RESUMO
Peptides are valuable for therapeutic development, with multicyclic peptides showing promise in mimicking antigen-binding potency of antibodies. However, our capability to engineer multicyclic peptide scaffolds, particularly for the construction of large combinatorial libraries, is still limited. Here, we study the interplay of disulfide pairing between three biscysteine motifs, and designed a range of triscysteine motifs with unique disulfide-directing capability for regulating the oxidative folding of multicyclic peptides. We demonstrate that incorporating these motifs into random sequences allows the design of disulfide-directed multicyclic peptide (DDMP) libraries with up to four disulfide bonds, which have been applied for the successful discovery of peptide binders with nanomolar affinity to several challenging targets. This study encourages the use of more diverse disulfide-directing motifs for creating multicyclic peptide libraries and opens an avenue for discovering functional peptides in sequence and structural space beyond existing peptide scaffolds, potentially advancing the field of peptide drug discovery.
Assuntos
Cisteína , Dissulfetos , Biblioteca de Peptídeos , Dissulfetos/química , Cisteína/química , Motivos de Aminoácidos , Descoberta de Drogas/métodos , Sequência de Aminoácidos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Humanos , Oxirredução , Dobramento de ProteínaRESUMO
Fibroblast growth factor 2 (FGF2) is an attractive biomaterial for pharmaceuticals and functional cosmetics. To improve the thermo-stability of FGF2, we designed two mutants harboring four-point mutations: FGF2-M1 (D28E/C78L/C96I/S137P) and FGF2-M2 (D28E/C78I/C96I/S137P) through bioinformatics, molecular thermodynamics, and molecular modeling. The D28E mutation reduced fragmentation of the FGF2 wild type during preparation, and the substitution of a whale-specific amino acid, S137P, enhanced the thermal stability of FGF2. Surface-exposed cysteines that participate in oligomerization through intermolecular disulfide bond formation were substituted with hydrophobic residues (C78L/C78I and C96I) using the in silico method. High-resolution crystal structures revealed at the atomic level that the introduction of mutations stabilizes each local region by forming more favorable interactions with neighboring residues. In particular, P137 forms CH-π interactions with the side chain indole ring of W123, which seems to stabilize a ß-hairpin structure, containing a heparin-binding site of FGF2. Compared to the wild type, both FGF2-M1 and FGF2-M2 maintained greater solubility after a week at 45 °C, with their Tm values rising by ~ 5 °C. Furthermore, the duration for FGF2-M1 and FGF2-M2 to reach 50% residual activity at 45 °C extended to 8.8- and 8.2-fold longer, respectively, than that of the wild type. Interestingly, the hydrophobic substitution of surface-exposed cysteine in both FGF2 mutants makes them more resistant to proteolytic cleavage by trypsin, subtilisin, proteinase K, and actinase than the wild type and the Cys â Ser substitution. The hydrophobic replacements can influence protease resistance as well as oligomerization and thermal stability. It is notable that hydrophobic substitutions of surface-exposed cysteines, as well as D28E and S137P of the FGF2 mutants, were designed through various approaches with structural implications. Therefore, the engineering strategies and structural insights adopted in this study could be applied to improve the stability of other proteins.
Assuntos
Cisteína , Fator 2 de Crescimento de Fibroblastos , Interações Hidrofóbicas e Hidrofílicas , Estabilidade Proteica , Cisteína/química , Cisteína/genética , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Mutação , Modelos Moleculares , Cristalografia por Raios X , Substituição de Aminoácidos , Humanos , TermodinâmicaRESUMO
L-cysteine is an essential component in pharmaceutical and agricultural industries, and synthetic biology has made strides in developing new metabolic pathways for its production, particularly in archaea with unique O-phosphoserine sulfhydrylases (OPSS) as key enzymes. In this study, we employed database mining to identify a highly catalytic activity OPSS from Acetobacterium sp. (AsOPSS). However, it was observed that the enzymatic activity of AsOPSS suffered significant feedback inhibition from the product L-cysteine, exhibiting an IC50 value of merely 1.2 mM. A semi-rational design combined with tunnel analysis strategy was conducted to engineer AsOPSS. The best variant, AsOPSSA218R was achieved, totally eliminating product inhibition without sacrificing catalytic efficiency. Molecular docking and molecular dynamic simulations indicated that the binding conformation of AsOPSSA218R with L-cys was altered, leading to a reduced affinity between L-cysteine and the active pocket. Tunnel analysis revealed that the AsOPSSA218R variant reshaped the landscape of the tunnel, resulting in the construction of a new tunnel. Furthermore, random acceleration molecular dynamics simulation and umbrella sampling simulation demonstrated that the novel tunnel improved the suitability for product release and effectively separated the interference between the product release and substrate binding processes. Finally, more than 45 mM of L-cysteine was produced in vitro within 2 h using the AsOPSSA218R variant. Our findings emphasize the potential for relieving feedback inhibition by artificially generating new product release channels, while also laying an enzymatic foundation for efficient L-cysteine production.
