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1.
Sci Rep ; 12(1): 7470, 2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35523830

RESUMO

Plant-derived products are frequently found as ingredients in cosmetics. However, the current data show non-neglectable skin sensitizing potential of these preparations suggesting an urgent need for data regarding their health safety profile. The aim of this study was to assess the skin sensitization potential of commercial essential oils by selected Lamiaceae species (Lavandula angustifolia, Melissa officinalis, Mentha longifolia, Thymus vulgaris, Salvia officinalis, and Rosmarinus officinalis) using a chemistry-based Direct Peptide Reactivity Assay (DPRA) in order to predict their potential allergic properties. In the DPRA assay, nucleophile-containing synthetic peptides (cysteine peptide and lysine peptide) were incubated with the test substance for 24 h. Depletion of the peptide in the reaction mixture was measured by high-pressure liquid chromatography (HPLC) using UV detection and the average peptide depletion data for cysteine and lysine was then calculated. Menthae longifoliae aetheroleum showed no or minimal reactivity with 4.48% cysteine depletion, Rosmarini aetheroleum and Salviae aetheroleum showed low reactivity with the 12.79% and 15.34% of cysteine depletion, respectively, while the other analyzed essential oils showed moderate reactivity with the cysteine depletion between 23.21 and 48.43%. According to DPRA predictive analysis, only Menthae longifoliae aetheroleum can be classified as negative, while all other essential oils may be classified as positive, thus having the potential to cause skin sensitization.


Assuntos
Cisteína , Óleos Voláteis , Cisteína/química , Lisina/química , Óleos Voláteis/farmacologia , Peptídeos/química , Pele
2.
J Gen Virol ; 103(5)2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35506985

RESUMO

CD163, a macrophage-specific membrane scavenger receptor, serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The removal of scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR5) of CD163 is sufficient to make transfected cells or genetically modified pigs resistant to PRRSV-1 and PRRSV-2 genotypes, and substitution of SRCR5 with SRCR8 from human CD163-like protein (hCD163L1) confers resistance to PRRSV-1 but not PRRSV-2 isolates. However, the specific regions within the SRCR5 polypeptide involved in PRRSV infection remain largely unknown. In this report, we performed mutational studies in order to identify which regions or amino acid sequences in the SRCR5 domain are critical for PRRSV infection. The approach used in this study was to make proline-arginine (PR) insertions along the SRCR5 polypeptide. Constructs were transfected into HEK293T cells, and then evaluated for infection with PRRSV-2 or PRRSV-1. For PRRSV-2, four PR insertions located after amino acids 8 (PR-9), 47 (PR-48), 54 (PR-55), and 99 (PR-100) had the greatest impact on infection. For PRRSV-1, insertions after amino acids 57 (PR-58) and 99 (PR-100) were critical. Computer simulations based on the crystal structure of SRCR5 showed that the mutations that affected infection localized to a similar region on the surface of the 3-D structure. Specifically, we found two surface patches that are essential for PRRSV infection. PR-58 and PR-55, which were separated by only three amino acids, had reciprocal effects on PRRSV-1 and PRRSV-2. Substitution of Glu-58 with Lys-58 reduced PRRSV-1 infection without affecting PRRSV-2, which partially explains the resistance to PRRSV-1 caused by the SRCR5 replacement with the homolog human SRCR8 previously observed. Finally, resistance to infection was observed following the disruption of any of the four conserved disulfide bonds within SRCR5. In summary, the results confirm that there are distinct differences between PRRSV-1 and PRRSV-2 on recognition of CD163; however, all mutations that affect infection locate on a similar region on the same face of SRCR5.


