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1.
Chem Commun (Camb) ; 56(10): 1537-1540, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31922154

RESUMO

Although the underlying cause of Alzheimer's disease (AD) is not known, the extracellular deposition of ß-amyloid (Aß) is considered as a hallmark of AD brains. Evidence has shown the occurrence of d-Asp, isoAsp, and d-Ser residues in Aß, which may be indicative of and/or contribute to the neurodegeneration in AD patients. Herein, we have developed the first high-throughput profiling technique for all 20 isobaric Aß peptide epimers containing Asp, isoAsp, and Ser isomers using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This new analytical strategy allows the direct detection and identification of all possible Asp, isoAsp, and Ser stereoisomers in Aß, and may contribute to a better understanding of the pathogenesis of AD.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/análise , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Ácido Aspártico/química , Cromatografia Líquida de Alta Pressão , Humanos , Serina/química , Estereoisomerismo , Espectrometria de Massas em Tandem
2.
Phys Chem Chem Phys ; 22(5): 3008-3016, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31957772

RESUMO

Infrared (IR) spectroscopy is commonly utilized for the investigation of protein structures and protein-mediated processes. While the amide I band provides information on protein secondary structures, amino acid side chains are used as IR probes for the investigation of protein reactions, such as proton pumping in rhodopsins. In this work, we calculate the IR spectra of the solvated aspartic acid, with both zwitterionic and protonated backbones, and of a capped form, i.e. mimicking the aspartic acid residue in proteins, by means of molecular dynamics (MD) simulations and the perturbed matrix method (PMM). This methodology has already proved its good modeling capabilities for the amide I mode and is here extended to the treatment of protein side chains. The computed side chain vibrational signal is in very good agreement with the experimental one, well reproducing both the peak frequency position and the bandwidth. In addition, the MD-PMM approach proposed here is able to reproduce the small frequency shift (5-10 cm-1) experimentally observed between the protonated and zwitterionic forms, showing that such a shift depends on the excitonic coupling between the modes localized on the side chain and on the backbone in the protonated form. The spectrum of the capped form, in which the amide I band is also calculated, agrees well with the corresponding experimental spectrum. The reliable calculation of the vibrational bands of carboxyl-containing side chains provides a useful tool for the interpretation of experimental spectra.


Assuntos
Aminoácidos/química , Simulação de Dinâmica Molecular , Proteínas/química , Espectrofotometria Infravermelho , Ácido Aspártico/química , Ácido Glutâmico/química , Teoria Quântica
3.
Biosci Biotechnol Biochem ; 84(3): 500-506, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31694479

RESUMO

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the separation and quantification of the enantiomers of N-methylaspartate and N-methylglutamate, after derivatization with Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide was established. The time required for the LC-ESI-MS/MS analysis was within 20 min and the detection limit was approximately 10 fmol per injection, demonstrating that this method can be used for the rapid determination of D-aspartate N-methyltransferase activity in the ark shell clam Scapharca broughtonii.Abbreviations: NMDA: N-methyl-D-aspartate; NMLA: N-methyl-L-aspartate; NMDG: N-methyl-D-glutamate; NMLG: N-methyl-L-glutamate; NMA: N-methylaspartate; NMG: N-methylglutamate; HPLC: high-performance liquid chromatography; SAM: S-adenosyl-L-methionine; OPA: o-phthalaldehyde; LC-ESI-MS/MS: liquid chromatography-electrospray ionization-tandem mass spectrometry; FDLA: Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide; FDAA: Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide; ESI: electrospray ionization; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry; MS/MS: tandem mass spectrometry.


Assuntos
Ácido Aspártico/química , Bivalves/metabolismo , Cromatografia Líquida/métodos , Metiltransferases/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Metiltransferases/química
4.
Anal Bioanal Chem ; 411(29): 7783-7789, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31705222

RESUMO

Evaluation of post-translational modifications of protein molecules is important for both basic and applied biomedical research. Mass spectrometric quantitative studies of modifications, which do not change the mass of the protein, such as isomerization of aspartic acid, do not necessarily require the use of isotope-labelled standards. However, the accurate solution of this problem requires a deep understanding of the relationship between the mole fractions of the isomers and the peak intensities in the mass spectra. In previous studies on the isomerization of aspartic acid in short beta-amyloid fragments, it has been shown that calibration curves used for such quantitative studies often have a non-linear form. The reason for the deviation in the shape of the calibration curves from linearity has not yet been established. Here, we propose an explanation for this phenomenon based on a probabilistic model of the fragmentation process and present a general approach for the selection of fragments that can be used for quantitative studies of the degree of isomerization. Graphical Abstract.


