RESUMO
The human StAR-related lipid transfer domain protein 2 (STARD2), also known as phosphatidylcholine (PC) transfer protein, is a single-domain lipid transfer protein thought to transfer PC lipids between intracellular membranes. We performed extensive µs-long molecular dynamics simulations of STARD2 of its apo and holo forms in the presence or absence of complex lipid bilayers. The simulations in water reveal ligand-dependent conformational changes. In the 2 µs-long simulations of apo STARD2 in the presence of a lipid bilayer, we observed spontaneous reproducible PC lipid uptake into the protein hydrophobic cavity. We propose that the lipid extraction mechanism involves one to two metastable states stabilized by choline-tyrosine or choline-tryptophane cation-π interactions. Using free energy perturbation, we evaluate that PC-tyrosine cation-π interactions contribute 1.8 and 2.5 kcal/mol to the affinity of a PC-STARD2 metastable state, thus potentially providing a significant decrease of the energy barrier required for lipid desorption.
Assuntos
Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Fosfatidilcolinas , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fosfatidilcolinas/química , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Termodinâmica , Interações Hidrofóbicas e Hidrofílicas , Tirosina/químicaRESUMO
Several studies have highlighted the presence of nitration damage following neuroinflammation in Alzheimer's disease (AD). Accordingly, post-transcriptional modifications of ß-amyloid (Aß), including peptide nitration, have been explored as a marker of the disease. However, the implications of Aß nitration in terms of aggregation propensity and neurotoxicity are still debated. Here, we show new data obtained using a photoactivatable peroxynitrite generator (BPT-NO) to overcome the limitations associated with chemical nitration methods. We found that the photoactivation of BPT-NO with the highly biocompatible red light selectively induces the nitration of tyrosine 10 of freshly solubilized full-length Aß1-42. Photonitrated Aß1-42 was, therefore, investigated for aggregation states and functions. It resulted that photonitrated Aß1-42 did not aggregate into small oligomers but rather self-assembled into large amorphous aggregates. When tested on neuronal-like SH-SY5Y cells and microglial C57BL/6 BV2 cells, photonitrated Aß1-42 showed to be free of neurotoxicity and able to induce phagocytic microglia cells. We propose that light-controlled nitration of the multiple forms in which Aß occurs (i.e., monomers, oligomers, fibrils) could be a tool to assess in real-time the impact of tyrosine nitration on the amyloidogenic and toxic properties of Aß1-42.
Assuntos
Peptídeos beta-Amiloides , Luz , Fragmentos de Peptídeos , Tirosina , Peptídeos beta-Amiloides/metabolismo , Tirosina/metabolismo , Tirosina/análogos & derivados , Tirosina/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Humanos , Animais , Microglia/metabolismo , Microglia/efeitos dos fármacos , Ácido Peroxinitroso/metabolismo , Camundongos , Agregados Proteicos/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacosRESUMO
Redox-active residues, such as tyrosine and tryptophan, play important roles in a wide range of biological processes. The α3Y de novo protein, which is composed of three α helices and a tyrosine residue Y32, provides a platform for investigating the redox properties of tyrosine in a well-defined protein environment. Herein, the proton-coupled electron transfer (PCET) reaction that occurs upon oxidation of tyrosine in this model protein by a ruthenium photosensitizer is studied by using a vibronically nonadiabatic PCET theory that includes hydrogen tunneling and excited vibronic states. The input quantities to the analytical nonadiabatic rate constant expression, such as the diabatic proton potential energy curves and associated proton vibrational wave functions, reorganization energy, and proton donor-acceptor distribution functions, are obtained from density functional theory calculations on model systems and molecular dynamics simulations of the solvated α3Y protein. Two possible proton acceptors, namely, water or a glutamate residue in the protein scaffold, are explored. The PCET rate constant is greater when glutamate is the proton acceptor, mainly due to the more favorable driving force and shorter equilibrium proton donor-acceptor distance, although contributions from excited vibronic states mitigate these effects. Nevertheless, water could be the dominant proton acceptor if its equilibrium constant associated with hydrogen bond formation is significantly greater than that for glutamate. Although these calculations do not definitively identify the proton acceptor for this PCET reaction, they elucidate the conditions under which each proton acceptor can be favored. These insights have implications for tyrosine-based PCET in a wide variety of biochemical processes.
