RESUMO
Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes1. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification2. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family1. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function3-9, and mutations in CCPs lead to human disease10-13. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant ß-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.
Assuntos
Carboxipeptidases , Glutamatos , Microtúbulos , Tubulina (Proteína) , Animais , Humanos , Sítios de Ligação , Encéfalo/metabolismo , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Carboxipeptidases/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Glutamatos/metabolismo , Glutamatos/química , Homeostase , Espectroscopia de Ressonância Magnética , Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Ligação Proteica , Células Sf9 , Especificidade por Substrato , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestruturaRESUMO
Microtubules are a major component of the cytoskeleton and can accumulate a plethora of modifications. The microtubule detyrosination cycle is one of these modifications; it involves the enzymatic removal of the C-terminal tyrosine of α-tubulin on assembled microtubules and the re-ligation of tyrosine on detyrosinated tubulin dimers. This modification cycle has been implicated in cardiac disease, neuronal development, and mitotic defects. The vasohibin and microtubule-associated tyrosine carboxypeptidase enzyme families are responsible for microtubule detyrosination. Their long-sought discovery allows to review and summarise differences and similarities between the two enzymes families and discuss how they interplay with other modifications and functions of the tubulin code.
Assuntos
Carboxipeptidases , Microtúbulos , Tubulina (Proteína) , Tirosina , Microtúbulos/metabolismo , Humanos , Animais , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Carboxipeptidases/metabolismo , Carboxipeptidases/genética , Carboxipeptidases/química , Tirosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/química , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Carboxypeptidase is an exopeptidase that hydrolyzes amino acids at the C-terminal end of the peptide chain and has a wide range of applications in food. However, in industrial applications, the relatively low catalytic efficiency of carboxypeptidases is one of the main limiting factors for industrialization. RESULTS: The study has enhanced the catalytic efficiency of Bacillus megaterium M32 carboxypeptidase (BmeCPM32) through semi-rational design. Firstly, the specific activity of the optimal mutant, BmeCPM32-M2, obtained through single-site mutagenesis and combinatorial mutagenesis, was 2.2-fold higher than that of the wild type (187.9 versus 417.8 U mg-1), and the catalytic efficiency was 2.9-fold higher (110.14 versus 325.75 s-1 mmol-1). Secondly, compared to the wild type, BmeCPM32-M2 exhibited a 1.8-fold increase in half-life at 60 °C, with no significant changes in its enzymatic properties (optimal pH, optimal temperature). Finally, BmeCPM32-M2 significantly increased the umami intensity of soy protein isolate hydrolysate by 55% and reduced bitterness by 83%, indicating its potential in developing tasty protein components. CONCLUSION: Our research has revealed that the strategy based on protein sequence evolution and computational residue mutation energy led to an improved catalytic efficiency of BmeCPM32. Molecular dynamics simulations have revealed that a smaller substrate binding pocket and increased enzyme-substrate affinity are the reasons for the enhanced catalytic efficiency. Furthermore the number of hydrogen bonds and solvent and surface area may contribute to the improvement of thermostability. Finally, the de-bittering effect of BmeCPM32-M2 in soy protein isolate hydrolysate suggests its potential in developing palatable protein components. © 2024 Society of Chemical Industry.
Assuntos
Bacillus megaterium , Proteínas de Bactérias , Carboxipeptidases , Paladar , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carboxipeptidases/metabolismo , Carboxipeptidases/genética , Carboxipeptidases/química , Bacillus megaterium/enzimologia , Bacillus megaterium/genética , Cinética , Humanos , Concentração de Íons de Hidrogênio , Estabilidade Enzimática , Engenharia de Proteínas , Biocatálise , Proteínas de Soja/química , Proteínas de Soja/metabolismo , Proteínas de Soja/genética , Mutagênese Sítio-Dirigida , Aromatizantes/química , Aromatizantes/metabolismo , CatáliseRESUMO
In mammals, a limited number of proteases catalyze with acidic amino acids as substrates. At present, there are only three known proteases: CCPs, carboxypeptidase O (CPO), and aspartate acylase (ASPA). Human CPO is a digestive enzyme that prefers glutamate as a substrate. It locates to the apical membrane of intestinal epithelial and is glycosylated protein. CPO is difficult to purify as it is a GPI-anchored protein. To obtain purified CPO, a truncated form called hCPOΔC was designed, which removed the C-terminal sequence of hCPO and was followed by His tag. Firstly, the truncated variant hCPOΔC (residues 1-349) was cloned into pFastBac vector to construct bacmid. Then the verified bacmid was transfected into Sf9 cells for expression. After the protein was successfully expressed, cell medium was collected and incubated with Ni resins. The target protein was eluted by imidazole through affinity chromatography. A purification method of human CPO with deglutamylation activity was successfully established using insect cells expression system. Purified hCPOΔC could hydrolyze glutamate in polypeptides.