Assuntos
Ácido Aspártico/análise , Modelos Teóricos , Peptídeos/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Ácido Aspártico/química , Isomerismo , Espectrometria de Massas/métodos , Probabilidade , Reprodutibilidade dos Testes
5.
Anal Bioanal Chem ; 411(27): 7105-7113, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31515585

RESUMO

A novel electrochemical approach for determination of arachidonic acid (ARA) was developed based on the linear arginine-glycine-aspartic-Au (RGD-Au) nanomaterial modified on glassy carbon electrode (GCE). The prepared material was characterized by transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), ultraviolet-visible spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The electrochemical signal was obtained from the reduction of 1,4-naphthoquinone and ARA served as a proton source. Under the optimum experimental conditions, the RGD-Au-based electrode was used to analyze ARA. Meanwhile, the electrochemical characteristics were also studied by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and linear sweep voltammetry (LSV). The sensor showed a wider linear range from 0.5 to 100 µM and the linear fitting equation was Ip (µA) = 0.0721 c + 2.4583 (R2 = 0.9987) with a detection limit of 80 nM. The application of the sensor in real samples was tested and compared with that of LC-MS/MS. This sensor would be a promising platform for detection of ARA in blood plasma. Graphical abstract.


Assuntos
Ácido Araquidônico/análise , Arginina/química , Ácido Aspártico/química , Glicina/química , Ouro/química , Nanopartículas/química , Ácido Araquidônico/sangue , Técnicas Eletroquímicas/métodos , Eletrólitos/química , Humanos , Limite de Detecção , Reprodutibilidade dos Testes , Análise Espectral/métodos
6.
Chemistry ; 25(63): 14370-14381, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31469197

RESUMO

Creating efficient and residue-directed artificial proteases is a challenging task due to the extreme inertness of the peptide bond, combined with the difficulty of achieving specific interactions between the catalysts and the protein side chains. Herein we report strictly site-selective hydrolysis of a multi-subunit globular protein, hemoglobin (Hb) from bovine blood, by a range of ZrIV -substituted polyoxometalates (Zr-POMs) in mildly acidic and physiological pH solutions. Among 570 peptide bonds in Hb, selective cleavage was observed at only eleven sites, each occurring at Asp-X peptide bonds located in the positive patches on the protein surface. The molecular origins of the observed Asp-X selectivity were rationalized by means of molecular docking, DFT-based binding, and mechanistic studies on model peptides. The proposed mechanism of hydrolysis involves coordination of the amide oxygen to ZrIV followed by a direct nucleophilic attack of the side chain carboxylate group on the C-terminal amide carbon atom with formation of a cyclic anhydride, which is further hydrolyzed to give the reaction products. The activation energy for the cleavage of the structurally related Glu-X sequence compared to Asp-X was calculated to be higher by 1.4 kcal mol-1 , which corresponds to a difference of about one order of magnitude in the rates of hydrolysis. The higher activation energy is attributed to the higher strain present in the six-membered ring of glutaric anhydride (Glu-X), as compared to the five-membered ring of the succinic anhydride (Asp-X) intermediate. Similarly, the cleavage at X-Asp and X-Glu bonds are predicted to be kinetically less likely as the corresponding activation energies were 6 kcal mol-1 higher, explaining the experimentally observed selectivity. The synergy between the negatively charged polyoxometalate cluster, which binds at positive patches on protein surfaces, and selective activation of Asp-X peptide bonds located in these regions by ZrIV ions, results in a novel class of artificial proteases with aspartate-directed reactivity, which is very rare among naturally occurring proteases.