Assuntos
Simulação de Dinâmica Molecular , Oxirredução , Prótons , Tirosina , Tirosina/química , Tirosina/metabolismo , Transporte de Elétrons , Teoria da Densidade FuncionalRESUMO
Photothermal therapy (PTT) of tumor would ineluctably cause oxidative stress and related inflammation in adjacent normal tissues, leading to a discounted therapeutic outcome. To address this issue, herein an innovative therapeutic strategy that integrates photothermal anticancer and normal cell protection is developed. A new type of nitrogen-doped carbon dot (ET-CD) has been synthesized in one step by hydrothermal method using ellagic acid and L-tyrosine as reaction precursors. The as-prepared ET-CD exhibits high photothermal conversion efficiency and good photothermal stability. After intravenous injection, ET-CD can accumulate at the tumor site and the hyperthermia generated under near infrared laser irradiation effectively ablates tumor tissues, thereby significantly inhibiting tumor growth. Importantly, owing to the inherited antioxidant activity from ellagic acid, ET-CD can remove reactive oxygen and nitrogen species produced in the body and reduce the levels of inflammatory factors induced by oxidative stress, so as to alleviate the damage caused by heat-induced inflammation to normal cells and tissues while photothermal anticancer. These attractive features of ET-CD may open the exploration of innovative therapeutic strategies to promote the clinical application of PTT.
Assuntos
Carbono , Ácido Elágico , Nitrogênio , Terapia Fototérmica , Tirosina , Carbono/química , Carbono/farmacologia , Nitrogênio/química , Ácido Elágico/farmacologia , Ácido Elágico/química , Ácido Elágico/uso terapêutico , Animais , Tirosina/química , Humanos , Camundongos , Terapia Fototérmica/métodos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Pontos Quânticos/química , Linhagem Celular Tumoral , Inflamação/tratamento farmacológico , Antineoplásicos/farmacologia , Antineoplásicos/química , Estresse Oxidativo/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Neoplasias/patologiaRESUMO
We report two methods for the preparation of peptide thioesters containing Tyr(SO3H) residue(s), without use of a protecting group for the sulfate moiety. The first was based on direct thioesterification using carbodiimide on a fully protected peptide acid, prepared on a 2-chlorotrityl (Clt) resin with fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis (Fmoc-SPPS). Subsequent deprotection of the protecting groups with trifluoroacetic acid (TFA) (0 °C, 4 h) yielded peptide thioesters containing Tyr(SO3H) residue(s). Peptide thioesters containing one to three Tyr(SO3H) residue(s), prepared by this method, were used as building blocks for the synthesis of the Nα-Fmoc-protected N-terminal part of P-selectin glycoprotein ligand 1 (PSGL-1) (Fmoc-PSGL-1(43-74)) via silver-ion mediated thioester segment condensation. The other method was based on the thioesterification of peptide azide, derived from a peptide hydrazide prepared on a NH2NH-Clt-resin with Fmoc-SPPS. Peptide thioester containing two Tyr(SO3H) residues, prepared via this alternative method, was used as a building block for the one-pot synthesis of the N-terminal extracellular portion of CC-chemokine receptor 5 (CCR5(9-26)) by native chemical ligation (NCL). The two methods for the preparation of peptide thioesters containing Tyr(SO3H) residue(s) described herein are applicable to the synthesis of various types of sulfopeptides.