Assuntos
Carboxipeptidases , Glutamatos , Animais , Humanos , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Peptídeo Hidrolases , Mamíferos/metabolismoRESUMO
Angiotensin-converting enzyme 2 (ACE2) is a metalloprotease that cleaves angiotensin II, a peptide substrate involved in the regulation of hypertension. Here, we identified a series of constrained bicyclic peptides, Bicycle, inhibitors of human ACE2 by panning highly diverse bacteriophage display libraries. These were used to generate X-ray crystal structures which were used to inform the design of additional Bicycles with increased affinity and inhibition of ACE2 enzymatic activity. This novel structural class of ACE2 inhibitors is among the most potent ACE2 inhibitors yet described in vitro, representing a valuable tool to further probe ACE2 function and for potential therapeutic utility.
Assuntos
Enzima de Conversão de Angiotensina 2 , Carboxipeptidases , Humanos , Carboxipeptidases/química , Peptidil Dipeptidase A , Ciclismo , Peptídeos/farmacologia , Angiotensina II , Fragmentos de PeptídeosRESUMO
As one of the most abundant bacteria in the human oral cavity, Fusobacterium nucleatum is closely involved in various oral diseases and is also a risk factor for other diseases. The peptidases of F. nucleatum can digest exogenous peptides into amino acids to satisfy its nutrient requirements. Here, a putative F. nucleatum peptidase, termed S9Cfn, which belongs to the S9C peptidase family was identified. Enzymatic activity assays combined with mass-spectrometric analysis revealed that S9Cfn is a carboxypeptidase, but not an aminopeptidase as previously annotated. The crystal structure of the S9Cfn tetramer was solved at 2.6â Å resolution and was found to contain a pair of oligomeric pores in the center. Structural analysis, together with site-directed mutagenesis and enzymatic activity assays, revealed a substrate-entrance tunnel that extends from each oligomeric pore to the catalytic triad, adjacent to which three conserved arginine residues are responsible for substrate binding. Moreover, comparison with other S9 peptidase structures indicated drastic conformational changes of the oligomeric pores during the catalytic cycle. Together, these findings increase the knowledge of this unique type of tetrameric carboxypeptidase and provide insight into the homeostatic control of microbiota in the human oral cavity.
Assuntos
Proteínas de Bactérias/metabolismo , Carboxipeptidases/metabolismo , Fusobacterium nucleatum/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Carboxipeptidases/química , Carboxipeptidases/genética , Domínio Catalítico , Cristalografia por Raios X , Humanos , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Conformação ProteicaRESUMO
BACKGROUND: Infectious diseases still affect large populations causing significant morbidity and mortality. Bacterial and fungal infections for centuries were the main factors of death and disability of millions of humans. Despite the progress in the control of infectious diseases, the appearance of resistance of microbes to existing drugs creates the need for the development of new effective antimicrobial agents. In an attempt to improve the antibacterial activity of previously synthesized compounds modifications to their structures were performed. METHODS: Nineteen thiazolidinone derivatives with 6-Cl, 4-OMe, 6-CN, 6-adamantan, 4-Me, 6-adamantan substituents at benzothiazole ring were synthesized and evaluated against panel of four bacterial strains S. aureus, L. monocytogenes, E. coli and S. typhimirium and three resistant strains MRSA, E. coli and P. aeruginosa in order to improve activity of previously evaluated 6-OCF3-benzothiazole-based thiazolidinones. The evaluation of minimum inhibitory and minimum bactericidal concentration was determined by microdilution method. As reference compounds ampicillin and streptomycin were used. RESULTS: All compounds showed antibacterial activity with MIC in range of 0.12-0.75 mg/mL and MBC at 0.25->1.00 mg/mL The most active compound among all tested appeared to be compound 18, with MIC at 0.10 mg/mL and MBC at 0.12 mg/mL against P. aeruginosa. as well as against resistant strain P. aeruginosa with MIC at 0.06 mg/mL and MBC at 0.12 mg/mL almost equipotent with streptomycin and better than ampicillin. Docking studies predicted that the inhibition of LD-carboxypeptidase is probably the possible mechanism of antibacterial activity of tested compounds. CONCLUSION: The best improvement of antibacterial activity after modifications was achieved by replacement of 6-OCF3 substituent in benzothiazole moiety by 6-Cl against S. aureus, MRSA and resistant strain of E. coli by 2.5 folds, while against L. monocytogenes and S. typhimirium from 4 to 5 folds.
