RESUMO
Lipase from Thermomyces lanuginosus (TLL) has been covalently immobilized on heterofunctional octyl-vinyl agarose. That way, the covalently immobilized enzymes will have identical orientation. Then, it has blocked using hexyl amine (HEX), ethylenediamine (EDA), Gly and Asp. The initial activity/stability of the different biocatalysts was very different, being the most stable the biocatalyst blocked with Gly. These biocatalysts had been utilized to analyze if the enzyme activity could decrease differently along thermal inactivation courses depending on the utilized substrate (that is, if the enzyme specificity was altered during its inactivation using 4 different substrates to determine the activity), and if this can be altered by the nature of the blocking agent and the inactivation conditions (we use pH 5, 7 and 9). Results show great changes in the enzyme specificity during inactivation (e.g., activity versus triacetin was much more quickly lost than versus the other substrates), and how this was modulated by the immobilization protocol and inactivation conditions. The difference in the changes induced by immobilization and inactivation were confirmed by fluorescence studies. That is, the functional and structural analysis of partially inactivated immobilized enzyme showed that their inactivation pathway is strongly depended on the support features and inactivation conditions.
Assuntos
Enzimas Imobilizadas/química , Eurotiales/enzimologia , Proteínas Fúngicas/química , Lipase/química , Microesferas , Sefarose/análogos & derivados , Ácido Aspártico/química , Enzimas Imobilizadas/metabolismo , Etilenodiaminas/química , Proteínas Fúngicas/metabolismo , Glicina/química , Lipase/metabolismo , Especificidade por Substrato , Sulfonas/química , Triacetina/químicaRESUMO
A simplified procedure to synthesize zwitterionic cellulose by means of N-protected aspartic anhydride under mild conditions was developed. The preparation of modified cellulose samples was carried out under heterogeneous, aqueous conditions by reacting NH4OH-activated cellulose with aspartic anhydrides N-protected with trifluoroacetyl (TFAc) and carbobenzyloxy (Cbz). Modified cellulose samples Cel-Asp-N-TFAc and Cel-Asp-N-Cbz were characterized by Fourier Transform Infrared (FTIR) and 13C solid state Nuclear Magnetic Resonance (NMR) spectroscopy. The functionalization degree of each cellulose sample was determined by the 13C NMR signal integration values corresponding to the cellulose C1 vs. the Cα of the aspartate residue and corroborated by elemental analysis. In agreement, both analytical methods averaged a grafting degree of 20% for Cel-Asp-N-TFAc and 16% for Cel-Asp-N-Cbz. Conveniently, Cel-Asp-N-TFAc was concomitantly partially N-deprotected (65%) as determined by the ninhydrin method. The zwitterion character of this sample was confirmed by a potentiometric titration curve and the availability of these amino acid residues on the cellulose was inspected by adsorption kinetics method with a 100 mg L-1 cotton blue dye solution. In addition, the synthesis reported in the present work involves environmentally related advantages over previous methodologies developed in our group concerning to zwitterionic cellulose preparation.
Assuntos
Anidridos/química , Ácido Aspártico/química , Celulose/química , Corantes/metabolismo , Adsorção , Anidridos/metabolismo , Ácido Aspártico/metabolismo , Celulose/metabolismoRESUMO
Arginase (EC 3.5.3.1) catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and requires a bivalent cation, especially Mn2+ for its catalytic activity. It is a component of the urea cycle and regulates the intracellular levels of l-arginine, which makes the arginase a target for treatment of vascular diseases and asthma. Mammalian arginases contain an unusual S-shaped motif located at the intermonomeric interface. Until now, the studies were limited to structural role of the motif. Then, our interest was focused on functional aspects and our hypothesis has been that the motif is essential for maintain the oligomeric state, having Arg308 as a central axis. Previously, we have shown that the R308A mutant is monomeric and re-associates to the trimeric-cooperative state in the presence of low concentrations of guanidine chloride. We have now mutated Asp204 that interacts with Arg308 in the neighbor subunit, and also we mutated Glu256, proposed as important for oligomerization. Concretely, the human arginase I mutants D204A, D204E, E256A, E256Q and E256D were generated and examined. No differences were observed in the kinetic parameters at pH 9.5 or in tryptophan fluorescence. However, the D204A and E256Q variants were monomeric. On the other hand, D204E and E256D proved to be trimeric and kinetically cooperative at pH 7.5, whereas hyperbolic kinetics was exhibited by E256A, also trimeric. The results obtained strongly support the importance of the interaction between Arg255 and Glu256 in the cooperative properties of arginase, and Asp204 would be relevant to maintain the oligomeric state through salt bridges with Arg255 and Arg308.