Assuntos
Ácido Aspártico/química , Materiais Biomiméticos/química , Complexos de Coordenação/química , Compostos de Tungstênio/química , Zircônio/química , Sequência de Aminoácidos , Sítios de Ligação , Materiais Biomiméticos/metabolismo , Catálise , Complexos de Coordenação/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Hidrólise , Espectroscopia de Ressonância Magnética , Conformação Molecular , Simulação de Acoplamento Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Termodinâmica
7.
J Chem Theory Comput ; 15(8): 4344-4350, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31318548

RESUMO

Uracil DNA glycosylase catalyzes the N-glycosidic bond cleavage of uracil, thereby initiating the base excision repair mechanism for this DNA lesion. Here we employ hybrid quantum mechanics/molecular mechanics calculations to investigate the exact mechanism of the nucleophile attack and the role of the conserved His148 residue. Our calculations suggest that the C1'-N1 bond dissociation proceeds by a migration of the electrophilic sugar in the direction of the water nucleophile, resulting in a planar, oxocarbenium-like transition state. The subsequent nucleophile addition and proton transfer to a nearby base occur without a barrier. We assign the role of a proton acceptor to His148 and elucidate why mutations of this residue curtail the enzymatic activity but do not fully suppress it.


Assuntos
Histidina/química , Uracila-DNA Glicosidase/química , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Histidina/metabolismo , Humanos , Modelos Moleculares , Prótons , Teoria Quântica , Termodinâmica , Uracila-DNA Glicosidase/metabolismo , Água/química
8.
J Chem Theory Comput ; 15(8): 4535-4546, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31264415

RESUMO

Anabaena Sensory Rhodopsin (ASR), a microbial photoactive protein featuring the retinal chromophore in two different conformations, exhibits a pH-dependent electronic absorption spectrum. Using the recently developed CpHMD-then-QM/MM multiscale protocol applied to ASR embedded in a membrane model, the pH-induced changes in its maximum absorption wavelength have been reproduced and analyzed. While the acidic tiny red-shift is essentially correlated with the deprotonation of an aspartic acid located on the ASR extracellular side, the larger blue-shift experimentally reported at pH values larger than 5 involves a cluster of titrating residues sitting on the cytoplasmic side. The ASR pH-dependent spectrum is the consequence of the competitive stabilization of retinal ground and excited states by the protein electrostatic potential.


Assuntos
Aminoácidos/química , Anabaena/química , Proteínas de Bactérias/química , Nostoc/química , Rodopsinas Sensoriais/química , Aminoácidos/análise , Ácido Aspártico/análise , Ácido Aspártico/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Prótons , Espectrofotometria , Eletricidade Estática
9.
Chem Commun (Camb) ; 55(60): 8880-8883, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31321399

RESUMO

We developed an artificial hydrolase based on the symmetrical Pizza6 ß-propeller protein for the metal-free hydrolysis of 4-nitrophenyl acetate and butyrate. Through site-specific mutagenesis and crystallisation studies, the catalytic mechanism was investigated and found to be dependent on a threonine-histidine dyad. The mutant with additional histidine residues generated the highest kcat values, forming a His-His-Thr triad and matched previously reported metalloenzymes. The highly symmetrical ß-propeller artificial enzymes and their protein-metal complexes have potential to be utilised in bioinorganic and supramolecular chemistry, as well as being developed further into 2D/3D catalytic materials.


Assuntos
Hidrolases/química , Sequência de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Butiratos/química , Catálise , Cobre/química , Histidina/química , Histidina/genética , Hidrolases/genética , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Nitrofenóis/química , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Treonina/química , Zinco/química
10.
J Pept Sci ; 25(7): e3193, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31309675

RESUMO

Aspartimide (Asi) formation is a notorious side reaction in peptide synthesis that is well characterized and described in literature. In this context, we observed significant amounts of chain termination in Fmoc-SPPS while synthesizing the N-terminal Xaa-Asp-Yaa motif. This termination was caused by the formation of piperazine-2,5-diones. We investigated this side reaction using a linear model peptide and independently synthesizing its piperazine-2,5-dione derivative. Nuclear magnetic resonance (NMR) data of the side product present in the crude linear peptide proves that exclusively the six-membered ring is formed whereas the theoretically conceivable seven-membered 1,4-diazepine-2,5-dione is not found. We propose a mechanism where nucleophilic attack of the N-terminal amino function takes place at the α-carbon of the carbonyl group of the corresponding Asi intermediate. In addition, we systematically investigated the impact of (a) different adjacent amino acid residues, (b) backbone protection, and (c) side chain protection of flanking amino acids. The side reaction is directly related to the Asi intermediate. Hence, hindering or avoiding Asi formation reduces or completely suppresses this side reaction.