Assuntos
Cisteína Sintase , Cisteína , Simulação de Dinâmica Molecular , Cisteína/química , Cisteína/metabolismo , Cisteína Sintase/química , Cisteína Sintase/metabolismo , Cisteína Sintase/genética , Simulação de Acoplamento Molecular , Engenharia de Proteínas/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genéticaRESUMO
Owing to the on-going emission of Hg into the global environment, new insight into their bioinorganic chemistry in mammals is urgently required to better understand their adverse health effects and analytical methods to quantify Hg2+ and MeHg+ in environmental samples are needed. Analytical separations can help to address both of these needs. While Hg2+ and MeHg+ have been most frequently separated by cation and reversed-phase (RP) HPLC, we here report on using anion-exchange (AEX) HPLC in conjunction with a flame atomic absorption spectrometer (FAAS) to observe the retention behavior of these mercury species in the pH range 5.0-8.0 using mobile phases comprised of 10 mM l-cysteine (Cys) in 100 mM phosphate buffer. The results obtained for pH 5.0 served as a starting point to develop a rapid HPLC separation for these mercurials. The addition of 5-20 % methanol (MeOH) to this mobile phase revealed that MeOH did not appreciably change the retention of Hg2+, but significantly reduced the retention of MeHg+. A 15 % MeOH-containing mobile phase offered the best compromise between achieving a rapid baseline separation in <400 s at affordable costs. To assess the suitability and robustness of the developed AEX-HPLC separation method for the analysis of environmental samples an inductively coupled plasma atomic emission spectrometer (ICP-AES) was employed as the mercury-specific detector. The developed AEX-HPLC-ICP-AES method allowed to achieve detection limits of 1.5 ppm for Hg2+ and 2.9 ppm for MeHg+ and was successfully applied to analyze wastewater that had been spiked with Hg2+ and MeHg+.
Assuntos
Cisteína , Mercúrio , Compostos de Metilmercúrio , Cromatografia Líquida de Alta Pressão/métodos , Cisteína/química , Mercúrio/análise , Mercúrio/química , Mercúrio/isolamento & purificação , Compostos de Metilmercúrio/análise , Compostos de Metilmercúrio/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Limite de Detecção , Espectrofotometria Atômica/métodos , Concentração de Íons de Hidrogênio , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/química , Poluentes Químicos da Água/isolamento & purificaçãoRESUMO
1,3-Butadiene (BD) is a carcinogenic air pollutant. N-acetyl-S-(4-hydroxy-2-buten-1-yl)-L-cysteine (MHBMA3 or 4HBeMA), an urinary BD metabolite with unspecified configuration, is considered the most sensitive BD biomarker and has been used in routine biomonitoring since 2012. However, two issues remain unaddressed: why its concentrations are unusually high relative to other urinary BD biomarkers and why some authors reported no detection of the biomarker whereas other authors readily quantitated it. To address the issues, we synthesized and structurally characterized the authentic trans- and cis-isomers of MHBMA3 (designated NE and NZ, respectively), developed an isotope-dilution LC-MS/MS method for their quantification, and examined 67 urine samples from barbecue restaurant personnel (n = 47) and hotel administrative staff (n = 20). The restaurant personnel were exposed to barbecue fumes, which contain relatively high concentrations of BD. The results showed that NE and NZ had highly similar NMR spectra, and were difficult to be well separated chromatographically. The NMR data showed that the MHBMA3 isomer investigated in most previous studies was NE. We did not detect NE and NZ in any samples; however, an interfering peak with varying heights was observed in most samples. Notably, under the chromatographic conditions used in the literature, the peak exhibited indistinguishable retention time from that of NE. Thus, it is highly likely that the interfering peak has been mis-identified as NE in previous studies, providing a reasonable explanation for the high MHBMA3 concentration in urine. The contradiction in the presence of MHBMA3 in urine was also caused by the mis-identification, because the researchers who reported the absence of MHBMA3 were actually detecting NZ. Thus, we clarified the confusion on MHBMA3 in previous studies through correctly identifying the two MHBMA3 isomers. The presence of NE and NZ in human urine warrants further investigations.