Assuntos
Arginase/ultraestrutura , Arginina/genética , Ácido Aspártico/genética , Conformação Proteica , Arginase/química , Arginase/genética , Arginina/química , Ácido Aspártico/química , Ácido Glutâmico/química , Ácido Glutâmico/genética , Humanos , Cinética , Substâncias Macromoleculares , Modelos Moleculares , Mutação/genética , Multimerização Proteica/genéticaRESUMO
Stable dimers aspartate-aspartate have been studied in aqueous and gas phase through theoretical simulations. The polarizable continuum model (PCM) has been applied to simulate the effect of the hydration on monomers and complexes. The quantum theory of atoms in molecules (QTAIM) and the interacting quantum atoms (IQA) scheme has been used to inquire into if, in the aqueous phase, individual hydrogen bonds have attractive electrostatic components. In all cases a spontaneous formation of the complexes in the aqueous phase are observed, while in the gas phase a considerable energy barrier must be overcome (between 100.8 to 263.2â kJ mol-1 ). The intermolecular distance at which this barrier is indicates when the hydrogen-bond interactions begin to take importance between the dimers and the corresponding molecular recognition among them. The IQA analysis shows that in aqueous phase, the hydrogen bonds N-Hâ â â O are mainly electrostatic in nature with a certain covalent character which increases linearly with the decrease of internuclear distances Hâ â â O. The Hâ â â H interactions observed are stabilizing and they are mainly quantum in nature.
Assuntos
Ácido Aspártico/química , Teoria Quântica , Ânions/química , Dimerização , Ligação de HidrogênioRESUMO
The aggregation of proteins in the brain is one of the main features of neurodegenerative diseases. In Alzheimer's disease, the abnormal aggregation of Aß-42 is due to intrinsic and extrinsic factors. The latter is due to variations in the environment, such as temperature, salt concentration, and pH. We evaluated the effect of protonation/deprotonation of residues that are part of trimeric and pentameric oligomers at pH 5, pH 6, and pH 7. Molecular dynamics simulation at 200 ns in the canonical ensemble was implemented. The results have revealed that histidine, glutamic acid, and aspartic acid residues showed a protonation/deprotonation effect in oligomers. The root mean square deviation analysis was used to analyze the structural stability at different pHs. We found an increase in hydrophobicity in the side chains of the trimer, while in the pentamer, the structural instability of a compact structure at pH 5 caused the hydrophobic core to open, revealing the hydrophobic region to the environment. At this point, we believe that conformational changes mediated by pH are essential in the aggregation of Aß-42 oligomers.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Fragmentos de Peptídeos/genética , Agregação Patológica de Proteínas/genética , Doença de Alzheimer/patologia , Sequência de Aminoácidos/genética , Peptídeos beta-Amiloides/química , Ácido Aspártico/química , Ácido Aspártico/genética , Histidina/química , Histidina/genética , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Agregação Patológica de Proteínas/patologia , Conformação Proteica , Multimerização Proteica/genéticaRESUMO
The development of polymers with low toxicity and efficient gene delivery remains a significant barrier of nonviral gene therapy. Modification and tuning of chemical structures of carriers is an attractive strategy for efficient nucleic acid delivery. Here, polyplexes consisting of plasmid DNA (pDNA) and dodecylated or non-dodecylated polysuccinimide (PSI)-based polycations are designed, and their transfection ability into HeLa cells is investigated by green fluorescent protein (GFP) expressing cells quantification. All cationic polymers show lower cytotoxicity than those of branched polyethyleneimine (bPEI). PSI and bPEI-based polyplexes have comparable physicochemical properties such as size and charge. Interestingly, a strong interaction between dodecylated polycations and pDNA caused by the hydrophobic moiety is observed in dodecylated PSI derivatives. Moreover, the decrease of GFP expression is associated with lower dissociation of pDNA from polyplexes according to the heparin displacement assay. Besides, a hydrophobization of PSI cationic derivatives with dodecyl side chains can modulate the integrity of polyplexes by hydrophobic interactions, increasing the binding between the polymer and the DNA. These results provide useful information for designing polyplexes with lower toxicity and greater stability and transfection performance.