Assuntos
Anti-Infecciosos/síntese química , Inibidores de Proteases/síntese química , Tiazolidinas/síntese química , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carboxipeptidases/antagonistas & inibidores , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Listeria monocytogenes/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Tiazolidinas/farmacologiaRESUMO
Channa striatus is high-protein food with many health functions. This study aimed to prepare, screen and identify the angiotensin-converting enzyme inhibition peptides (ACEIPs) from C. striatus hydrolysates by response surface methodology and bioaffinity ultrafiltration coupled with LC-Orbitrap-MS/MS and molecular docking. The optimal conditions for preparing ACEIPs were hydrolysis temperature 55 °C, hydrolysis time 3 h, pH 9, solid-liquid ratio 1:20 g/mL, and enzyme addition 5%, the ACE inhibition and molecular weight distribution of obtained hydrolysate was 54.35% and 8770-160 Da, respectively. Seven novel ACEIPs were screened through the established high-throughput screening approach, among which, EYFR and LPGPGP showed the strongest ACE inhibition with the IC50 value of 179.2 and 186.3 µM, respectively (P > 0.05). Molecular docking revealed that three and ten hydrogen bonds were formed between ACE and LPGPGP and EYFR, respectively, S1 and S2 were the major active pockets, but the major driving forces varied.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/análise , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Proteínas de Peixes/química , Peptídeos/análise , Peptídeos/isolamento & purificação , Hidrolisados de Proteína/química , Ultrafiltração , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Animais , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Hidrólise , Simulação de Acoplamento Molecular , Peso Molecular , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Espectrometria de Massas em TandemRESUMO
The helical morphology of Campylobacter jejuni, a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located â¼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δpgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape.
Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/citologia , Carboxipeptidases/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Peptidoglicano/metabolismo , Campylobacter jejuni/metabolismo , Carboxipeptidases/química , Domínio Catalítico , Humanos , Conformação ProteicaRESUMO
Pyrrolidone carboxypeptidases (Pcps) (E.C. 3.4.19.3) can cleave the peptide bond adjacent to pyro-glutamic acid (pGlu), an N-terminal modification observed in some proteins that provides protection against common proteases. Pcp derived from extremely thermophilic Fervidobacterium islandicum AW-1 (FiPcp), that belongs to the cysteine protease family, is involved in keratin utilization under stress conditions. Although an irreversible oxidative modification of active cysteine to its sulfonic acid derivative (Cys-SO3H) renders the enzyme inactive, the molecular details for the sulfonic acid modification in inactive Pcp remain unclear. Here, we determined the crystal structure of FiPcp at 1.85 Å, revealing the oxidized form of cysteine sulfonic acid (C156-SO3H) in the catalytic triad (His-Cys-Glu), which participates in the hydrolysis of pGlu residue containing peptide bond. The three oxygen atoms of cysteine sulfonic acid were stabilized by hydrogen bonds with H180, carbonyl backbone of Q83, and water molecules, resulting in inactivation of FiPcp. Furthermore, FiPcp demonstrated a unique 139KKKK142 motif involved in inter-subunit electrostatic interactions whose mutation significantly affects the thermostability of tetrameric FiPcp. Thus, our high-resolution structure of the first inactive FiPcp with irreversible oxidative modification of active cysteine provides not only the molecular basis of the redox-dependent catalysis of Pcp, but also the structural features of its thermostability.