Assuntos
Aminoácidos/química , Ácido Aspártico/análogos & derivados , Fluorenos/química , Peptídeos/síntese química , Piperazinas/síntese química , Técnicas de Síntese em Fase Sólida , Sequência de Aminoácidos , Ácido Aspártico/química , Estrutura Molecular , Peptídeos/química , Piperazinas/química
11.
Biochim Biophys Acta Proteins Proteom ; 1867(9): 831-839, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31226490

RESUMO

With age, long-lived proteins in the human body deteriorate, which can have consequences both for aging and disease. The aging process is often associated with the formation of covalently crosslinked proteins. Currently our knowledge of the mechanism of formation of these crosslinks is limited. In this study, proteomics was used to characterize sites of covalent protein-protein crosslinking and identify a novel mechanism of protein-protein crosslinking in the adult human lens. In this mechanism, Lys residues are crosslinked to C-terminal Asp residues that are formed by non-enzymatic protein truncation. Ten different crosslinks were identified in major lens proteins such as αA-crystallin, αB-crystallin and AQP0. Crosslinking in AQP0 increased significantly with age and also increased significantly in cataract lenses compared with normal lenses. Using model peptides, a mechanism of formation of the Lys-Asp crosslink was elucidated. The mechanism involves spontaneous peptide cleavage on the C-terminal side of Asp residues which can take place in the pH range 5-7.4. Cleavage appears to involve attack by the side chain carboxyl group on the adjacent peptide bond, resulting in the formation of a C-terminal Asp anhydride. This anhydride intermediate can then either react with water to form Asp, or with a nucleophile, such as a free amine group to form a crosslink. If an ε-amino group of Lys or an N-terminal amine group attacks the anhydride, a covalent protein-protein crosslink will be formed. This bi-phasic mechanism represents the first report to link two spontaneous events: protein cleavage and crosslinking that are characteristic of long-lived proteins.


Assuntos
Aquaporinas/química , Ácido Aspártico/química , Proteínas do Olho/química , Modelos Moleculares , Peptídeos/química , Cadeia A de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/química , Aquaporinas/metabolismo , Ácido Aspártico/metabolismo , Proteínas do Olho/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cristalino/química , Cristalino/metabolismo , Peptídeos/metabolismo , Cadeia A de alfa-Cristalina/metabolismo , Cadeia B de alfa-Cristalina/metabolismo
12.
Biochim Biophys Acta Proteins Proteom ; 1867(7-8): 722-731, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31100408

RESUMO

Kynureninase from Pseudomonas fluorescens (Pfkynase) catalyzes the pyridoxal-5'-phosphate (PLP) dependent hydrolytic cleavage of L-kynurenine to give anthranilate and L-alanine. Asp-132 and Asp-201 are located in the structure near the pyridine NH of the PLP, with Asp-201 forming a hydrogen bond. Mutation of Asp-132 to alanine and glutamate and Asp-201 to glutamate results in reduced catalytic activity with L-kynurenine and ß-benzoyl-L-alanine, but not O-benzoyl-l-serine. D132A, D132E D201E and S36A mutant Pfkynases all can form quinonoid and vinylogous amide intermediates with ß-benzoyl-L-alanine, similar to wild-type enzyme. D132A, D132E, and D201E Pfkynase react more slowly with ß-benzoyl-L-alanine and benzaldehyde to form an aldol product absorbing at 490 nm than wild-type, with D132E reacting the slowest. The 1H NMR spectra of wild-type and D201E Pfkynase are very similar in the low field region from 10 to 18 ppm, but that of D132A Pfkynase is missing a resonance at 13.1 ppm. These results show that these residues modulate the reactivity of the PLP at different stages during the reaction cycle. Ser-36 is located near the expected location of the carbonyl oxygen of the substrate. Mutation of Ser-36 to alanine results in a 230-fold reduction of kcat and 30-fold reduction in kcat/Km with L-kynurenine, but very little effect on the reaction of O-benzoyl-l-serine. Thus, the rate-determining step in the reaction of S36A Pfkynase is the Cß-Cγ bond cleavage. These results support the hypothesis that Ser-36 together with Tyr-226 is part of an oxyanion hole that polarizes the carbonyl of the substrate in the catalytic mechanism of Pfkynase.