Assuntos
Ésteres , Peptídeos , Técnicas de Síntese em Fase Sólida , Peptídeos/química , Peptídeos/síntese química , Ésteres/química , Ésteres/síntese química , Sulfatos/química , Tirosina/química , Tirosina/síntese química , Compostos de Sulfidrila/química , Compostos de Sulfidrila/síntese química , Estrutura Molecular , Glicoproteínas de MembranaRESUMO
The plasma protein α1-acid glycoprotein (AGP) primarily affects the pharmacokinetics of basic drugs. There are two AGP variants in humans, A and F1*S, exhibiting distinct drug-binding selectivity. Elucidation of the drug-binding selectivity of human AGP variants is essential for drug development and personalized drug therapy. Herein, we aimed to establish the contribution of amino acids 112 and 114 of human AGP to drug-binding selectively. Both amino acids are located in the drug-binding region and differ between the variants. Phe112/Ser114 of the A variant and its equivalent residues in the F1*S variant (Leu112/Phe114) were swapped with each other. Binding experiments were then conducted using the antiarrhythmic drug disopyramide, which selectively binds to the A variant. A significant decrease in the bound fraction was observed in each singly mutated A protein (Phe112Leu or Ser114Phe). Moreover, the bound fraction of the double A mutant (Phe112Leu/Ser114Phe) was decreased to that of wild-type F1*S. Intriguingly, the double F1*S mutant (Leu112Phe/Phe114Ser), in which residues were swapped with those of the A variant, showed only partial restoration in binding. The triple F1*S mutant (Leu112Phe/Phe114Ser/Asp115Tyr), where position 115 is thought to contribute to the difference in pocket size between variants, showed a further recovery in binding to 70% of that of wild-type A. These results were supported by thermodynamic analysis and acridine orange binding, which selectively binds the A variant. Together, these data indicate that, in addition to direct interaction with Phe112 and Ser114, the binding pocket size contributed by Tyr115 is important for the drug-binding selectivity of the A variant.
Assuntos
Orosomucoide , Ligação Proteica , Orosomucoide/metabolismo , Orosomucoide/genética , Orosomucoide/química , Humanos , Sítios de Ligação , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Tirosina/química , Tirosina/metabolismo , Tirosina/genética , Mutação , Serina/metabolismo , Serina/genética , Serina/química , Antiarrítmicos/química , Antiarrítmicos/metabolismoRESUMO
Pyridyloxy-directed Ir(III)-catalyzed diacylmethylation of protected tyrosines was achieved with alkyl and (hetero)aryl sulfoxonium ylides, furnishing tyrosine-based unnatural amino acids in good yields. Furthermore, the late stage exemplification of the strategy was successfully accomplished in tyrosine-containing dipeptides, tripeptides and tetrapeptides in moderate yields. This methodology is distinguished by its site-selectivity, tolerance of sensitive functional groups, scalability, and retention of the chiral configuration for tyrosine motifs.
Assuntos
Irídio , Peptídeos , Tirosina , Irídio/química , Catálise , Tirosina/química , Metilação , Peptídeos/química , Compostos de Sulfônio/química , Estrutura MolecularRESUMO
Although the disease caused by chikungunya virus (CHIKV) is of great interest to public health organizations around the world, there are still no authorized antivirals for its treatment. Previously, dihalogenated anti-CHIKV compounds derived from L-tyrosine (dH-Y) were identified as being effective against in vitro infection by this virus, so the objective of this study was to determine the mechanisms of its antiviral action. Six dH-Y compounds (C1 to C6) dihalogenated with bromine or chlorine and modified in their amino groups were evaluated by different in vitro antiviral strategies and in silico tools. When the cells were exposed before infection, all compounds decreased the expression of viral proteins; only C4, C5 and C6 inhibited the genome; and C1, C2 and C3 inhibited infectious viral particles (IVPs). Furthermore, C1 and C3 reduce adhesion, while C2 and C3 reduce internalization, which could be related to the in silico interaction with the fusion peptide of the E1 viral protein. Only C3, C4, C5 and C6 inhibited IVPs when the cells were exposed after infection, and their effect occurred in late stages after viral translation and replication, such as assembly, and not during budding. In summary, the structural changes of these compounds determine their mechanism of action. Additionally, C3 was the only compound that inhibited CHIKV infection at different stages of the replicative cycle, making it a compound of interest for conversion as a potential drug.
