RESUMO
VAMP/synaptobrevin-associated protein B (VAP-B) is a type II ER membrane protein, but its N-terminal MSP domain (MSPd) can be cleaved and secreted. Mutations preventing the cleavage and secretion of MSPd have been implicated in cases of human neurodegenerative diseases. The site of VAP cleavage and the tissues capable in releasing the processed MSPd are not understood. In this study, we analyze the C. elegans VAP-B homolog, VPR-1, for its processing and secretion from the intestine. We show that intestine-specific expression of an N-terminally FLAG-tagged VPR-1 rescues underdeveloped gonad and sterility defects in vpr-1 null hermaphrodites. Immunofluorescence studies reveal that the tagged intestinal expressed VPR-1 is present at the distal gonad. Mass spectrometry analysis of a smaller product of the N-terminally tagged VPR-1 identifies a specific cleavage site at Leu156. Mutation of the leucine results in loss of gonadal MSPd signal and reduced activity of the mutant VPR-1. Thus, we report for the first time the cleavage site of VPR-1 and provide direct evidence that intestinally expressed VPR-1 can be released and signal in the distal gonad. These results establish the foundation for further exploration of VAP cleavage, MSPd secretion, and non-cell-autonomous signaling in development and diseases.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Helminto/metabolismo , Proteínas de Membrana/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/metabolismo , Genes de Helmintos , Gônadas/química , Gônadas/crescimento & desenvolvimento , Gônadas/metabolismo , Proteínas de Helminto/química , Organismos Hermafroditas/genética , Organismos Hermafroditas/metabolismo , Organismos Hermafroditas/fisiologia , Infertilidade , Intestinos/citologia , Intestinos/fisiologia , Leucina/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Fenótipo , Mutação Puntual , Domínios Proteicos , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: The transparent epidermis of Caenorhabditis elegans makes it an attractive model to study sperm motility and migration within an intact reproductive tract. C elegans synthesize specific F-series prostaglandins (PGFs) that are important for guiding sperm toward the spermatheca. These PGFs are synthesized from polyunsaturated fatty acid (PUFA) precursors, such as arachidonic acid (AA), via a novel pathway, independent of the classical cyclooxygenases (Cox) responsible for most PG synthesis. While the enzyme(s) responsible for PG synthesis has yet to be identified, the DAF-7 TGFß pathway has been implicated in modulating PG levels and sperm guidance. RESULTS: We find that the reduced PGF levels in daf-1 type I receptor mutants are responsible for the sperm guidance defect. The lower level of PGs in daf-1 mutants is due in part to the inaccessibility of AA. Finally, lipid analysis and assessment of sperm guidance in daf-1;daf-3 double mutants suggest DAF-3 suppresses PG production and sperm accumulation at the spermatheca. Our data suggest that DAF-3 functions in the nervous system, and possibly the germline, to affect sperm guidance. CONCLUSION: The C elegans TGFß pathway regulates many pathways to modulate PG metabolism and sperm guidance. These pathways likely function in the nervous system and possibly the germline.
Assuntos
Prostaglandinas/biossíntese , Motilidade dos Espermatozoides/genética , Fator de Crescimento Transformador beta/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Feminino , Masculino , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
There are no therapies to prevent emphysema progression. Chymotrypsin-like elastase 1 (CELA1) is a serine protease that binds and cleaves lung elastin in a stretch-dependent manner and is required for emphysema in a murine antisense oligonucleotide model of α-1 antitrypsin (AAT) deficiency. This study tested whether CELA1 is important in strain-mediated lung matrix destruction in non-AAT-deficient emphysema and the efficacy of CELA1 neutralization. Airspace simplification was quantified after administration of tracheal porcine pancreatic elastase (PPE), after 8 months of cigarette smoke (CS) exposure, and in aging. In all 3 models, Cela1-/- mice had less emphysema and preserved lung elastin despite increased lung immune cells. A CELA1-neutralizing antibody was developed (KF4), and it inhibited stretch-inducible lung elastase in ex vivo mouse and human lung and immunoprecipitated CELA1 from human lung. In mice, systemically administered KF4 penetrated lung tissue in a dose-dependent manner and 5 mg/kg weekly prevented emphysema in the PPE model with both pre- and postinjury initiation and in the CS model. KF4 did not increase lung immune cells. CELA1-mediated lung matrix remodeling in response to strain is an important contributor to postnatal airspace simplification, and we believe that KF4 could be developed as a lung matrix-stabilizing therapy in emphysema.