Assuntos
Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Acetilcisteína/urina , Acetilcisteína/análogos & derivados , Acetilcisteína/química , Isomerismo , Limite de Detecção , Butadienos/química , Butadienos/urina , Reprodutibilidade dos Testes , Cisteína/urina , Cisteína/análogos & derivados , Cisteína/química , Biomarcadores/urina , MasculinoRESUMO
Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.
Assuntos
Cisteína , Dissulfetos , Simulação de Dinâmica Molecular , Anticorpos de Domínio Único , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Animais , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Estabilidade Proteica , Receptores de Antígenos/química , Receptores de Antígenos/metabolismo , Receptores de Antígenos/genética , Receptores de Antígenos/imunologia , Afinidade de Anticorpos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Muramidase/química , Muramidase/metabolismo , Muramidase/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , MutaçãoRESUMO
Cys is one of the least abundant amino acids in proteins. However, it is often highly conserved and is usually found in important structural and functional regions of proteins. Its unique chemical properties allow it to undergo several post-translational modifications, many of which are mediated by reactive oxygen, nitrogen, sulfur, or carbonyl species. Thus, in addition to their role in catalysis, protein stability, and metal binding, Cys residues are crucial for the redox regulation of metabolism and signal transduction. In this review, we discuss Cys post-translational modifications (PTMs) and their role in plant metabolism and signal transduction. These modifications include the oxidation of the thiol group (S-sulfenylation, S-sulfinylation and S-sulfonylation), the formation of disulfide bridges, S-glutathionylation, persulfidation, S-cyanylation S-nitrosation, S-carbonylation, S-acylation, prenylation, CoAlation, and the formation of thiohemiacetal. For each of these PTMs, we discuss the origin of the modifier, the mechanisms involved in PTM, and their reversibility. Examples of the involvement of Cys PTMs in the modulation of protein structure, function, stability, and localization are presented to highlight their importance in the regulation of plant metabolic and signaling pathways.
Assuntos
Cisteína , Proteínas de Plantas , Plantas , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Cisteína/metabolismo , Cisteína/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Plantas/metabolismo , OxirreduçãoRESUMO
Biothiols, characterized by thiol groups, exhibit remarkable affinity for certain metals, playing pivotal roles in intracellular and extracellular biological processes. Fluctuations in their levels profoundly impact overall physiological health. Despite the development of various probes for biothiol detection and quantification, their inability to monitor thiol-to-disulfide state transitions persists as a limitation. Given their association with pathologies, early detection remains imperative. Gold nanorod (AuNR)-based colorimetric probes have garnered attention for their utility in visual diagnostic assays. Herein, we present a cost-effective, and sensitive multicolor ratio measuring probe enabling on-site simultaneous identification, discrimination, and quantification of essential biothiolsâcysteine (CYS), glutathione (GSH), cystine (CYSS), and glutathione disulfide (GSSG)âwhile also quantifying thiol-to-disulfide ratios. Our investigation clarifies the probe's functionality, elucidating etching and antietching mechanisms based on sulfhydryl group coordination with Hg2+. This coordination impedes gold amalgam formation, facilitating discriminative detection via AuNR size and aspect ratio modulation, validated by transmission electron microscopy. Notably, distinct rainbow-like fingerprint patterns were discernible both visually and spectroscopically for the aforementioned biothiols and their respective thiol-to-disulfide ratios. Subsequent qualitative and quantitative analyses via linear discriminant analysis (LDA) and partial least squares regression revealed linear correlations over broad concentration ranges (CYS: 1.9-40 µmol L-1, GSH: 3.2-200.0 µmol L-1, CYSS: 2.0-70.0 µmol L-1, GSSG: 3.7-100.0 µmol L-1), with detection limits of 0.66 µmol L-1 (CYS), 1.07 µmol L-1 (GSH), 0.69 µmol L-1 (CYSS), and 1.24 µmol L-1 (GSSG). Moreover, thiol-to-disulfide ratios exhibited linear patterns within 0.2-5 µmol L-1, with detection limits of 0.13 and 0.09 µmol L-1, and exceptional analytical sensitivities of 32.648 and 49.782 for (CYS/CYSS) and (GSH/GSSG), respectively. Lastly, we evaluated the probe's performance in complex matrices relative to aqueous media, both quantitatively and qualitatively.