Assuntos
Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Antígenos CD , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Cisteína/genética , Células HEK293 , Humanos , Mutação , Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Domínios Proteicos , Receptores de Superfície Celular , Receptores Depuradores/genética , Suínos
3.
Biochemistry (Mosc) ; 87(2): 170-178, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35508908

RESUMO

The review considers the reasons and consequences of post-transcriptional tyrosine substitutions for cysteine residues. Main attention is paid to the Tyr/Cys substitutions that arise during gene expression in bacterial systems at the stage of protein translation as a result of misrecognition of the similar mRNA codons. Notably, translation errors generally occur relatively rarely - from 10-4 to 10-3 errors per codon for E. coli cells, but in some cases the error rate increases significantly. For example, this is typical for certain pairs of codons, when the culture conditions change or in the presence of antibiotics. Thus, with overproduction of the recombinant human alpha-synuclein in E. coli cells, the content of the mutant form with the replacement of Tyr136 (UAC codon) with a cysteine residue (UGC codon) can reach 50%. Possible reasons for the increased production of alpha-synuclein with the Tyr136Cys substitution are considered, as well as consequences of the presence of mutant forms in preparations of amyloidogenic proteins when studying their pathological transformation in vitro. A separate section is devoted to the Tyr/Cys substitutions occurring due to mRNA editing by adenosine deaminases, which is typical for eukaryotic organisms, and the possible role of this process in the amyloid transformation of proteins associated with neurodegenerative diseases.


Assuntos
Proteínas Amiloidogênicas , alfa-Sinucleína , Códon , Cisteína/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Tirosina , alfa-Sinucleína/metabolismo
4.
Sci Rep ; 12(1): 7262, 2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35508689

RESUMO

Next-generation site-specific cysteine-based antibody-drug-conjugates (ADCs) broaden therapeutic index by precise drug-antibody attachments. However, manufacturing such ADCs for clinical validation requires complex full reduction and reoxidation processes, impacting product quality. To overcome this technical challenge, we developed a novel antibody manufacturing process through cysteine (Cys) metabolic engineering in Chinese hamster ovary cells implementing a unique cysteine-capping technology. This development enabled a direct conjugation of drugs after chemoselective-reduction with mild reductant tris(3-sulfonatophenyl)phosphine. This innovative platform produces clinical ADC products with superior quality through a simplified manufacturing process. This technology also has the potential to integrate Cys-based site-specific conjugation with other site-specific conjugation methodologies to develop multi-drug ADCs and exploit multi-mechanisms of action for effective cancer treatments.


Assuntos
Antineoplásicos , Imunoconjugados , Animais , Anticorpos , Antineoplásicos/uso terapêutico , Células CHO , Cricetinae , Cricetulus , Cisteína , Dissulfetos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Engenharia Metabólica
5.
J Phys Chem A ; 126(18): 2818-2824, 2022 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-35500128

RESUMO

An improved understanding of the P450 structure is relevant to the development of biomimetic catalysts and inhibitors for controlled CH-bond activation, an outstanding challenge of synthetic chemistry. Motivated by the experimental findings of an unusually short Fe-S bond of 2.18 Šfor the wild-type (WT) OleT P450 decarboxylase relative to a cysteine pocket mutant form (A369P), a computational model that captures the effect of the thiolate axial ligand on the iron-sulfur distance is presented. With the computational efficiency and streamlined analysis in mind, this model combines a cluster representation of the enzyme─40-110 atoms, depending on the heme and ligand truncation level─with a density functional theory (DFT) description of the electronic structure (ES) and is calibrated against the experimental data. The optimized Fe-S distances show a difference of 0.25 Šbetween the low and high spin states, in agreement with the crystallographic structures of the OleT WT and mutant forms. We speculate that this difference is attributable to the packing of the ligand; the mutant is bulkier due to an alanine-to-proline replacement, meaning that it is excluded from the energetically favored low-spin minimum because of steric constraints. The presence of pure spin-state pairs and the intersection of the low/high spin states for the enzyme model is indicative of the limitations of single-reference ES methods in such systems and emphasizes the significance of using the proper state when modeling the hydrogen atom transfer (HAT) reaction catalyzed by OleT. At the same time, the correct characterization of both the short and long Fe-S bonds within a small DFT-based model of 42 atoms paves the way for quantum dynamics modeling of the HAT step, which initiates the OleT decarboxylation reaction.