Assuntos
Enfisema , Enfisema Pulmonar , Animais , Humanos , Camundongos , Envelhecimento , Elastina , Elastase Pancreática , Enfisema Pulmonar/prevenção & controle , SuínosRESUMO
The extracellular cellular matrix (ECM) maintains tissue structure and regulates signaling functions by continuous degradation and remodeling. Inflammation or other disease conditions activate proteases including matrix metalloproteinases (MMPs) that degrade ECM proteins and in particular generate fragments of collagen and elastin, some of which are biologically active ECM peptides or matrikines. Stepwise degradation of collagen by MMP 8, 9 and prolyl endopeptidase release the matrikine proline-glycine-proline (PGP) and its product acetyl-PGP (AcPGP). These peptides are considered as potential biomarkers and therapeutic targets for many disease conditions such as chronic lung disease, heart disease, and cancer. However, there is no published, validated method for the measurement of PGP and AcPGP in plasma and therefore, we developed a sensitive, selective and reliable, isotope dilution LC-multiple reaction monitoring MS method for their determination in human plasma. The chromatographic separation of PGP and AcPGP was achieved in 3 min using Jupiter column with a gradient consisting of acidified acetonitrile and water at a flow rate of 0.5 ml/min. The limit of detection (LOD) for PGP and AcPGP was 0.01 ng/ml and the limit of quantification (LOQ) was 0.05 ng/ml and 0.1 ng/ml, respectively. Precision and accuracy values for all analytes were within 20 % except for the lowest QC of 0.01 ng/ml. The mean extraction recoveries of these analytes were > 90 % using a Phenomenex Phree cartridge and the matrix effect was < 15 % for all the QCs for PGP and AcPGP except the lowest QC. The stability of PGP and AcPGP was > 90 % in several tested conditions including autosampler use, storage at -80 °C, and after 6 times freeze-thaw cycles. Using this method, we successfully extracted and determined PGP levels in human plasma from healthy and COPD subjects. Therefore, this method is suitable for quantification of these peptides in the clinical setting.
Assuntos
Glicina , Prolina , Humanos , Cromatografia Líquida , Espectrometria de Massas em Tandem , Peptídeos , ColágenoRESUMO
Prostaglandins comprise a family of lipid signaling molecules derived from polyunsaturated fatty acids and are involved in a wide array of biological processes, including fertilization. Prostaglandin-endoperoxide synthase (a.k.a. cyclooxygenase or Cox) initiates prostaglandin synthesis from 20-carbon polyunsaturated fatty acids, such as arachidonic acid. Oocytes of Caenorhabditis elegans (C. elegans) have been shown to secrete sperm-guidance cues prostaglandins, independent of Cox enzymes. Both prostaglandin synthesis and signal transduction in C. elegans are environmentally modulated pathways that regulate sperm guidance to the fertilization site. Environmental factors such as food triggers insulin and TGF-ß secretion and their levels regulate tissue-specific prostaglandin synthesis in C. elegans. This novel PG pathway is abundant in mouse and human ovarian follicular fluid, where their functions, mechanism of synthesis and pathways remain to be established. Given the importance of prostaglandins in reproductive processes, a better understanding of how diets and other environmental factors influence their synthesis and function may lead to new strategies towards improving fertility in mammals.
RESUMO
Monomeric 30 kDa gamma-glutamyl transpeptidase (GGT(30)) was purified from culture broth of Bacillus licheniformis ER-15 along with a heterodimeric 67 kDa GGT (GGT(67)). In presence of subtilisin, GGT(30) had improved catalytic efficiency (V(max)/K(m)) of 59 min(-1), altered pH and temperature optima of pH 11 and 70 degrees C and had salt-tolerant glutaminase activity. Glutaminase activity was retained even in protease-inhibited condition in presence of 2 mM PMSF. GGT(30) and subtilisin complexation was also confirmed by relative electrophoretic mobility and fluorescence quenching experiment.