Assuntos
Ouro , Nanotubos , Compostos de Sulfidrila , Ouro/química , Nanotubos/química , Compostos de Sulfidrila/química , Compostos de Sulfidrila/análise , Aprendizado de Máquina , Glutationa/química , Glutationa/análise , Cisteína/análise , Cisteína/química , Limite de Detecção , Humanos , Colorimetria/métodos , Mercúrio/análise , Mercúrio/química , Cistina/química , Cistina/análiseRESUMO
Sulforaphene (4-methylsufinyl-3-butenyl isothiocyanate, SFE), produced by myrosinase hydrolysis of glucoraphenin (4-methylsulfinyl-3-butenyl glucosinolate) found in radish seeds, is strongly associated with cancer prevention. In this study, we investigated the stability of SFE (purity above 98%) under various thiol-containing compounds at 25 °C, such as sodium hydrosulfide (NaHS), glutathione (GSH), and cysteine (Cys). We observed that the degradation of SFE was closely related to the presence and dissociation capacity of thiol-containing compounds in the solution, particularly the thiol group. We found that the degradation rate of SFE was influenced by incubation with NaHS, GSH, and Cys, with distinct degradation products detected for each of these thiol-containing compounds. Compared to GSH, sulfide and Cys played important roles in promoting the degradation of SFE. Furthermore, we found substantial quantities of hydrogen sulfide in conjunction with SFE during the hydrolysis process of seeds, and a heat treatment of the seeds resulted in increased production of SFE. However, the introduction of sulfide-oxidizing bacteria to the hydrolytic system did not exhibit any inhibitory effect on the degradation of SFE. These results provided a guideline for industries to improve the stability of SFE during preparation.
Assuntos
Isotiocianatos , Compostos de Sulfidrila , Compostos de Sulfidrila/química , Hidrólise , Isotiocianatos/química , Isotiocianatos/farmacologia , Cisteína/química , Cisteína/análogos & derivados , Sementes/química , Glutationa/metabolismo , Glutationa/química , Raphanus/química , Sulfeto de Hidrogênio/químicaRESUMO
This study aimed to explore the effects of S-nitrosylation on caspase-3 modification and its subsequent effects on beef myofibril degradation in vitro. Recombinant caspase-3 was reacted with different concentrations of S-nitrosoglutathione (GSNO, nitric oxide donor) at 37 °C for 30 min and subsequently incubated with purified myofibrillar protein from bovine semimembranosus muscle. Results indicated that the activity of caspase-3 was significantly reduced after GSNO treatments (P < 0.05) and showed a dose-dependent inhibitory effect, which was attributed to the increased S-nitrosylation extent of caspase-3. LC-MS/MS analysis revealed that caspase-3 was S-nitrosylated at cysteine sites 116, 170, 184, 220, and 264. Moreover, the degradation of desmin and troponin-T was notably suppressed by S-nitrosylated caspase-3 (P < 0.05). To conclude, protein S-nitrosylation could modify the cysteine residues of caspase-3, which accounts for the reduced caspase-3 activity and further represses its proteolytic ability on beef myofibrillar protein.