Assuntos
Ácido Aspártico/análogos & derivados , DNA , Vetores Genéticos , Plasmídeos , Transfecção , Ácido Aspártico/química , Ácido Aspártico/farmacologia , DNA/química , DNA/farmacologia , Vetores Genéticos/química , Vetores Genéticos/farmacologia , Células HeLa , Humanos , Plasmídeos/química , Plasmídeos/farmacologiaRESUMO
DAPT is a potent γ-secretase (GS) inhibitor that blocks the production of short amyloid-ß (Aß) peptides. Aggregation and oligomerization of Aß peptides have been associated with the development and progression of Alzheimer's disease. A recent cryo-electron microscopy density map disclosed DAPT binding at the GS active site. In this study, we employed the density map data to assign a possible binding pose of DAPT to characterize its dynamic behavior through different molecular dynamics simulation approaches. Our simulations showed a high preference of DAPT for the intramembrane region of the protein and that its entry site is located between TM2 and TM3 of PS1. DAPT interaction with the active site led to a decreased flexibility of key PS1 regions related to the recognition and internalization of GS substrates. Moreover, our study showed that the proximity of DAPT to the catalytic aspartic acids should be able to modify its protonation states, preventing the enzyme from reaching its active form. These results provide valuable information toward understanding the molecular mechanism of a GS inhibitor for the development of novel Alzheimer's disease treatments.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Diaminas/química , Inibidores Enzimáticos/química , Tiazóis/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Ácido Aspártico/química , Domínio Catalítico , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
BACKGROUND: Targeting specific tissues remains a major challenge to the promise of gene therapy. For example, several strategies have failed to target adeno-associated virus 2 (AAV2) vectors, to bone. We have evaluated in vitro and in vivo the affinity of an AAV2 vector to bone matrix, hydroxyapatite (HA) to treat Mucopolysacccharidosis IVA. METHODS: To increase vector affinity to HA, an aspartic acid octapeptide (D8) was inserted immediately after the N-terminal region of the VP2 capsid protein. The modified vector had physical titers and transduction efficiencies comparable to the unmodified vector. RESULTS: The bone-targeting vector had significantly higher HA affinity and vector genome copies in bone than the unmodified vector. The modified vector was also released from HA, and its enzyme activity in bone, 3 months post infusion, was 4.7-fold higher than the unmodified vector. CONCLUSION: Inserting a bone-targeting peptide into the vector capsid increases gene delivery and expression in the bone without decreasing enzyme expression. This approach could be a novel strategy to treat systemic bone diseases.
Assuntos
Osso e Ossos/metabolismo , Proteínas do Capsídeo/química , Durapatita/química , Vetores Genéticos , Mucopolissacaridose IV/terapia , Animais , Ácido Aspártico/química , Medula Óssea/metabolismo , Encéfalo/metabolismo , Capsídeo , Dependovirus , Perfilação da Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Células HEK293 , Humanos , Hidroxiapatitas/química , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Parvovirinae , Domínios Proteicos , TransgenesRESUMO
The Arc (anoxic redox control) two-component system of Escherichia coli, comprising ArcA as the response regulator and ArcB as the sensor histidine kinase, modulates the expression of numerous genes in response to respiratory growth conditions. Under reducing growth conditions, ArcB autophosphorylates at the expense of ATP, and transphosphorylates ArcA via a His292 â Asp576 â His717 â Asp54 phosphorelay, whereas under oxidizing growth conditions, ArcB catalyzes the dephosphorylation of ArcA-P by a reverse Asp54 â His717 â Asp576 â Pi phosphorelay. However, the exact phosphoryl group transfer routes and the molecular mechanisms determining their directions are unclear. Here, we show that, during signal propagation, the His292 â Asp576 and Asp576 â His717 phosphoryl group transfers within ArcB dimers occur intra- and intermolecularly, respectively. Moreover, we report that, during signal decay, the phosphoryl group transfer from His717 to Asp576 takes place intramolecularly. In conclusion, we present a mechanism that dictates the direction of the phosphoryl group transfer within ArcB dimers and that enables the discrimination of the kinase and phosphatase activities of ArcB.
Assuntos
Ácido Aspártico/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Histidina/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Proteínas Quinases/metabolismo , Ácido Aspártico/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Histidina/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/genética , Transdução de SinaisRESUMO
A new set of applications can be achieved when using high stability proteases. Industrially, high costs can be related to production medium and purification process. Magnetic nanoparticles have been successfully used for rapid and scalable purification. In this work, azocasein were immobilized on magnetite nanoparticles and applied in a single step purification of protease produced by Penicillium aurantiogriseum using soybean flour medium, and the new purified enzyme was characterized. Glutaraldehyde activated nanoparticles were used in azocasein immobilization and then incubated with dialyzed 60-80% saline precipitation fraction of crude extract for purification. Adsorbents were washed 7 times (0.1â¯M NaCl solution) and eluted 3 times (1â¯M NaCl solution), these final elutions contained the purified protease. This protease was purified 55.68-fold, retaining 46% of its original activity. Presented approximately 40â¯kDa on SDS-PAGE and optimum activity at 45⯰C and pH 9.0. Maintained over 60% of activity from pH 6.0 to 11.0. Kept more than 50% activity from 15 to 55⯰C, did not lose any activity over 48â¯hâ¯at 25⯰C. Inhibitors assay suggested a serine protease with aspartic residues on its active site. Results report a successful application of an alternative purification method and novel broad pH tolerant protease.