Assuntos
Bacillus/enzimologia , Ativação Enzimática/efeitos dos fármacos , Subtilisina/farmacologia , gama-Glutamiltransferase/metabolismo , Bacillus/efeitos dos fármacos , Catálise , Eletroforese em Gel de PoliacrilamidaRESUMO
We previously discovered that Caenorhabditis elegans synthesizes Cox-independent F-series prostaglandins (PGs). To delineate the Cox-independent prostaglandin pathways and evaluate their role in sperm motility in C. elegans, we developed a novel biochemical method for the rapid production of F-series PGs using arachidonic acid as the substrate and worm lysate as source of enzyme(s). Among the four F2-series PGs produced in the reaction, three of them were identified as 8-isoPGF2α, 5iPF2 VI, and PGF2α based on their retention times and MS/MS spectral comparison with standards using LC-MS/MS. PG production was not markedly affected by specific antioxidants, or Cox, Lox, and Cyp inhibitors, suggesting that these PGs are formed through a novel, biologically regulated mechanism in C. elegans. This study also assessed the ability of 8-isoPGF2α, 5iPF2 VI, PGF2α, and a mixture containing these PGs in a 0.5/0.08/1 ratio that reflects their synthetic composition to modulate sperm motility in fat-2 mutants. PGF2α and the PG mixture at 25 µM concentration significantly stimulated sperm velocity by 28% and 38%, whereas 8-isoPGF2α and 5iPF2 VI reduced the velocity by 21% and 30%, respectively, compared to vehicle control. These results indicate that the sperm motility effects of PGs are structure- and composition-dependent in C. elegans.
Assuntos
Caenorhabditis elegans/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/metabolismo , Motilidade dos Espermatozoides , Animais , Caenorhabditis elegans/enzimologia , Cromatografia Líquida , Masculino , Espectrometria de Massas em TandemRESUMO
Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and ß-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with ßA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with ßA3-crystallin. The deamidated mutants of αA-crystallin (αA N101D and αA N123D) displayed lower adherence propensity for ßA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for ßA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with ßA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with ßA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with ßA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor ßA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.
Assuntos
Amidas/metabolismo , Cristalinas/metabolismo , Processamento de Proteína Pós-Traducional , Cadeia B de alfa-Cristalina/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalinas/química , Cristalinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Cristalino/química , Cristalino/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Cadeia B de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/genética , Cadeia A de beta-Cristalina/química , Cadeia A de beta-Cristalina/genéticaRESUMO
PURPOSE: The CRYAAN101D transgenic mouse model expressing deamidated αA-crystallin (deamidation at N101 position to D) develops cortical cataract at the age of 7 to 9 months. The present study was carried out to explore the molecular mechanism that leads to the development of cortical opacity in CRYAAN101D lenses. METHODS: RNA sequence analysis was carried out on 2- and 4-month-old αA-N101D and wild type (WT) lenses. To understand the biologic relevance and function of significantly altered genes, Ingenuity Pathway Analysis (IPA) was done. To elucidate terminal differentiation defects, immunohistochemical, and Western blot analyses were carried out. RESULTS: RNA sequence and IPA data suggested that the genes belonging to gene expression, cellular assembly and organization, and cell cycle and apoptosis networks were altered in N101D lenses. In addition, the tight junction signaling and Rho A signaling were among the top three canonical pathways that were affected in N101D mutant. Immunohistochemical analysis identified a series of terminal differentiation defects in N101D lenses, specifically, increased proliferation and decreased differentiation of lens epithelial cells (LEC) and decreased denucleation of lens fiber cells (LFC). The expression of Rho A was reduced in different-aged N101D lenses, and, conversely, Cdc42 and Rac1 expressions were increased in the N101D mutants. Moreover, earlier in development, the expression of major membrane-bound molecular transporter Na,K-ATPase was drastically reduced in N101D lenses. CONCLUSIONS: The results suggest that the terminal differentiation defects, specifically, increased proliferation and decreased denucleation are responsible for the development of lens opacity in N101D lenses.