Assuntos
Bactérias/enzimologia , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Queratinas/metabolismo , Pirrolidinonas/metabolismo , Motivos de Aminoácidos , Bactérias/classificação , Domínio Catalítico , Cristalografia por Raios X , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Estabilidade Enzimática , Ligação de Hidrogênio , Hidrólise , Modelos Moleculares , Oxirredução , Oxigênio/metabolismo , Eletricidade Estática , Água/metabolismoRESUMO
Carboxypeptidase T (CPT) from Thermoactinomyces vulgaris (EC 3.4.17.18) has a broad substrate specificity, the mechanism of which remains unclear. It cleaves off arginine residues by 10, and lysine residues by 100 times worse than hydrophobic leucine residues despite the presence of negatively charged Asp260 at the bottom of the primary specificity pocket. To study the relationship between the structure and specificity the 3D structure of CPT in complex with the stable transition state analog N-sulfamoyl-l-lysine (SLys) was determined in which the S-atom imitates the sp3-hybridized carbon in the scissile-bond. Crystals grown in microgravity has the symmetry of space group P6322. The present complex structure was compared with the previously reported complex structure of CPT and N-sulfamoyl-L-arginine (SArg). The location/binding of SLys in the active site of CPT very closely resembled that of SArg, and the positively charged N-atom of SLys was at the same position as the corresponding positively charged N-atom of SArg. The SLys complex is stabilized by the hydrogen bond between the nitrogen atom and OH-group of Thr257. The contact areas of the residues Tyr255, Leu211, and Thr262 with SLys were reduced in comparison with the same of SArg. This difference in bonding of SArg and SLys side chains in the primary specificity pocket induces shifts differences within the catalytic center (especially Tyr255-O20 and S18-Arg129 N1 gap) that may influence the enzyme's catalytic reaction. Therefore, this information may be useful for the design of carboxypeptidases with improved selectivity towards Arg/Lys for biotechnological applications.
Assuntos
Proteínas de Bactérias/química , Carboxipeptidases/química , Thermoactinomyces/enzimologia , Proteínas de Bactérias/metabolismo , Carboxipeptidases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Lisina/análogos & derivados , Lisina/metabolismo , Modelos Moleculares , Especificidade por Substrato , Thermoactinomyces/química , Thermoactinomyces/metabolismoRESUMO
Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1' position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions.
Assuntos
Carboxipeptidases/química , Humanos , Domínios Proteicos , Especificidade por SubstratoRESUMO
Mycobacterial peptidoglycan (PG) is an unsolved puzzle due to its complex structure and involvement of multiple enzymes in the process of its remodelling. dd-Carboxypeptidases are low molecular mass penicillin-binding proteins (LMM-PBPs) that catalyzes the cleavage of terminal d-Ala of muramyl pentapeptide branches and thereby helps in the PG remodelling process. Here, we have assigned the function of a putative LMM-PBP, MSMEG_2432 of Mycobacterium smegmatis, by showing that it exhibits both dd-CPase and ß-lactamase activities. Like conventional dd-CPase (PBP5 from E. coli), upon ectopic complementation in a deformed seven PBP deletion mutant of E. coli, MSMEG_2432 has manifested its ability to restore ~75â% of the cell population to their normal rod shape. Further, in vitrodd-CPase assay has confirmed its ability to release terminal d-Ala from the synthetic tripeptide and the peptidoglycan mimetic pentapeptide substrates ending with d-Ala-d-Ala. Also, elevated resistance against penicillins and cephalosporins upon ectopic expression of MSMEG_2432 suggests the presence of ß-lactamase activity, which is further confirmed in vitro through nitrocefin hydrolysis assay. Moreover, it is found apparent that D169A substitution in MSMEG_2432 influences both of its in vivo and in vitrodd-CPase and ß-lactamase activities. Thus, we infer that MSMEG_2432 is a dual function enzyme that possesses both dd-CPase and ß-lactamase activities.