Assuntos
Heme , Ferro , Cisteína/química , Teoria da Densidade Funcional , Heme/química , Ferro/química , Ligantes
6.
Mol Cell ; 82(9): 1613-1615, 2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35523127

RESUMO

Jouandin et al. (2022) show that lysosomal-derived cysteine serves as a signal to promote the tricarboxylic acid (TCA) cycle and suppress TORC1 signaling for Drosophila to endure starvation periods.


Assuntos
Proteínas de Drosophila , Inanição , Adaptação Psicológica , Animais , Cisteína , Drosophila , Proteínas de Drosophila/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética
7.
Front Immunol ; 13: 870679, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35514966

RESUMO

Many immunological diseases can be treated by regulating neurobehavior, in which extracellular ATP is a vital member of endogenous danger-associated molecular pattern signaling molecule that plays a crucial part in innate neuro-related immunity. It is actively released through pannexin (Panx) and connexin (Cx) hemichannels from activated or stressed cells during inflammation, injury, or apoptosis. In addition to participating in ATP release, Panxs and Cxs also have crucial immune functions. In this study, pannexin1, three connexin32 isoforms and connexin43 were identified and characterized in spotted sea bass (Lateolabrax maculatus), which were named LmPanx1, LmCx32.2, LmCx32.3, LmCx32.7, and LmCx43. Their similar topological structures were discovered by sequence analysis: a relatively unconserved C-terminal region and four highly conserved transmembrane (TM) domains, and so on. Each extracellular (ECL) region of Panx1 has two conserved cysteine residues. Unlike Panx1, each ECL region of Cx32 and Cx43 contains three conserved cysteine residues, forming two conserved motifs: CX6CX3C motif in ECL1 and CX4CX5C motif in ECL2. Furthermore, Panx1 and Cx43 share similar genomic organization and synteny with their counterparts in selected vertebrates. Cx32 and CX43 were located in the same locus in fish, but diverged into two loci from amphibian. Moreover, despite varying expression levels, the identified genes were constitutively expressed in all examined tissues. All genes were upregulated by PAMP [lipopolysaccharide and poly(I:C)] stimulation or bacterial infection in vivo and in vitro, but they were downregulated in the brain at 6 or 12 h after stimulation. Especially, the three LmCx32 isoforms and LmCx43 were upregulated by ATP stimulation in primary head kidney leukocytes; however, downregulation of LmCx32.3 and LmCx43 expression were noted at 12 h. Conversely, ATP treatment inhibited the expression of LmPanx1. Importantly, we showed that the spotted sea bass Panx1, Cx43, and Cx32 were localized on the cellular membrane and involved in inflammation-induced ATP release. Taken together, our results demonstrated that Panx1, Cx32, and Cx43 are important neuro-related immune response genes involved in inflammation-induced ATP release.


Assuntos
Bass , Doenças dos Peixes , Trifosfato de Adenosina/metabolismo , Animais , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Cisteína , Doenças dos Peixes/genética , Proteínas de Peixes/metabolismo , Imunidade Inata/genética , Inflamação/genética
8.
J Chromatogr A ; 1671: 463040, 2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35428031