Assuntos
Proteínas de Bactérias/química , Hidrolases/química , Pseudomonas fluorescens/enzimologia , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Proteínas de Bactérias/genética , Hidrolases/genética , Mutação de Sentido Incorreto , Pseudomonas fluorescens/genética , Serina/química , Serina/genética
13.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096657

RESUMO

Aspartic acid (Asp) residues are prone to non-enzymatic stereoinversion, and Asp-residue stereoinversion is believed to be mediated via a succinimide (SI) intermediate. The stereoinverted Asp residues are believed to cause several age-related diseases. However, in peptides and proteins, few studies have reported the stereoinversion of glutamic acid (Glu) residues whose structures are similar to that of Asp. We previously presumed that Glu-residue stereoinversion proceeds via a glutarimide (GI) intermediate and showed that the calculated activation barriers of SI- and GI-intermediate stereoinversion are almost equivalent in the gas phase. In this study, we investigated the stereoinversion pathways of the l-GI intermediate in the aqueous phase using B3LYP density functional methods. The calculated activation barrier of l-GI-intermediate stereoinversion in the aqueous phase was approximately 36 kcal·mol-1, which was much higher than that in the gas phase. Additionally, as this activation barrier exceeded that of Asp-residue stereoinversion, it is presumed that Glu-residue stereoinversion has a lower probability of proceeding under physiological conditions than Asp-residue stereoinversion.


Assuntos
Ácido Aspártico/química , Resistência a Medicamentos , Ácido Glutâmico/química , Piperidonas/química , Estereoisomerismo , Água/química , Catálise , Estrutura Molecular , Peptídeos/química , Proteínas/química , Succinimidas/química
14.
Soft Matter ; 15(20): 4200-4207, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31070656

RESUMO

Herein, we have designed and synthesized a novel forky peptide D3F3 that transforms into a hydrogel through crosslinking induced by ZIs stimuli. We have employed D3F3 as a suitable drug carrier that is conjugated with DOX. Since the concentration of zinc ions necessary for triggering gelation falls into the physiological range present in prostate tissue, while other cationic ions fail to trigger at physiological concentrations, the peptide-based drug delivery system (DDS) is injectable and would achieve prostate tissue-specific self-assembly in situ. The D3F3 hydrogels exhibited an optimal gelation time, satisfactory mechanical strength (can be enhanced after incorporation of DOX) as well as excellent thixotropic properties. The DDS reserved some DOX in the prostate 24 h after the injection, making local sustained release possible. In addition, the peptide materials demonstrated no cytotoxicity against normal fibroblast cells and no damage was observed to the prostate tissue of rats. The drug release followed a non-Fickian diffusion model, with no burst release observed. Importantly, the DOX-hydrogel system exhibited good anti-cancer efficacy when incubated with prostate cancer cells DU-145. Therefore, this study lays the groundwork for the future design of tissue-specific DDSs that are triggered by cationic ions (e.g. zinc ions), and the platform could be further developed to incorporate other potent drugs utilized in the field of prostate cancer therapy, thereby increasing their potency and reducing their side effects.


Assuntos
Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Hidrogéis/química , Oligopeptídeos/síntese química , Neoplasias da Próstata/tratamento farmacológico , Animais , Ácido Aspártico/química , Cátions Bivalentes , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Reagentes para Ligações Cruzadas/química , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Células NIH 3T3 , Próstata , Zinco/química
15.
Bioengineered ; 10(1): 71-77, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30982422

RESUMO

ß-N-Acetylglucosaminidases (GlcNAcases) possess many important biological functions and are used for promising applications that are often hampered by low-activity enzymes. We previously demonstrated that most GlcNAcases of the glycoside hydrolase (GH) family 20 showed higher activities than those of other GH families, and we presented two novel GH 20 GlcNAcases that showed higher activities than most GlcNAcases. A highly flexible structure, which was attributed to the presence of to a high proportion of random coils and flexible amino acid residues, was presumed to be a factor in the high activity of GH 20 GlcNAcases. In this study, we further hypothesized that two special positions might play a key role in catalytic activity. The increase in GH 20 GlcNAcase activity might correspond to the increased structural flexibility and substrate affinity of the two positions due to an increase in random coils and amino acid residues, notably acidic Asp and Glu.