Assuntos
Proteínas de Bactérias/metabolismo , Carboxipeptidases/metabolismo , Mycobacterium smegmatis/enzimologia , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carboxipeptidases/química , Carboxipeptidases/genética , Mycobacterium smegmatis/química , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Penicilinas/farmacologia , Peptidoglicano/metabolismo , beta-Lactamases/química , beta-Lactamases/genéticaRESUMO
Efficient communication between cells requires the ability to process precursor proteins into their mature and biologically active forms, prior to secretion into the extracellular space. Eukaryotic cells achieve this via a suite of enzymes that involve a signal peptidase complex, prohormone convertases and carboxypeptidases. Using genome and transcriptome data of the demosponge Amphimedon queenslandica, a universal ancestor to metazoan multicellularity, we endeavour to bridge the evolution of precursor processing machinery from single-celled eukaryotic ancestors through to the complex multicellular organisms that compromise Metazoa. The precursor processing repertoire as defined in this study of A. queenslandica consists of 3 defined signal peptidase subunits, 6 prohormone convertases and 1 carboxypeptidase, with 2 putative duplicates identified for signal peptidase complex subunits. Analysis of their gene expression levels throughout the sponge development enabled us to predict levels of activity. Some A. queenslandica precursor processing components belong to established functional clades while others were identified as having novel, yet to be discovered roles. These findings have clarified the presence of precursor processing machinery in the poriferans, showing the necessary machinery for the removal of precursor sequences, a critical post-translational modification required by multicellular organisms, and further sets a foundation towards understanding the molecular mechanism for ancient protein processing.
Assuntos
Carboxipeptidases/metabolismo , Poríferos/metabolismo , Pró-Proteína Convertases/metabolismo , Subunidades Proteicas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Carboxipeptidases/química , Carboxipeptidases/genética , Filogenia , Poríferos/classificação , Poríferos/genética , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Subunidades Proteicas/químicaRESUMO
Mycotoxin removers include enzymes and adsorbents that may be used in animal feeds to eliminate the toxic effects of mycotoxins. This study aimed to determine the removability of two different types of mycotoxin removers, adsorbents and enzyme degradation reagents (EDRs), in the simulated gastrointestinal conditions of pigs and poultry. Seven commercial mycotoxin removers, including five EDRs and two adsorbents, were tested in vitro. In this study, the supplemented dosages of mycotoxin removers used in pig and poultry feeds were the commercial recommendation ranging from 0.05% to 0.2%. For pigs, the in vitro gastric and small intestinal simulations were performed by immersing the mycotoxin-tainted feed in artificial gastric juice (AGJ) at pH 2.5 for 5 h or in artificial intestinal juice (AIJ) at pH 6.5 for 2 h to mimick in vivo conditions. For poultry, mycotoxin-tainted feeds were immersed in AGJ for 2 h at pH 4.5 and 0.5 h at pH of 2.5, respectively, to simulate crop/glandular stomach and gizzard conditions; the small intestinal simulation was in AIJ for 2 h at pH 6.5. For the pig, EDRs and adsorbents had deoxynivalenol (DON) removability (1 mg/kg) of 56% to 100% and 15% to 19%, respectively. Under the concentration of 0.5 mg/kg, the zearalenone (ZEN) removability by EDRs and adsorbents was 65% to 100% and 0% to 36%, respectively. For the simulation in poultry, the removability of DON by EDRs and adsorbents (5 mg/kg) was 56% to 79% and 1% to 36%, respectively; for the concentration of 0.5 mg/kg, the removability of ZEN by EDRs and adsorbents was 38% to 69% and 7% to 9%, respectively. These results suggest that EDRs are more effective in reducing DON and ZEN contamination compared to the adsorbent methods in the simulated gastrointestinal tracts of pig and poultry. The recoveries of DON and ZEN of pig in vitro gastrointestinal simulations were higher than 86.4% and 84.7%, respectively, with 88.8% and 85.9%, respectively, in poultry. These results demonstrated the stability and accuracy of our mycotoxin extraction process and in vitro simulation efficiency.