RESUMO

Mechanistic understanding of immunoglobulin G (IgG) binding to protein A is crucial for the design and development of high-performance protein A chromatography. In this work, the IgG binding domain (Z) of protein A from Staphylococcus aureus was genetically modified by introducing a cysteine residue at the N-terminus (Cys-Z) or a cysteine-lysine dipeptide at the C-terminus (Z-Cys), and the two ligands were used to unravel the IgG binding mechanism by means of binding kinetics and different single molecule measurements. Surface plasma resonance (SPR) measurement of the binding kinetics of mouse myeloma IgG2a (mIgG2a) to the two ligands indicated that oriented ligand immobilization significantly increased the association rate constant of mIgG2a, and Z-Cys had the highest binding affinity to mIgG2a among the three ligands (Cys-Z, Z-Cys and Z). This was attributed to the synergistic contribution of the high association rate constant and low dissociation rate constant to mIgG2a. Furthermore, quartz crystal microbalance with energy dissipation monitoring (QCM-D) measurement provided the maximum adsorption densities of IgGs on the Z-Cys-immobilized chip as zeta potentials of IgGs were nearly zero. The QCM-D investigation revealed that the adsorbed layer was dependent on ligand type and density, and IgG. Moreover, Z-Cys and Cys-Z induced IgG binding in flipped orientations, as evidenced by the antigen-antibody reaction. Finally, rectangular DNA origami tiles were introduced to analyze the molecular orientation of adsorbed IgG. Single-molecule imaging showed that mIgG2a was associated with flexible Z-Cys on the tiles predominantly in side-on and end-on orientations. The research has provided molecular insight into the binding mechanism of IgG molecules at liquid-solid interfaces and would help design new protein A-based ligands and high-capacity adsorbents.


Assuntos
Imunoglobulina G , Proteína Estafilocócica A , Animais , Cisteína/química , Imunoglobulina G/química , Cinética , Ligantes , Camundongos , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Estafilocócica A/química
9.
Trends Biochem Sci ; 47(5): 372-374, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35427478

RESUMO

Modifications of cysteine residues in redox-sensitive proteins are key to redox signaling and stress response in all organisms. A novel type of redox switch was recently discovered that comprises lysine and cysteine residues covalently linked by an nitrogen-oxygen-sulfur (NOS) bridge. Here, we discuss chemical and biological implications of this discovery.


Assuntos
Cisteína , Lisina , Cisteína/química , Lisina/metabolismo , Oxirredução , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Proteínas/química
10.
Biomolecules ; 12(4)2022 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-35454145

RESUMO

Unlike its shorter analog, cysteine, and its methylated derivative, methionine, homocysteine is not today a proteinogenic amino acid. However, this thiol containing amino acid is capable of forming an activated species intramolecularly. Its thiolactone could have made it an interesting molecular building block at the origin of life on Earth. Here we study the cyclization of homocysteine in water and show theoretically and experimentally that in an acidic medium the proportion of thiolactone is significant. This thiolactone easily reacts with amino acids to form dipeptides. We envision that these reactions may help interpret why a methionine residue is introduced at the start of all protein synthesis.


Assuntos
Homocisteína , Peptídeos , Cisteína/metabolismo , Metionina/metabolismo , Compostos de Sulfidrila
11.
J Med Chem ; 65(8): 5902-5925, 2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35412827

RESUMO

Protein S-nitrosation (SNO), a posttranslational modification (PTM) of cysteine (Cys) residues elicited by nitric oxide (NO), regulates a wide range of protein functions. As a crucial form of redox-based signaling by NO, SNO contributes significantly to the modulation of physiological functions, and SNO imbalance is closely linked to pathophysiological processes. Site-specific identification of the SNO protein is critical for understanding the underlying molecular mechanisms of protein function regulation. Although careful verification is needed, SNO modification data containing numerous functional proteins are a potential research direction for druggable target identification and drug discovery. Undoubtedly, SNO-related research is meaningful not only for the development of NO donor drugs but also for classic target-based drug design. Herein, we provide a comprehensive summary of SNO, including its origin and transport, identification, function, and potential contribution to drug discovery. Importantly, we propose new views to develop novel therapies based on potential protein SNO-sourced targets.