Assuntos
Catarata/genética , Regulação da Expressão Gênica , Córtex do Cristalino/metabolismo , RNA Mensageiro/genética , Cadeia A de alfa-Cristalina/genética , Animais , Western Blotting , Catarata/metabolismo , Catarata/patologia , Proliferação de Células , Modelos Animais de Doenças , Imuno-Histoquímica , Córtex do Cristalino/patologia , Camundongos , Camundongos Transgênicos , Cadeia A de alfa-Cristalina/biossínteseRESUMO
Pro-sequences were swapped in cis between keratinases from Bacillus licheniformis (Ker BL) and Bacillus pumilus (Ker BP) to construct Ker ProBP-BL and Ker ProBL-BP, respectively. Expression of these keratinases was carried out constitutively by E. coli HB101-pEZZ18 system. They were characterized with respect to their parent enzymes, Ker BL and Ker BP, respectively. Ker ProBP-BL became more thermostable with a t(1/2) of 45 min at 80°C contrary to Ker BL which was not stable beyond 60°C. Similarly, the activity of Ker ProBP-BL on keratin and casein substrate, i.e. K:C ratio increased to 1.2 in comparison to 0.1 for Ker BL. Hydrolysis of insulin B-chain revealed that the cleavage sites increased to six from four in case of Ker ProBP-BL in comparison to Ker BL. However, cleavage sites decreased from seven to four in case of Ker ProBL-BP in comparison to the parent keratinase, Ker BP. Likewise, Ker ProBL-BP revealed altered pH and temperature kinetics with optima at pH 10 and 60°C in comparison to Ker BP which had optima at pH 9 and 70°C. It also cleaved soluble substrates with better efficiency in comparison to Ker BP with K:C ratio of 1.6. Pro-sequence mediated conformational changes were also observed in trans and were almost similar to the features acquired by the chimeras constructed in cis by swapping the pro-sequence region.
Assuntos
Sequência de Aminoácidos , Bacillus/enzimologia , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Estabilidade Enzimática , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Bacillus/classificação , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Precursores Enzimáticos/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética/métodos , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Peptídeo Hidrolases/genética , Dobramento de Proteína , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Gamma glutamyl transpeptidase from Bacillus pumilus KS12 (GGTBP) was cloned, expressed in pET-28-E. coli expression system as a heterodimeric enzyme with molecular weights of 45 and 20 kDa for large and small subunit, respectively. It was purified by nickel affinity chromatography with hydrolytic and transpeptidase activity of 1.82 U/mg and 4.35 U/mg, respectively. Sequence analysis revealed that GGTBP was most closely related to Bacillus licheniformis GGT and had all the catalytic residues and nucleophiles for autoprocessing recognized from E. coli. It was optimally active at pH 8 and 60°C. It exhibited pH stability from pH 6-9 and high thermostability with t(1/2) of 15 min at 70°C. It had K(m), V(max) of 0.045 mM, 4.35 µmol/mg/min, respectively. Decoupling of autoprocessing by co-expressing large and small subunit in pET-Duet1-E. coli expression system yielded active enzyme with transpeptidase activity of 5.31 U/mg. Though N-terminal truncations of rGGTBP upto 95 aa did not affect autoprocessing of GGT however activity was lost with truncation beyond 63 aa.