Assuntos
Antivirais , Febre de Chikungunya , Vírus Chikungunya , Tirosina , Replicação Viral , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/fisiologia , Tirosina/farmacologia , Tirosina/análogos & derivados , Tirosina/metabolismo , Tirosina/química , Antivirais/farmacologia , Antivirais/química , Febre de Chikungunya/tratamento farmacológico , Febre de Chikungunya/virologia , Animais , Replicação Viral/efeitos dos fármacos , Chlorocebus aethiops , Células Vero , Humanos , Internalização do Vírus/efeitos dos fármacos , Proteínas Virais/metabolismoRESUMO
The utility of antibody therapeutics is hampered by potential cross-reactivity with healthy tissue. Over the past decade, significant advances have been made in the design of activatable antibodies, which increase, or create altogether, the therapeutic window of a parent antibody. Of these, antibody prodrugs (pro-antibodies) are masked antibodies that have advanced the most for therapeutic use. They are designed to reveal the active, parent antibody only when encountering proteases upregulated in the microenvironment of the targeted disease tissue, thereby minimizing off-target activity. However, current pro-antibody designs are relegated to fusion proteins that append masking groups restricted to the use of only canonical amino acids, offering excellent control of the site of introduction, but with no authority over where the masking group is installed other than the N-terminus of the antibody. Here, we present a palladium-based bioconjugation approach for the site-specific introduction of a masked tyrosine mimic in the complementary determining region of the FDA approved antibody therapeutic ipilimumab used as a model system. The approach enables the introduction of a protease cleavable group tethered to noncanonical polymers (polyethylene glycol (PEG)) resulting in 47-fold weaker binding to cells expressing CTLA-4, the target antigen of ipilimumab. Upon exposure to tumor-associated proteases, the masking group is cleaved, unveiling a tyrosine-mimic (dubbed hydroxyphenyl cysteine (HPC)) that restores (>90% restoration) binding affinity to its target antigen.
Assuntos
Pró-Fármacos , Tirosina , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Humanos , Tirosina/química , Paládio/química , Estrutura Molecular , Imunoconjugados/químicaRESUMO
Aromatic amino acid decarboxylases (AAADs) are pyridoxal-5'-phosphate (PLP)-dependent enzymes that catalyze the decarboxylation of aromatic amino acid l-amino acids. In plants, apart from canonical AAADs that catalyze the straightforward decarboxylation reaction, other members of the AAAD family function as aromatic acetaldehyde synthases (AASs) and catalyze more complex decarboxylation-dependent oxidative deamination. The interconversion between a canonical AAAD and an AAS can be achieved by a single tyrosine-phenylalanine mutation in the large catalytic loop of the enzymes. In this work, we report implicit ligand sampling (ILS) calculations of the canonical l-tyrosine decarboxylase from Papaver somniferum (PsTyDC) that catalyzes l-tyrosine decarboxylation and its Y350F mutant that instead catalyzes the decarboxylation-dependent oxidative deamination of the same substrate. Through comparative analysis of the resulting three-dimensional (3D) O2 free energy profiles, we evaluate the impact of the key tyrosine/phenylalanine mutation on oxygen accessibility to both the wild type and Y350F mutant of PsTyDC. Additionally, using molecular dynamics (MD) simulations of the l-tryptophan decarboxylase from Catharanthus roseus (CrTDC), we further investigate the dynamics of a large catalytic loop known to be indispensable to all AAADs. Results of our ILS and MD calculations shed new light on how key structural elements and loop conformational dynamics underlie the enzymatic functions of different members of the plant AAAD family.