Assuntos
Acetilglucosaminidase/química , Ácido Aspártico/química , Proteínas de Bactérias/química , Ácido Glutâmico/química , Acetilglucosaminidase/classificação , Acetilglucosaminidase/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Biocatálise , Ácido Glutâmico/metabolismo , Hidrólise , Cinética , Micrococcaceae/química , Micrococcaceae/enzimologia , Paenibacillus/química , Paenibacillus/enzimologia , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Rhizobiaceae/química , Rhizobiaceae/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serratia marcescens/química , Serratia marcescens/enzimologia , Streptomyces/química , Streptomyces/enzimologia , Relação Estrutura-Atividade , Especificidade por Substrato
16.
mBio ; 10(2)2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992347

RESUMO

Biogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. In Enterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that in Pseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those from Escherichia coli The P. aeruginosa lipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either the E. coli or P. aeruginosa LolCDE. We further demonstrate that an inhibitor of E. coli LolCDE is active against P. aeruginosa only when expressing the E. coli orthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCE Gram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways of Escherichia coli and Pseudomonas aeruginosa are interchangeable, with the E. coli orthologues correctly sorting the P. aeruginosa lipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified in E. coli as aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Escherichia coli/genética , Lipoproteínas/metabolismo , Transporte Proteico , Pseudomonas aeruginosa/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Ácido Aspártico/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Lipoproteínas/genética , Pseudomonas aeruginosa/genética
17.
Toxicol In Vitro ; 59: 126-134, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30986424

RESUMO

The present study aimed to explore the potential of hydroxylated carbon nanotubes (CNTnols) conjugated with aspartic acid for the delivery of docetaxel (DTX) to breast cancer cells. The conjugate was well-characterized by FT-IR, NMR, XRD and FE-SEM. The nanoconjugate offered a hydrodynamic diameter of 86.31 ±â€¯1.02 nm, with a PDI of 0.113 and zeta potential of -41.6 ±â€¯0.17 mV. The designed nanosystem offered a controlled & pH dependent release vouching release of drug in the cancerous cytosol, not in blood, assuring delivery of the pay-load to the site of action. The carriers offered substantial hemocompatibility and lower plasma protein binding, ensuring more drug available at the site of action. The in-vitro cell viability studies in MDA MB-231 cells inferred approx. 2.8 times enhancement in the cytotoxicity potential of the conjugate vis-à-vis plain drug. Pharmacokinetic studies also corroborated the superiority of the designed nanoconjugate in terms of enhanced bioavailable fractions, reduced clearance and longer bioresidence to that of plain docetaxel. The present studies, successfully provide a workable nanomedicine, loaded with a BCS class-IV drug, for improved efficacy and safety in breast cancer.


Assuntos
Antineoplásicos/administração & dosagem , Ácido Aspártico/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Docetaxel/administração & dosagem , Portadores de Fármacos/administração & dosagem , Nanotubos de Carbono , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Ácido Aspártico/química , Ácido Aspártico/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Docetaxel/química , Docetaxel/farmacocinética , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Liberação Controlada de Fármacos , Sinergismo Farmacológico , Humanos , Nanotubos de Carbono/química , Ratos Wistar
18.
Leg Med (Tokyo) ; 38: 25-31, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30925381

RESUMO

Age estimation in adults based on aspartic acid racemization (AAR) provides fewer errors and higher precision than that based on bone morphology for the identification of cadavers. The technique has been established in some labs as a routine method. However, as the essential requisites for the technique, a wide age range of teeth of the same type as the target tooth must be collected for calibration for each examination. We investigated whether dentin standard samples could be prepared by increasing the AAR rate via heat. Powdered dentin was prepared from a maxillary first premolar (13 years) and heated for 0-72 h at 110 °C. The extent of AAR increased significantly with heating time and the correlation was strong (r = 0.913; p < 0.01). Similar results were found for a mandibular canine (24 years, r = 0.948; p < 0.01) and a maxillary third molar (20 years, r = 0.944; p < 0.01). We attempted to estimate the age of four maxillary first premolars of persons aged 25-58 years by using the heated samples (18 years, 12 h to 7 days). The differences between the actual and estimated ages were within ±5 years. The stability of the AAR rates in the powdered dentin during storage at 22-25 °C, 4 °C, and -30 °C was examined after 1 year and no significant changes had occurred. We were able to prepare dentin standard samples and created a calibration curve. This is a pilot study that needs to be validated before it can be used in forensic practice.