Assuntos
Bacillus/enzimologia , Biotecnologia/métodos , Catálise , Escherichia coli/metabolismo , gama-Glutamiltransferase/genética , gama-Glutamiltransferase/metabolismo , Sequência de Aminoácidos , Bacillus/classificação , Bacillus/genética , Bacillus/crescimento & desenvolvimento , Cromatografia de Afinidade , Clonagem Molecular , Dimerização , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , gama-Glutamiltransferase/química , gama-Glutamiltransferase/isolamento & purificaçãoRESUMO
Keratinase (ker BL) from Bacillus licheniformis ER-15 was cloned into vector pEZZ18 for extracellular expression in Escherichia coli HB101. Recombinant keratinase was secreted with high specific activity (75 units/mg) under non-inducible conditions after 36 h at 37 degrees C and 300 rpm in a shake flask. Protein was concentrated and, subsequently, purified by ion-exchange chromatography using Q-sepharose with 95.8% yield. The recombinant keratinase was a serine protease and most active in the pH range of 8-12 and at 60 degrees C. The enzyme was stable over a wide pH range of 4-12 for 3 h. ker BL degraded bovine serum albumin, casein, azocasein, gelatin, and feather. E. coli HB101 harboring pEZZ18 ker BL2 degraded chicken feather completely within 24 h at 37 degrees C.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/genética , Escherichia coli/genética , Espaço Extracelular/enzimologia , Expressão Gênica , Peptídeo Hidrolases/genética , Animais , Bacillus/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bovinos , Galinhas , Estabilidade Enzimática , Escherichia coli/metabolismo , Espaço Extracelular/genética , Espaço Extracelular/metabolismo , Plumas/química , Cinética , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Soroalbumina Bovina/química , Especificidade por SubstratoRESUMO
A feather degrading strain of Bacillus licheniformis ER-15 was isolated which also degraded α-keratin of hooves. A detailed analysis revealed that a novel monomeric γ-glutamyl transpeptidase (GGT(30)), a proteolytic product of heterodimeric 67 kDa γ-glutamyl transpeptidase (GGT(67)), assists subtilisin during its action on α keratin. An equimolar combination of subtilisin and GGT(30) was designated as KerN and was used as ungual enhancer for topical application. KerN was effective in releasing proteins from nail plate surface and 300 µg of enzyme could release 41 µg protein/mg of nail after 24 h treatment. Scanning electron micrograph (SEM) revealed loosening of nail matrix confirming the action of KerN on nail keratin. Drug permeation studies revealed permeation of clotrimazole through both enzymatically pretreated nail plates and also through nail plates in presence of KerN. Nearly 58% drug could be retained by nail plates after 24 h of 300 µg/mL KerN which further enhanced up to 97% by prolonging the enzyme application. The enzyme was found to be stable in presence of drug even after 72 h. Thus, KerN can be used as an additive in formulation of topical drug for onchomycosis.
Assuntos
Unhas/efeitos dos fármacos , Unhas/metabolismo , Subtilisina/farmacologia , gama-Glutamiltransferase/metabolismo , Absorção , Administração Tópica , Animais , Antifúngicos/administração & dosagem , Bacillus/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Química Farmacêutica , Clotrimazol/metabolismo , Clotrimazol/farmacologia , Estabilidade Enzimática , Casco e Garras/metabolismo , Humanos , Hidrólise , Queratinas/metabolismo , Queratinas/farmacologia , Doenças da Unha/tratamento farmacológico , Unhas/ultraestrutura , Estudos Prospectivos , Subtilisina/metabolismo , Fatores de Tempo , gama-Glutamiltransferase/farmacologiaRESUMO
A novel dimeric 58 kDa keratinase is reported from Bacillus licheniformis ER-15. The bacterium produced 244 U/ml keratinase in 48 h which was increased by eight fold (1962 U/ml) after medium optimization by one-variable-at-a-time and response surface methodology. Enzyme was concentrated by ultrafiltration followed by acetone precipitation and purified by gel filtration chromatography. It had subunit of 30 and 28 kDa and pI of 8.4. Enzyme was maximally active at pH 11 and 70 degrees C. It hydrolyzed various complex proteins viz. haemoglobin, feather, hooves, fibrin and meat protein. It was a thiol activated serine protease and 6.25-fold enhancement in activity was observed in presence of 5mM mercaptoethanol. Nearly 1200 U keratinase degraded 1.5 g feather in 12h at pH 8, 50 degrees C in redox free environment. This enzyme also dehaired buffalo hide within 16 h in presence of 3% Ca (OH)(2).