Assuntos
Tricotecenos/química , Zearalenona/química , Adsorção , Silicatos de Alumínio , Animais , Bacillus subtilis/enzimologia , Carboxilesterase/química , Carboxipeptidases/química , Parede Celular/química , Contaminação de Alimentos , Suco Gástrico , Hidrolases/química , Indicadores e Reagentes , Oxirredutases/química , Aves Domésticas , Suínos , LevedurasRESUMO
Recently, we have observed a relationship between poor breadmaking quality and protease activities related to fungal infection. This study aims to identify potential gluten-degrading proteases secreted by fungi and to analyze effects of these proteases on rheological properties of dough and gluten. Fusarium graminearum-infected grain was used as a model system. Zymography showed that serine-type proteases secreted by F. graminearum degrade gluten proteins. Zymography followed by liquid chromatography-mass spectrometry (MS)/MS analysis predicted one serine carboxypeptidase and seven serine endo-peptidases to be candidate fungal proteases involved in gluten degradation. Effects of fungal proteases on the time-dependent rheological properties of dough and gluten were analyzed by small amplitude oscillatory shear rheology and large deformation extensional rheology. Our results indicate that fungal proteases degrade gluten proteins not only in the grain itself, but also during dough preparation and resting. Our study gives new insights into fungal proteases and their potential role in weakening of gluten.
Assuntos
Carboxipeptidases/metabolismo , Endopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Glutens/metabolismo , Doenças das Plantas/microbiologia , Triticum/microbiologia , Carboxipeptidases/química , Endopeptidases/química , Farinha/análise , Farinha/microbiologia , Proteínas Fúngicas/química , Fusarium/fisiologia , Glutens/análise , Espectrometria de Massas , Reologia , Triticum/química , Triticum/metabolismoRESUMO
Zearalenone (ZEA) is an estrogenic and ochratoxin A (OTA) is a hepatotoxic Fusarium mycotoxin commonly seen in cereals and fruits products. No previous investigation has studied on a single platform for the multi degradation mycotoxin. The current study aimed to investigate the bifunctional activity of a novel fusion recombinant. We have generated a recombinant fusion enzyme (ZHDCP) by combining two single genes named zearalenone hydrolase (ZHD) and carboxypeptidase (CP) in frame deletion by crossover polymerase chain reaction (PCR). We identified enzymatic properties and cell cytotoxicity assay of ZHDCP enzyme. Our findings have demonstrated that ZEA was completely degraded to the non-toxic product in 2 h by ZHDCP enzyme at an optimum pH of 7 and a temperature of 35 °C. For the first time, it was found out that ZEA 60% was degraded by CP degrades in 48 h. Fusion ZHDCP and CP enzyme were able to degrade 100% OTA in 30 min at pH 7 and temperature 30 °C. ZEA- and OTA-induced cell death and increased cell apoptosis rate and regulated mRNA expression of Sirt1, Bax, Bcl2, Caspase3, TNFα, and IL6 genes. Our novel findings demonstrated that the fusion enzyme ZHDCP possess bifunctional activity (degrade OTA and ZEA), and it could be used to degrade more mycotoxins.
Assuntos
Carboxipeptidases/química , Hidrolases/química , Enzimas Multifuncionais/química , Ocratoxinas/química , Proteínas Recombinantes de Fusão/química , Zearalenona/química , Carboxipeptidases/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Hidrolases/genética , Ocratoxinas/toxicidade , Zearalenona/toxicidadeRESUMO
Penicillin-binding proteins (PBPs) share the namesake because of their ability to bind penicillin or any beta-lactam antibiotic. In other words, PBPs are the targets of ß-lactam antibiotics that hold nearly 60% of the global antibiotic market. These enzymes catalyze the final stages of peptidoglycan (PG) biosynthesis by acting as transglycosylases and transpeptidases. PBPs are also involved in PG remodeling by catalyzing DD-carboxypeptidase (DD-CPase) and endopeptidase reactions. Though the cross-linking abilities of PBPs are well known, the process of remodeling is still unclear, thereby drawing attention toward the DD-CPase enzymes. Here, we describe the step-by-step procedures for isolation of the bacterial cell membrane and detection of PBPs in it, followed by the purification of PBPs (DD-CPases) by both ampicillin-affinity and nickel-nitrilotriacetic acid (Ni-NTA) chromatography. The protocols to determine the enzymatic efficiency are also elucidated. The assays are aimed to determine the kinetic parameters for the interaction of the PBP with BOCILLIN, to evaluate its acylation and deacylation rates, and with its peptide substrates, to assess its DD-CPase activity.
