundamental changes in the substrate specificity of the mutant enzyme due to the differences in the structure of the mobile loop located at the active site entrance that affects the substrate-induced conformational rearrangements of the active site.


Assuntos
Carboxipeptidase B/química , Carboxipeptidase B/metabolismo , Carboxipeptidases A/química , Carboxipeptidases A/metabolismo , Domínio Catalítico , Especificidade por Substrato , Thermoactinomyces/enzimologia
18.
Int J Mol Med ; 40(5): 1397-1404, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28949379

RESUMO

A reduction in pancreatic islet ß-cells leads to the onset of diabetes. Hence, the identification of the mechanisms inducing ß-cell proliferation is important for developing a treatment course against the disease. It has been well established that post-translational modifications (PTMs) of proteins affect their functionality. In addition, PTMs have been suggested to play important roles in organ regeneration. Therefore, in this study, we investigated PTMs associated with pancreatic regeneration using two-dimensional electrophoresis. Four carboxypeptidase B1 (CPB1) proteins were identified at different isoelectric points, with the same molecular weight. The motif of CPB1 PTMs was identified by mass spectrophotometry, and the downregulation of CPB1 phosphorylation in pancreatectomy was confirmed. The dephosphorylation of CPB1 induced ß-cell proliferation. We thus surmise that the altered PTM of CPB1 is associated with pancreatic regeneration.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Carboxipeptidase B/metabolismo , Ativação Linfocitária/imunologia , Animais , Carboxipeptidase B/química , Carboxipeptidase B/genética , Proliferação de Células , Sobrevivência Celular/genética , Células Cultivadas , Imuno-Histoquímica , Células Secretoras de Insulina/metabolismo , Ativação Linfocitária/genética , Masculino , Pancreatectomia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteoma , Proteômica/métodos , Ratos , Regeneração
19.
Microb Cell Fact ; 16(1): 117, 2017 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-28693509

RESUMO

BACKGROUND: Industrial processes for recombinant protein production challenge production hosts, such as the yeast Pichia pastoris, on multiple levels. During a common P. pastoris fed-batch process, cells experience strong adaptations to different metabolic states or suffer from environmental stresses due to high cell density cultivation. Additionally, recombinant protein production and nutrient limitations are challenging in these processes. RESULTS: Pichia pastoris producing porcine carboxypeptidase B (CpB) was cultivated in glucose or methanol-limited fed-batch mode, and the cellular response was analyzed using microarrays. Thereby, strong transcriptional regulations in transport-, regulatory- and metabolic processes connected to sulfur, phosphorus and nitrogen metabolism became obvious. The induction of these genes was observed in both glucose- and methanol- limited fed batch cultivations, but were stronger in the latter condition. As the transcriptional pattern was indicative for nutrient limitations, we performed fed-batch cultivations where we added the respective nutrients and compared them to non-supplemented cultures regarding cell growth, productivity and expression levels of selected biomarker genes. In the non-supplemented reference cultures we observed a strong increase in transcript levels of up to 89-fold for phosphorus limitation marker genes in the late fed-batch phase. Transcript levels of sulfur limitation marker genes were up to 35-fold increased. By addition of (NH4)2SO4 or (NH4)2HPO4, respectively, we were able to suppress the transcriptional response of the marker genes to levels initially observed at the start of the fed batch. Additionally, supplementation had also a positive impact on biomass generation and recombinant protein production. Supplementation with (NH4)2SO4 led to 5% increase in biomass and 52% higher CpB activity in the supernatant, compared to the non-supplemented reference cultivations. In (NH4)2HPO4 supplemented cultures 9% higher biomass concentrations and 60% more CpB activity were reached. CONCLUSIONS: Transcriptional analysis of P. pastoris fed-batch cultivations led to the identification of nutrient limitations in the later phases, and respective biomarker genes for indication of limitations. Supplementation of the cultivation media with those nutrients eliminated the limitations on the transcriptional level, and was also shown to enhance productivity of a recombinant protein. The biomarker genes are versatily applicable to media and process optimization approaches, where tailor-made solutions are envisioned.


Assuntos
Técnicas de Cultura Celular por Lotes , Pichia/genética , Pichia/fisiologia , Proteínas Recombinantes/biossíntese , Sulfato de Amônio/farmacologia , Animais , Biomarcadores , Biomassa , Carboxipeptidase B/biossíntese , Carboxipeptidase B/genética , Meios de Cultura/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Glucose/metabolismo , Glucose/farmacologia , Metanol/metabolismo , Análise em Microsséries , Pichia/efeitos dos fármacos , Proteínas Recombinantes/isolamento & purificação , Suínos
20.
Klin Padiatr ; 229(2): 59-66, 2017 Mar.
Artigo em Alemão | MEDLINE | ID: mdl-28444650

RESUMO

Background For the new cystic fibrosis (CF) newborn screening program in Germany the Federal Joint Committee (G-BA) implemented a new screening protocol using immunoreactive trypsinogen (IRT) as first and pancreatitis associated protein (PAP) as second tier. Gene analysis with a panel of 31 CFTR-mutations is used as third tier to increase the positive predictive value (PPV) which is known to be low in pure biochemical IRT/PAP protocols. Methods For post hoc analysis the data pool (n=372 906) of a study evaluating a pure biochemical IRT/PAP protocol was used for assessment of the 3-step G-BA protocol in comparison with an alternative screening protocol recommended by the authors. The difference between the 2 protocols is the procedure when IRT>99.9th percentile. In the G BA protocol PAP and DNA analysis will be by-passed while in the alternative protocol only the PAP step will be circumvented. Results Both 3-tier IRT/PAP+SN/DNA protocols did not lose sensitivity due to addition of genetic analysis when the results were compared to those of the 2-tier biochemical IRT/PAP protocol. However, the protocols provide different results regarding PPV. The G-BA protocol showed with 351 a much higher number of false-positively detected newborns (PPV 20.2%) when compared to 31 false-positively detected newborns in the alternative protocol (PPV 69.6%). Conclusions The G-BA protocol had a worse performance when compared with the alternative protocol recommended by the authors. The higher number of false-positively detected newborns using the G-BA protocol will lead to more consultations including sweat tests, will create more anxiety in parents, and will result in higher costs after screening.


Assuntos
Fibrose Cística/epidemiologia , Fibrose Cística/prevenção & controle , Triagem Neonatal/métodos , Carboxipeptidase B/genética , Estudos Transversais , Fibrose Cística/genética , Análise Mutacional de DNA , Reações Falso-Positivas , Feminino , Testes Genéticos , Alemanha , Humanos , Recém-Nascido , Masculino , Tripsina/genética
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