Assuntos
Descarboxilases de Aminoácido-L-Aromático , Domínio Catalítico , Simulação de Dinâmica Molecular , Oxigênio , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Descarboxilases de Aminoácido-L-Aromático/genética , Descarboxilases de Aminoácido-L-Aromático/química , Oxigênio/metabolismo , Oxigênio/química , Papaver/enzimologia , Papaver/genética , Papaver/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Tirosina/metabolismo , Tirosina/química , Tirosina/genéticaRESUMO
Different from conventional synthetic polymers, polypeptides exhibit a distinguishing characteristic of adopting specific secondary structures, including random coils, α-helixes, and ß-sheets. The conformation determines the rigidity and solubility of polypeptide chains, which further direct the self-assembly and morphology of the nanostructures. We studied the effect of distinct secondary structures on the self-assembly behavior of polytyrosine (PTyr)-derived amphiphilic copolymers. Two block copolymers of enantiopure poly(ethylene glycol)-b-poly(l-tyrosine) (PEG-b-P(l-Tyr)) and racemic poly(ethylene glycol)-b-poly(dl-tyrosine) (PEG-b-P(dl-Tyr)) were synthesized through the ring-opening polymerization of l-tyrosine N-thiocarboxyanhydride (l-Tyr-NTA) and dl-tyrosine N-thiocarboxyanhydride (dl-Tyr-NTA), respectively, by using poly(ethylene glycol) amine as the initiator. PEG44-b-P(l-Tyr)10 adopts a ß-sheet conformation and self-assembles into rectangular nanosheets in aqueous solutions, while PEG44-b-P(dl-Tyr)9 is primarily in a random coil conformation with a tiny content of ß-sheet structures, which self-assembles into sheaf-like nanofibrils. A pH increase results in the ionization of phenolic hydroxyl groups, which decreases the ß-sheet content and increases the random coil content of the PTyr segments. Accordingly, PEG44-b-P(l-Tyr)10 and PEG44-b-P(dl-Tyr)9 self-assemble to form slender nanobelts and twisted nanoribbons, respectively, in alkaline aqueous solutions. The secondary structure-driven self-assembly of PTyr-derived copolymers is promising to construct filamentous nanostructures, which have potential for applications in controlled drug release.
Assuntos
Peptídeos , Polietilenoglicóis , Polietilenoglicóis/química , Concentração de Íons de Hidrogênio , Peptídeos/química , Estrutura Secundária de Proteína , Tirosina/química , Polimerização , Nanoestruturas/químicaRESUMO
The wildtype H-Ras protein functions as a molecular switch in a variety of cell signaling pathways, and mutations to key residues result in a constitutively active oncoprotein. However, there is some debate regarding the mechanism of the intrinsic GTPase activity of H-Ras. It has been hypothesized that ordered water molecules are coordinated at the active site by Q61, a highly transforming amino acid site, and Y32, a position that has not previously been investigated. Here, we examine the electrostatic contribution of the Y32 position to GTP hydrolysis by comparing the rate of GTP hydrolysis of Y32X mutants to the vibrational energy shift of each mutation measured by a nearby thiocyanate vibrational probe to estimate changes in the electrostatic environment caused by changes at the Y32 position. We further compared vibrational energy shifts for each mutation to the hydration potential of the respective side chain and demonstrated that Y32 is less critical for recruiting water molecules into the active site to promote hydrolysis than Q61. Our results show a clear interplay between a steric contribution from Y32 and an electrostatic contribution from Q61 that are both critical for intrinsic GTP hydrolysis.
Assuntos
Guanosina Trifosfato , Eletricidade Estática , Tiocianatos , Hidrólise , Tiocianatos/química , Tiocianatos/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/química , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Tirosina/química , Tirosina/metabolismo , Tirosina/genética , Mutação , Domínio Catalítico , Água/química , Água/metabolismo , Modelos MolecularesRESUMO
Surface-enhanced Raman scattering (SERS) is a powerful technique primarily due to its high sensitivity and signal-enhancing properties, which enable the identification of unique vibrational fingerprints. These fingerprints can be used for the diagnosis and monitoring of diseases such as cancer. It is crucial to selectively identify cancer biomarkers for early diagnosis. A correlation has been established between the reduction in the concentration of specific amino acids and the stage of the disease, particularly tryptophan (TPP) and tyrosine (TRS) in individuals diagnosed with prostate cancer. In this work, we present a strategy to analyze TPP and TRS amino acids using molecularly imprinted polymer nanoparticles (nanoMIPs), which selectively detect target molecules in a SERS sensor. NanoMIPs are synthesized using the solid-phase molecular imprinting method with TPP and TRS as templates. These are then immobilized on a SERS substrate with gold nanoparticles to measure samples prepared from tryptophan and tyrosine in phosphate-buffered saline. The detection and quantification limits of the designed sensor are 7.13 µM and 23.75 µM for TPP, and 22.11 µM and 73.72 µM for TRS, respectively. Our study lays the groundwork for future investigations utilizing nanoMIPs in SERS assessments of TPP and TRS as potential biomarkers for prostate cancer detection.