Assuntos
Caspase 3 , Miofibrilas , Animais , Bovinos , Miofibrilas/química , Miofibrilas/metabolismo , Caspase 3/metabolismo , Caspase 3/química , Caspase 3/genética , S-Nitrosoglutationa/química , S-Nitrosoglutationa/metabolismo , S-Nitrosoglutationa/farmacologia , Espectrometria de Massas em Tandem , Cisteína/metabolismo , Cisteína/química , Proteólise/efeitos dos fármacos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Óxido Nítrico/metabolismo , Troponina T/metabolismo , Troponina T/química , Proteínas Musculares/metabolismo , Proteínas Musculares/químicaRESUMO
Oxidative stress on cysteine (Cys)-containing proteins has been associated with physiological disorders, as suggested for the human cofilin-1 (CFL-1) protein, in which the oxidized residues are likely implicated in the aggregation process of α-synuclein, leading to severe neuronal injuries. Considering the relevance of the oxidation state of cysteine, quantification of thiols may offer a guide for the development of effective therapies. This work presents, for the very first time, thiol quantification within CFL-1 in solution and on the surface following classic and adapted versions of Ellman's assay. The 1:1 stoichiometric Ellman's reaction occurs between 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), and the free thiol of the cysteine residue, producing two 2-nitro-5-thiobenzoate (TNB2-) ions, one of which is released into the medium. While in solution, the thiol concentration was determined by the absorbance of the released TNB2-, on the surface, the mass of the attached TNB2- ion to the protein allowed the quantification by means of the multiparametric surface plasmon resonance (MP-SPR) technique. The SPR angle change after the interaction of DTNB with immobilized CFL-1 gave a surface coverage of 26.5 pmol cm-2 for the TNB2- ions (ΓTNB2-). The ratio of this value to the surface coverage of CFL-1, ΓCFL-1 = 6.5 ± 0.6 pmol cm-2 (also determined by MP-SPR), gave 4.1 as expected for this protein, i.e., CFL-1 contains four Cys residues in its native form (reduced state). A control experiment with adsorbed oxidized protein showed no SPR angle change, thus proving the reliability of adapting Ellman's assay to the surface using the MP-SPR technique. The results presented in this work provide evidence of the heterogenization of Ellman's assay, offering a novel perspective for studying thiol-containing species within proteins. This may be particularly useful to ensure further studies on drug-like molecules that can be carried out with validated oxidized or reduced CFL-1 or other analogous systems.
Assuntos
Cofilina 1 , Compostos de Sulfidrila , Ressonância de Plasmônio de Superfície , Ressonância de Plasmônio de Superfície/métodos , Humanos , Compostos de Sulfidrila/química , Compostos de Sulfidrila/análise , Cofilina 1/química , Cofilina 1/metabolismo , Ácido Ditionitrobenzoico/química , Propriedades de Superfície , Cisteína/química , Cisteína/análiseRESUMO
OBJECTIVE: Breast cancer is the most frequently diagnosed cancer and the second cause of death worldwide. The drug often used for chemotherapy is cisplatin. However, the drug cisplatin has a number of problems, including lack of selectivity, undesirable side effects, resistance, and toxicity in the body. So research is carried out on new drug compounds with low toxicity by designing in silico with molecular docking. METHODS: Mn(II) Cysteine-Tyrosine dithiocarbamate is a new complex molecule whose research involves several steps, such as in-silico molecular docking testing with target proteins, ADMET then synthesis, characterization and in-vitro MCF-7 cells for anticancer drugs. The synthesis process involves the reaction of manganese metal with tyrosine, cysteine, CS2 and KOH. Characterization tests have been carried out including FT-IR spectroscopy, SEM-EDS, UV Vis, conductivity, melting point and XRD. RESULT: Confirm the structure of the compound using UV Vis, obtained orbitals π to π* and n to π* in the group N = C = S is represented by the absorption at 400 nm and 600 nm, FT-IR with the results obtained by the functional groups O-H, N-H, C =N and C=S. In vitro test results showed morphological changes (apoptosis) in MCF-7 cancer cells starting from 250 µg/mL and an IC50 value of 416.90 µg/mL. Molecular docking studies of the Mn(II)Cysteine-Tyrosine dithiocarbamate complex were identified with 4,4',4''-[(2R)-butane-1,1,2-triyl]triphenol - Estrogen α which showed an active site with amino acid residues GLU323, GLU385, VAL446, ILE514, TRP360, LYS449, MET388, MET357, PHE445, VAL392 and ILE389. Hydrophobic and hydrophobic bonds are seen in Mn(II)Cysteine-Tyrosine dithiocarbamate - Estrogen α has a bond energy of -77.5372 kJ/mol. CONCLUSION: Despite having a high H-bond interaction intensity, the chemical does not have a powerful enough anticancer impact. Despite the produced compound's low bioactivity, this study should offer important new understandings into how molecular structure affects anticancer activity.