Assuntos
Bioensaio , Carboxipeptidases/química , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/isolamento & purificação , Acilação , Bioensaio/métodos , Membrana Celular/metabolismo , Cromatografia de Afinidade , Clonagem Molecular , Expressão Gênica , Hidrólise , Cinese , Proteínas de Ligação às Penicilinas/genética , SolubilidadeRESUMO
Decoding the interaction between coordination compounds and proteins is of fundamental importance in biology, pharmacy, and medicine. In this context, protein- ligand docking represents a particularly interesting asset to predict how small compounds could interact with biomolecules, but to date, very little information is available to adapt these methodologies to metal-containing ligands. Here, we assessed the predictive capability of a metal-compatible parameter set for the docking program GOLD for metallo ligands with multiple vacant sites and different geometries. The study first presents a benchmark of 25 well-characterized X-ray metallo ligand-protein adducts. In 100% of the cases, the docking solutions are superimposable to the X-ray determination, and in 92% the value of the root-mean-square deviation between the experimental and calculated structures is lower than 1.5 Å. After the validation step, we applied these methods to five case studies for the prediction of the binding of pharmacological active metal species to proteins: (i) the anticancer copper(II) complex [CuII(Br)(2-hydroxy-1-naphthaldehyde benzoyl hydrazine)(indazole)] to human serum albumin (HSA); (ii) one of the active species of antidiabetic and antitumor vanadium compounds, VIVO2+ ion, to carboxypeptidase; (iii) the antiarthritic species [AuI(PEt3)]+ to HSA; (iv) the antitumor oxaliplatin to ubiquitin; (v) the antitumor ruthenium(II) compound RAPTA-PentaOH to cathepsin B. The calculations suggested that the binding modes are in good agreement with the partial information retrieved from spectroscopic and spectrometric analysis and allowed us, in certain cases, to propose additional hypotheses. This method is an important update in protein-metallo ligand docking, which could have a wide field of application, from biology and inorganic biochemistry to medicinal chemistry and pharmacology.
Assuntos
Carboxipeptidases/metabolismo , Catepsina B/metabolismo , Complexos de Coordenação/metabolismo , Albumina Sérica Humana/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação , Carboxipeptidases/química , Catepsina B/química , Complexos de Coordenação/química , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Albumina Sérica Humana/química , Ubiquitina/químicaRESUMO
Complement system orchestrates the innate and adaptive immunity via the activation, recruitment, and regulation of immune molecules to destroy pathogens. However, regulation of the complement is essential to avoid injuries to the autologous tissues. The present study unveils the characteristic features of an important complement component, anaphylatoxin inactivator from red lip mullet at its molecular and functional level. Mullet carboxypeptidase N1 (MuCPN1) cDNA sequence possessed an open reading frame of 1347 bp, which encoded a protein of 449 amino acids with a predicted molecular weight of 51â¯kDa. In silico analysis discovered two domains of PM14-Zn carboxypeptidase and a C-terminal domain of M14 N/E carboxypeptidase, two zinc-binding signature motifs, and an N-glycosylation site in the MuCPN1 sequence. Homology analysis revealed that most of the residues in the sequence are conserved among the other selected homologs. Phylogeny analysis showed that MuCPN1 closely cladded with the Maylandia zebra CPN1 and clustered together with the teleostean counterparts. A challenge experiment showed modulated expression of MuCPN1 upon polyinosinic:polycytidylic acid and Lactococcus garviae in head kidney, spleen, gill, and liver tissues. The highest upregulation of MuCPN1 was observed 24â¯h post infection against poly I:C in each tissue. Moreover, the highest relative expressions upon L. garviae challenge were observed at 24â¯h post infection in head kidney tissue and 48â¯h post infection in spleen, gill, and liver tissues. MuCPN1 transfected cells triggered a 2.2-fold increase of nitric oxide (NO) production upon LPS stimulation compared to the un-transfected controls suggesting that MuCPN1 is an active protease which releases arginine from complement C3a, C4a, and C5a. These results have driven certain way towards enhancing the understanding of immune role of MuCPN1 in the complement defense mechanism of red lip mullet.