Assuntos
Nanopartículas Metálicas , Impressão Molecular , Análise Espectral Raman , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/química , Impressão Molecular/métodos , Nanopartículas Metálicas/química , Ouro/química , Propriedades de Superfície , Humanos , Triptofano/análise , Triptofano/química , Tirosina/análise , Tirosina/química , Neoplasias da Próstata/diagnóstico , MasculinoRESUMO
Chemokines are an important family of small proteins integral to leukocyte recruitment during inflammation. Dysregulation of the chemokine-chemokine receptor axis is implicated in many diseases, and both chemokines and their cognate receptors have been the targets of therapeutic development. Analysis of the antigen-binding regions of chemokine-binding nanobodies revealed a sequence motif suggestive of tyrosine sulfation. Given the well-established importance of post-translational tyrosine sulfation of receptors for chemokine affinity, it was hypothesized that the sulfation of these nanobodies may contribute to chemokine binding and selectivity. Four nanobodies (16C1, 9F1, 11B1, and 11F2) were expressed using amber codon suppression to incorporate tyrosine sulfation. The sulfated variant of 16C1 demonstrated significantly improved chemokine binding compared to the non-sulfated counterpart, while the other nanobodies displayed equipotent or reduced affinity upon sulfation. The ability of tyrosine sulfation to modulate chemokine binding, both positively and negatively, could be leveraged for chemokine-targeted sulfo-nanobody therapeutics in the future.
Assuntos
Quimiocinas , Anticorpos de Domínio Único , Tirosina , Tirosina/metabolismo , Tirosina/química , Tirosina/análogos & derivados , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Quimiocinas/metabolismo , Quimiocinas/química , Humanos , Ligação Proteica , Sulfatos/metabolismo , Sulfatos/químicaRESUMO
Copper amine oxidases (CAOs) catalyze the oxidative deamination of primary amines to aldehyde, ammonia, and hydrogen peroxide as products and are widely distributed in bacteria, plants, and eukaryotes. These enzymes initiate the single turnover, post-translational conversion of an active site tyrosine to the redox cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ), subsequently employing TPQ to catalyze steady-state amine oxidation. The mechanisms of TPQ biogenesis and steady-state amine oxidation have been studied extensively, with consensus mechanisms proposed for both reactions. One unresolved issue has been whether the Cu2+ center must undergo formal reduction to Cu1+ in the course of the reaction. Herein, we investigate the properties of the active site of a yeast (Hansenula polymorpha) amine oxidase (HPAO) that has undergone site-specific insertion of a para-aminophenylalanine (pAF) into the position of either the precursor tyrosine to TPQ (Y405) or the two strictly conserved neighboring tyrosines (Y305 and Y407). While our original intention was to interrogate cofactor biogenesis using a precursor unnatural amino acid (UAA) of altered redox potential and pKa, we instead observe an unanticipated reaction assigned to an intramolecular electron transfer from pAF to the active site copper ion. We establish the generality of the observed active site chemistry using exogenously added, aniline-containing substrates under conditions that prevent side chain amine oxidation. The results support previous proposals that the activation of the TPQ precursor occurs in the absence of a formal valence change at the active site copper site. The described reaction of pAFs with the active site redox Cu2+ center of HPAO provides a prototype for either the engineering of the enzymatic oxidation of exogenous anilines or the insertion of site-specific free radical probes within proteins.