Assuntos
Antineoplásicos , Neoplasias da Mama , Cisteína , Manganês , Simulação de Acoplamento Molecular , Tiocarbamatos , Tirosina , Humanos , Tiocarbamatos/farmacologia , Tiocarbamatos/química , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Células MCF-7 , Cisteína/química , Cisteína/farmacologia , Manganês/química , Manganês/farmacologia , Tirosina/química , Tirosina/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Feminino , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/síntese química , Apoptose/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Quinones are among the most important components in natural organic matter (NOM) for redox reactions; however, no quinones in complex environmental media have been identified. To aid the identification of quinone-containing molecules in ultracomplex environmental samples, we developed a chemical tagging method that makes use of a Michael addition reaction between quinones and thiols (-SH) in cysteine (Cys) and cysteine-contained peptides (CCP). After the tagging, candidates of quinones in representative aqueous environmental samples (water extractions of biochar) were identified through high-resolution mass spectrometry (HRMS) analysis. The MS and UV spectra analysis showed rapid reactions between Cys/CCP and model quinones with ß-carbon from the same benzene ring available for Michael addition. The tagging efficiency was not influenced by other co-occurring nonquinone representative compounds, including caffeic acid, cinnamic acid, and coumaric acid. Cys and CCP were used to tag quinones in water extractions of biochars, and possible candidates of quinones (20 and 53 based on tagging with Cys and CCP, respectively) were identified based on the HRMS features for products of reactions with Cys/CCP. This study has successfully demonstrated that such a Michael addition reaction can be used to tag quinones in complex environmental media and potentially determine their identities. The method will enable an in-depth understanding of the redox chemistry of NOM and its critical chemical compositions and structures.
Assuntos
Cisteína , Espectrometria de Massas , Peptídeos , Quinonas , Cisteína/química , Peptídeos/química , Quinonas/química , Carvão Vegetal/químicaRESUMO
Developing NIR-IIb luminescence probes with rapid visualization and a high penetration depth is essential for diabetes research. Combining a sensitizing switch with lanthanide-doped nanoparticles (LnNPs) has been employed to fabricate the NIR-IIb probes. However, these probes mainly adopt heptamethine cyanine dye as the antenna, and the NIR-IIb signal is activated by inhibiting the photoinduced electron transfer (PET) of the dye. Due to limited recognition units, this strategy makes many biomolecules undetectable, such as cysteine (Cys), which is closely related to diabetes. Herein, in this article, hemicyanine dye, NFL-OH, was verified as a new antenna to sensitize NIR-IIb emission from LnNPs. Unlike traditional cyanine dyes, hemicyanine's fluorescence intensity can also be modulated by intramolecular charge transfer (ICT), thereby expanding the range of detectable targets for NIR-IIb probes based on sensitization mechanism. Through switching the hemicyanine-sensitized NIR-IIb emission, we successfully fabricated an NFL-Cys-LnNPs' nanoprobe, which can effectively monitor Cys concentration in the liver of diabetic mice during diabetes progression and evaluate the efficacy of diabetic drugs. Our work not only presents an excellent tool for Cys imaging but also introduces new concepts for designing NIR-IIb probes.
Assuntos
Cisteína , Diabetes Mellitus Experimental , Corantes Fluorescentes , Animais , Camundongos , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Diabetes Mellitus Experimental/induzido quimicamente , Cisteína/química , Cisteína/análise , Raios Infravermelhos , Imagem Óptica , Nanopartículas/química , Carbocianinas/química , Progressão da Doença , Humanos , MasculinoRESUMO
The significance of HER-1 and CA 125 lies in their ability to guide cancer diagnosis, treatment, and monitoring, improving personalized care and enhancing prognostic accuracy. The utilization of HER-1 and CA 125 as screening biomarkers for the anticipation of early-stage cancer and monitoring cancer progression is expanding due to the invasive and costly nature of present techniques. In this study, a novel stochastic sensor was developed for the simultaneous determination of HER-1 and CA 125 in whole blood, saliva, and gastric tumor tissue samples using a fast, easy, inexpensive, and portable method. The stochastic sensor was prepared by electropolymerization of cysteine on the surface of the Au-TiO2@rGO/SPCE sensor. The Au-TiO2@rGO nanocomposite was synthesized using a simple chemical reduction process. The proposed sensor showed wide linear concentration ranges and very low limits of quantification (LOQ). The concentration ranges were from 3.9 × 10-14 to 3.9 × 10-8 µg mL-1, with a LOQ of 3.9 × 10-14 µg mL-1 for HER-1. For CA 125, the concentration ranges were from 8.3 × 10-14 to 8.3 × 10-10 U mL-1, with a LOQ of 8.3 × 10-14 U mL-1. Both biomarkers exhibit precise discrimination in different biological samples, with recoveries above 96.78% and RSD values below 0.04%. With a confidence level of 99%, the Student t-test revealed that there is no statistically significant difference between the outcomes obtained by using the poly-Cys/Au-TiO2@rGO/SPCE sensor for screening examinations of biological samples. This was determined because the results were not significantly different from one another.