Assuntos
Determinação da Idade pelos Dentes/métodos , Ácido Aspártico/química , Dentina/química , Temperatura Alta , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Dente Pré-Molar , Feminino , Humanos , Masculino , Maxila , Pessoa de Meia-Idade , Dente Serotino , Projetos Piloto , Fatores de Tempo , Adulto Jovem
19.
Front Biosci (Landmark Ed) ; 24: 648-687, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844704

RESUMO

Glutamate carboxypeptidases II and III (GCPII and GCPIII) are highly homologous di-zinc metallopeptidases belonging to the M28 family. These enzymes are expressed in a variety of tissues, including the brain, prostate, kidney, testis and jejunum. GCPII has been recognized as a neuropeptidase in the central nervous system, as a folate hydrolase participating in absorption of folates in the jejunum and, most importantly, as a prostate-specific membrane antigen that is highly expressed in prostate adenocarcinoma. Furthermore, it has been identified in the neovasculature of most human solid tumors. In contrast, GCPIII has not been associated with any specific physiological function or pathology, and its expression, activity and inhibition have not been as well-studied. In this review, we provide an overview of the current understanding of the structure, enzymatic activity, substrate specificity, and tissue distribution of these two homologous enzymes. We discuss their potential physiological functions and describe the available animal models, including genetically modified mice. We also review the potential use of specific monoclonal antibodies and small-molecule inhibitors recognizing GCPII/III for diagnosis, imaging and experimental therapy of human cancers and other pathologies.


Assuntos
Antígenos de Superfície/metabolismo , Biomarcadores Tumorais/metabolismo , Carboxipeptidases/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Neuropeptídeos/química , Peptídeo Hidrolases/metabolismo , Adenocarcinoma/metabolismo , Animais , Anticorpos Monoclonais/química , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Encéfalo/metabolismo , Modelos Animais de Doenças , Glutamatos/química , Humanos , Hidrólise , Doenças Inflamatórias Intestinais/metabolismo , Intestino Delgado/metabolismo , Jejuno/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Fenótipo , Neoplasias da Próstata/metabolismo , Ratos
20.
Nano Lett ; 19(3): 1467-1478, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30730145

RESUMO

Sustaining blood retention for theranostic nanoparticles is a big challenge. Various approaches have been attempted and have demonstrated some success but limitations remain. We hypothesized that peptides capable of increasing blood residence time for M13 bacteriophage, a rod-shaped nanoparticle self-assembled from proteins and nucleic acids, should also prolong blood circulation for engineered nanoparticles. Here we demonstrate the feasibility of this approach by identifying a series of blood circulation-prolonging (BCP) peptides through in vivo screening of an M13 peptide phage display library. Intriguingly, the majority of the identified BCP peptides contained an arginine-glycine-aspartic acid (RGD) motif, which was necessary but insufficient for the circulation-prolonging activity. We further demonstrated that the RGD-mediated specific binding to platelets was primarily responsible for the enhanced blood retention of BCP1. The utility of the BCP1 peptide was demonstrated by fusion of the peptide to human heavy-chain ferritin (HFn), leading to significantly improved pharmacokinetic profile, enhanced tumor cell uptake and optimum anticancer efficacy for doxorubicin encapsulated in the HFn nanocage. Our results provided a proof-of-concept for an innovative yet simple strategy, which utilizes phage display to discover novel peptides with the capability of substantially prolonging blood circulation for engineered theranostic nanoparticles.


Assuntos
Doxorrubicina/farmacologia , Ferritinas/química , Nanopartículas/química , Peptídeos/química , Sequência de Aminoácidos/genética , Arginina/química , Ácido Aspártico/química , Bacteriófago M13/química , Transporte Biológico/genética , Técnicas de Visualização da Superfície Celular , Doxorrubicina/química , Glicina/química , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Biblioteca de Peptídeos , Peptídeos/sangue
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