Assuntos
Amina Oxidase (contendo Cobre) , Compostos de Anilina , Cobre , Tirosina , Amina Oxidase (contendo Cobre)/metabolismo , Amina Oxidase (contendo Cobre)/química , Tirosina/metabolismo , Tirosina/química , Tirosina/análogos & derivados , Cobre/química , Cobre/metabolismo , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Radicais Livres/metabolismo , Radicais Livres/química , Oxirredução , Domínio Catalítico , Fenilalanina/metabolismo , Fenilalanina/química , Fenilalanina/análogos & derivadosRESUMO
PURPOSE: There is currently no ideal radiotracer for imaging bacterial infections. Radiolabelled D-amino acids are promising candidates because they are actively incorporated into the peptidoglycan of the bacterial cell wall, a structural feature which is absent in human cells. This work describes fluorine-18 labelled analogues of D-tyrosine and D-methionine, O-(2-[18F]fluoroethyl)-D-tyrosine (D-[18F]FET) and S-(3-[18F]fluoropropyl)-D-homocysteine (D-[18F]FPHCys), and their pilot evaluation studies as potential radiotracers for imaging bacterial infection. PROCEDURES: D-[18F]FET and D-[18F]FPHCys were prepared in classical fluorination-deprotection reactions, and their uptake in Staphylococcus aureus and Pseudomonas aeruginosa was evaluated over 2 h. Heat killed bacteria were used as controls. A clinically-relevant foreign body model of S. aureus infection was established in Balb/c mice, as well as a sterile foreign body to mimic inflammation. The ex vivo biodistribution of D-[18F]FPHCys in the infected and inflamed mice was evaluated after 1 h, by dissection and gamma counting. The uptake was compared to that of [18F]FDG. RESULTS: In vitro uptake of both D-[18F]FET and D-[18F]FPHCys was specific to live bacteria. Uptake was higher in S. aureus than in P. aeruginosa for both radiotracers, and of the two, higher for D-[18F]FPHCys than D-[18F]FET. Blocking experiments with non-radioactive D-[19F]FPHCys confirmed specificity of uptake. In vivo, D-[18F]FPHCys had greater accumulation in S. aureus infection compared with sterile inflammation, which was statistically significant. As anticipated, [18F]FDG showed no significant difference in uptake between infection and inflammation. CONCLUSIONS: D-[18F]FPHCys uptake was higher in infected tissues than inflammation, and represents a fluorine-18 labelled D-AA with potential to detect a S. aureus reference strain (Xen29) in vivo. Additional studies are needed to evaluate uptake of this radiotracer in clinical isolates.
Assuntos
Camundongos Endogâmicos BALB C , Tomografia por Emissão de Pósitrons , Staphylococcus aureus , Tirosina , Animais , Tomografia por Emissão de Pósitrons/métodos , Projetos Piloto , Tirosina/análogos & derivados , Tirosina/química , Distribuição Tecidual , Homocisteína/metabolismo , Homocisteína/análogos & derivados , Homocisteína/química , Pseudomonas aeruginosa , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Radioisótopos de Flúor/química , Feminino , Camundongos , Infecções Bacterianas/diagnóstico por imagem , Infecções Bacterianas/microbiologiaRESUMO
Fluorescent gold nanoclusters (AuNCs) have shown promise as metal ion sensors. Further research into surface ligands is crucial for developing sensors that are both selective and sensitive. Here, we designed simple tripeptides to form fluorescent AuNCs, capitalizing on tyrosine's reduction capability under alkaline conditions. We investigated tyrosine's role in both forming AuNCs and sensing metal ions. Two tripeptides, tyrosine-cysteine-tyrosine (YCY) and serine-cysteine-tyrosine (SCY), were used to form AuNCs. YCY peptides produced AuNCs with blue and red fluorescence, while SCY peptides produced blue-emitting AuNCs. The blue fluorescence of YCY- and SCY-AuNCs was selectively quenched by Fe3+ and Cu2+, whereas red-emitting YCY-AuNC fluorescence remained stable with 13 different metal ions. The number of tyrosine residues influenced the sensor response. DLS measurements revealed different aggregation propensities in the presence of various metal ions, indicating that chelation between the peptide and target ions led to aggregation and fluorescence quenching. Highlighting the innovation of our approach, our study demonstrates the feasibility of the rational design of peptides for the formation of fluorescent AuNCs that serve as highly selective and sensitive surface ligands for metal ion sensing. This method marks an advancement over existing methods due to its dual capability in both synthesizing gold nanoclusters and detecting analytes, specifically Fe3+ and Cu2+.