Assuntos
Biomarcadores Tumorais , Antígeno Ca-125 , Ouro , Grafite , Neoplasias Gástricas , Titânio , Humanos , Antígeno Ca-125/sangue , Antígeno Ca-125/análise , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/sangue , Titânio/química , Ouro/química , Grafite/química , Biomarcadores Tumorais/sangue , Limite de Detecção , Técnicas Biossensoriais/métodos , Cisteína/sangue , Cisteína/química , Saliva/química , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Processos Estocásticos , Nanocompostos/química , Detecção Precoce de Câncer/métodos , Proteínas de MembranaRESUMO
Human lactoferrin (hLf) is an innate host defense protein that inhibits microbial H+-ATPases. This protein includes an ancestral structural motif (i.e., γ-core motif) intimately associated with the antimicrobial activity of many natural Cys-rich peptides. Peptides containing a complete γ-core motif from hLf or other phylogenetically diverse antimicrobial peptides (i.e., afnA, SolyC, PA1b, PvD1, thanatin) showed microbicidal activity with similar features to those previously reported for hLf and defensins. Common mechanistic characteristics included (1) cell death independent of plasma membrane (PM) lysis, (2) loss of intracellular K+ (mediated by Tok1p K+ channels in yeast), (3) inhibition of microbicidal activity by high extracellular K+, (4) influence of cellular respiration on microbicidal activity, (5) involvement of mitochondrial ATP synthase in yeast cell death processes, and (6) increment of intracellular ATP. Similar features were also observed with the BM2 peptide, a fungal PM H+-ATPase inhibitor. Collectively, these findings suggest host defense peptides containing a homologous γ-core motif inhibit PM H+-ATPases. Based on this discovery, we propose that the γ-core motif is an archetypal effector involved in the inhibition of PM H+-ATPases across kingdoms of life and contributes to the in vitro microbicidal activity of Cys-rich antimicrobial peptides.
Assuntos
Motivos de Aminoácidos , ATPases Translocadoras de Prótons , Humanos , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/química , Lactoferrina/farmacologia , Lactoferrina/química , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Cisteína/metabolismo , Cisteína/química , Candida albicans/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacosRESUMO
The fragment molecular orbital (FMO) method is an efficient quantum chemical calculation technique for large biomolecules, dividing each into smaller fragments and providing interfragment interaction energies (IFIEs) that support our understanding of molecular recognition. The ab initio fragment MO method (ABINIT-MP), an FMO processing program, can automatically divide typical proteins and nucleic acids. In contrast, small molecules such as ligands and heterosystems must be manually divided. Thus, we developed a graphical user interface to easily handle such manual fragmentation as a library for the Molecular Operating Environment (MOE) that preprocesses and visualizes FMO calculations. We demonstrated fragmentation with IFIE analyses for the two following cases: (1) covalent cysteine-ligand bonding inside the SARS-CoV-2 main protease (Mpro) and nirmatrelvir (Paxlovid) complex and (2) the metal coordination inside a zinc-bound cyclic peptide. IFIE analysis successfully identified the key amino acid residues for the molecular recognition of nirmatrelvir with Mpro and the details of their interactions (e.g., hydrogen bonds and CH/π interactions) via ligand fragmentation of functional group units. In metalloproteins, we found an efficient and accurate scheme for the fragmentation of Zn2+ ions with four histidines coordinated to the ion. FMOe simplifies manual fragmentation, allowing users to experiment with various fragmentation patterns and perform in-depth IFIE analysis with high accuracy. In the future, our findings will provide valuable insight into complicated cases, such as ligand fragmentation in modality drug discovery, especially for medium-sized molecules and metalloprotein fragmentation around metals.