Assuntos
Cobre , Ouro , Ferro , Nanopartículas Metálicas , Ouro/química , Cobre/química , Cobre/análise , Nanopartículas Metálicas/química , Ferro/química , Ferro/análise , Oligopeptídeos/química , Oligopeptídeos/análise , Tirosina/química , Tirosina/análise , Espectrometria de Fluorescência/métodosRESUMO
Tyr-derived cyclic peptide natural products are formed by enzymatic manifolds that oxidatively cross-link embedded phenolic side chains of tyrosine (Tyr) and 4-hydroxyphenylglycine residues during their controlled production. Bioactive Tyr-derived cyclic peptides, such as the arylomycins and vancomycins, continue to motivate the development of enzymatic and chemical strategies for their de novo assembly and modification. However, chemical access to these structurally diverse natural cycles can be challenging and step intensive. Therefore, we developed an oxidative procedure to selectively convert Tyr-containing N4-substituted 1,2,4-triazolidine-3,5-dione peptides (urazole peptides) into stable Tyr-linked cyclic peptides. We show that Tyr-containing urazole peptides are simple to prepare and convert into reactive N4-substituted 1,2,4-triazoline-3,5-dione peptides by oxidation, which then undergo spontaneous cyclization under mildly basic aqueous conditions to form a cross-linkage with the phenol side chain of embedded Tyr residues. Using this approach, we have demonstrated access to over 25 Tyr-linked cyclic peptides (3- to 11-residue cycles) with good tolerance of native residue side chain functionalities. Importantly, this method is simple to perform, and product formation can be quickly confirmed by mass spectrometric and 1H NMR spectroscopic analyses.
Assuntos
Peptídeos Cíclicos , Tirosina , Tirosina/química , Ciclização , Peptídeos Cíclicos/química , Peptídeos Cíclicos/síntese química , Triazóis/química , OxirreduçãoRESUMO
Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.
Assuntos
Fibrinogênio , Espectrometria de Massas em Tandem , Tirosina , Tirosina/química , Tirosina/análogos & derivados , Espectrometria de Massas em Tandem/métodos , Fibrinogênio/química , Fibrinogênio/metabolismo , Cromatografia Líquida/métodos , Humanos , Processamento de Proteína Pós-Traducional , Tripsina/química , Tripsina/metabolismo , Sulfatos/química , Sequência de Aminoácidos , Peptídeos/química , Peptídeos/análise , ElétronsRESUMO
The coordination of enzymes and regulatory proteins for eukaryotic DNA replication and repair is largely achieved by Proliferating Cell Nuclear Antigen (PCNA), a toroidal homotrimeric protein that embraces the DNA duplex. Many proteins bind PCNA through a conserved sequence known as the PCNA interacting protein motif (PIP). PCNA is further regulated by different post-translational modifications. Phosphorylation at residue Y211 facilitates unlocking stalled replication forks to bypass DNA damage repair processes but increasing nucleotide misincorporation. We explore here how phosphorylation at Y211 affects PCNA recognition of the canonical PIP sequences of the regulatory proteins p21 and p15, which bind with nM and µM affinity, respectively. For that purpose, we have prepared PCNA with p-carboxymethyl-L-phenylalanine (pCMF, a mimetic of phosphorylated tyrosine) at position 211. We have also characterized PCNA binding to the non-canonical PIP sequence of the catalytic subunit of DNA polymerase δ (p125), and to the canonical PIP sequence of the enzyme ubiquitin specific peptidase 29 (USP29) which deubiquitinates PCNA. Our results show that Tyr211 phosphorylation has little effect on the molecular recognition of p21 and p15, and that the PIP sequences of p125 and USP29 bind to the same site on PCNA as other PIP sequences, but with very low affinity.