Assuntos
Proteases 3C de Coronavírus , Metaloproteínas , Ligantes , Metaloproteínas/química , Metaloproteínas/metabolismo , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , SARS-CoV-2 , Teoria Quântica , Modelos Moleculares , Zinco/química , Cisteína/química , Software , Peptídeos Cíclicos/química , COVID-19/virologiaRESUMO
Peptides are receiving significant attention in pharmaceutical sciences due to their applications as anti-inflammatory drugs; however, many aspects of their interactions and mechanisms at the molecular level are not well-known. This work explores the molecular structure of two peptides-(i) cysteine (Cys)-asparagine (Asn)-serine (Ser) (CNS) as a molecule in the gas phase and solvated in water in zwitterion form, and (ii) the crystal structure of the dipeptide serine-asparagine (SN), a reliable peptide indication whose experimental cell parameters are well known. A search was performed by means of atomistic calculations based on density functional theory (DFT). These calculations matched the experimental crystal structure of SN, validating the CNS results and useful for assignments of our experimental spectroscopic IR bands. Our calculations also explore the intercalation of CNS into the interlayer space of montmorillonite (MNT). Our quantum mechanical calculations show that the conformations of these peptides change significantly during intercalation into the confined interlayer space of MNT. This intercalation is energetically favorable, indicating that this process can be a useful preparation for therapeutic anti-inflammatory applications and showing high stability and controlled release processes.
Assuntos
Anti-Inflamatórios , Bentonita , Cisteína , Teoria da Densidade Funcional , Serina , Bentonita/química , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Cisteína/química , Serina/química , Asparagina/química , Modelos Moleculares , Peptídeos/química , Substâncias Intercalantes/químicaRESUMO
Cysteine is the most reactive naturally occurring amino acid due to the presence of a free thiol, presenting a tantalizing handle for covalent modification of peptides/proteins. Although many mass spectrometry experiments could benefit from site-specific modification of Cys, the utility of direct arylation has not been thoroughly explored. Recently, Spokoyny and co-workers reported a Au(III) organometallic reagent that robustly arylates Cys and tolerates a wide variety of solvents and conditions. Given the chromophoric nature of aryl groups and the known susceptibility of carbon-sulfur bonds to photodissociation, we set out to identify an aryl group that could efficiently cleave Cys carbon-sulfur bonds at 266 nm. A streamlined workflow was developed to facilitate rapid examination of a large number of aryls with minimal sample using a simple test peptide, RAAACGVLK. We were able to identify several aryl groups that yield abundant homolytic photodissociation of the adjacent Cys carbon-sulfur bonds with short activation times (<10 ms). In addition, we characterized the radical products created by photodissociation by subjecting the product ions to further collisional activation. Finally, we tested Cys arylation with human hemoglobin, identified reaction conditions that facilitate efficient modification of intact proteins, and evaluated the photochemistry and activation of these large radical ions.
Assuntos
Cisteína , Peptídeos , Cisteína/química , Peptídeos/química , Ouro/química , Processos Fotoquímicos , Proteínas/química , Compostos Organoáuricos/química , HumanosRESUMO
Biomolecular condensates (BCs) are membraneless hubs enriched with proteins and nucleic acids that have emerged as important players in many cellular functions. Uncovering the sequence determinants of proteins for phase separation is essential in understanding the biophysical and biochemical properties of BCs. Despite significant discoveries in the past decade, the role of cysteine residues in BC formation and dissolution has remained unknown. Here, to uncover the involvement of disulfide cross-links and their redox sensitivity in BCs, we designed a "stickers and spacers" model of phase-separating peptides interspersed with cysteines. Through biophysical investigations, we learned that cysteines promote liquid-liquid phase separation in oxidizing conditions and perpetuate liquid condensates through disulfide cross-links, which can be reversibly tuned with redox chemistry. By varying the composition of cysteines, subtle but distinct changes in the viscoelastic behavior of the condensates were observed. Empirically, we conclude that cysteines function neither as stickers nor spacers but as covalent nodes to lower the effective concentrations for sticker interactions and inhibit system-spanning percolation networks. Together, we unmask the possible role of cysteines in the formation of biomolecular condensates and their potential use as tunable covalent cross-linkers in developing redox-sensitive viscoelastic materials.