Assuntos
Cisteína , Proteína S , Cisteína/química , Óxido Nítrico/metabolismo , Nitrosação , Processamento de Proteína Pós-Traducional , Proteína S/metabolismo , Proteínas/metabolismo
12.
Viruses ; 14(4)2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35458499

RESUMO

Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus. The infection of HeLa cells with protein 3A cysteine 38 to alanine mutants (C38A) attenuates virus replication, resulting in comparably lower virus particle formation. Consistently, in a mouse infection model, the enhanced virus propagation of CVB3-3A wt in comparison to the CVB3-3A[C38A] mutant was confirmed and found to promote severe liver tissue damage. In contrast, infection with the CVB3-3A[C38A] mutant mitigated hepatic tissue injury and ameliorated the signs of systemic inflammatory responses, such as hypoglycemia and hypothermia. Based on these data and our previous report on the C38-mediated stabilization of the CVB3-3A protein, we conclude that the highly conserved amino acid C38 in protein 3A enhances the virulence of CVB3.


Assuntos
Infecções por Coxsackievirus , Infecções por Enterovirus , Enterovirus , Animais , Cisteína , Enterovirus Humano B/fisiologia , Células HeLa , Humanos , Camundongos , Virulência , Replicação Viral
13.
Anal Bioanal Chem ; 414(14): 4217-4225, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35462599

RESUMO

Nanozymes have both the high catalytic activity of natural enzymes and the stability and economy of mimetic enzymes. Research on nanozymes is rapidly emerging, and the continuous development of highly catalytic active nanozymes is of far-reaching significance. This work reports heteroatomic nitrogen (N) and phosphorus (P) double-doped mesoporous carbon structures and metallic Fe coordination generated sponge-like nanozymes (Fe/NPCs) with good peroxidase activity. On this basis, we constructed a highly sensitive colorimetric sensor with cysteine and phenol as simulated analytes using Fe/NPCs nanozymes, and the response limits reached 53.6 nM and 5.4 nM, respectively. Besides, the method has high accuracy in the detection of cysteine and phenol at low concentrations in serum and tap water, which lays a foundation for application in the fields of environmental protection and biosensors.


Assuntos
Colorimetria , Fenol , Catálise , Cisteína , Peróxido de Hidrogênio/química , Peroxidases/química , Fenóis
14.
Water Res ; 218: 118479, 2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35477064

RESUMO

A novel approach for the enhancement of phosphorus (P) recovery from Fe bound P compounds (FePs)-bearing sludge by co-fermentation with protein-rich biomass (PRB) is reported. Four PRBs (silkworm chrysalis meal, fish meal, corn gluten meal, and soya bean meal) were used for co-fermentation. The results revealed that PRBs with strong surface hydrophobicity and loose structure favored the hydrolysis and acidogenesis processes. Sulfide produced by PRB could react with FePs to form FeS and promote P release. Due to the neutralization of volatile fatty acids (VFAs) by a relatively high concentration of ammonia, the pH was maintained near neutral and thus prevented the dissolution of metal ions (e.g., Fe and Ca). This was beneficial to save the cost of subsequent P recovery and form high-purity struvite. Compared with the control, the soluble orthophosphate and VFAs increased by 88.3% and 531.3%, respectively, in the co-fermentation system with silkworm chrysalis meal. Cysteine was the important intermediate. The metagenomics analysis indicated that the gene abundances of phosphate acetyltransferase and acetate kinase, which were key enzymes in the acetate metabolism, increased by 117.7% and 52.2%, respectively. The gene abundances of serine O-acetyltransferase and cysteine synthase increased by 63.4% and 54.4%, respectively. Cysteine was primarily transformed to pyruvate and sulfide. This study provides an environment-friendly strategy to simultaneously recover P and VFAs resources from FePs-bearing sludge and PRB waste.


Assuntos
Fósforo , Esgotos , Animais , Biomassa , Cisteína/metabolismo , Ácidos Graxos Voláteis/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Proteínas , Esgotos/química , Sulfetos
15.
Cell Chem Biol ; 29(5): 785-798.e19, 2022 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-35364007

RESUMO

Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection.


Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Cisteína , Citomegalovirus/fisiologia , Humanos , Peptídeo Hidrolases , Proteases Virais
16.
Curr Pharm Des ; 28(8): 661-670, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35366767

RESUMO

BACKGROUND: S-Allylcysteine (SAC), an organosulfur phytochemical sourced from aged garlic extract, is well known for its varied biomedical applications, such as anti-oxidant, anti-inflammatory, and detoxification mechanisms. Despite this, the scientific findings on the defensive impact of SAC against kidney failure (KF) are still unclear. Therefore, in the current investigation, the animal model of KF was induced by adenine in Wistar rats, and the animals were divided into four groups as control, KF induction using adenine, SAC treated KF rats for an experimental duration of 8 weeks. METHODS: KF progression was assessed by various serum and tissue markers, and the results demonstrated that the renal functions' markers, KIM-1 (kidney injury molecule-1), cystatin, NGAL (neutrophil gelatinase-associated lipocalin), were found increased in adenine-treated rats compared to control. In addition, the inflammatory markers, matrix proteins, and fibrosis signatures explicated by RT-PCR, ELISA demonstrated a profound increase. On the other hand, rats received SAC mitigated KF considerably (p < 0.001) with restored cellular functions. Besides, SAC pre-treatment abrogated the cytokines and pro-inflammatory signals (COX-2 and PGE2) in a dose-dependent manner. CONCLUSION: Furthermore, the fibrosis signaling markers mediators, such as SMAD-2,-3 were increased with associated matrix proteins. Thus, the present study substantiated that SAC possesses a significant renoprotective effect that might have been demonstrated by the inhibition of the TGF-ß1/Smad3 signaling pathway.


Assuntos
Falência Renal Crônica , Insuficiência Renal Crônica , Adenina/farmacologia , Idoso , Animais , Cisteína/análogos & derivados , Fibrose , Humanos , Ratos , Ratos Wistar , Proteínas Smad/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
17.
Commun Biol ; 5(1): 331, 2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35393494

RESUMO

Cerebral small vessel disease (SVD) is a prevalent disease of aging and a major contributor to stroke and dementia. The most commonly inherited SVD, CADASIL, is caused by dominantly acting cysteine-altering mutations in NOTCH3. These mutations change the number of cysteines from an even to an odd number, but the impact of these alterations on NOTCH3 protein structure remain unclear. Here, we prepared wildtype and four mutant recombinant NOTCH3 protein fragments to analyze the impact of CADASIL mutations on oligomerization, thiol status, and protein stability. Using gel electrophoresis, tandem MS/MS, and collision-induced unfolding, we find that NOTCH3 mutant proteins feature increased amounts of inappropriate disulfide bridges, reduced cysteines, and structural instability. Presence of a second protein factor, an N-terminal fragment of NOTCH3 (NTF), is capable of further altering disulfide statuses of both wildtype and mutant proteins, leading to increased numbers of reduced cysteines and further destabilization of NOTCH3 structure. In sum, these studies identify specific cysteine residues alterations and quaternary structure induced by CADASIL mutations in NOTCH3; further, we validate that reductive factors alter the structure and stability of this small vessel disease protein.


Assuntos
CADASIL , Demência Vascular , Receptor Notch3 , CADASIL/genética , CADASIL/metabolismo , Cisteína/genética , Dissulfetos , Humanos , Proteínas Mutantes , Receptor Notch3/genética , Receptores Notch/metabolismo , Espectrometria de Massas em Tandem
18.
Analyst ; 147(9): 1808-1814, 2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35404374

RESUMO

Enzyme-based sensing platforms have undergone rapid development in the field of diagnosis and bioanalysis. Here we present a novel fluorescent artificial enzyme-based detection strategy for L-cysteine (Cys) and H2O2 by fabricating a series of Au-Ag bimetallic nanoparticles with peroxidase-like activity. Taking advantage of the enhanced performance of catalysts by optimizing the surface structure, the sensitive detection of Cys with an ultralow detection limit of 0.035 µM and accurate quantification in the range of 0.075-2 µM were achieved. It was revealed that the mechanism of the catalytic process on the Au-Ag surface follows the electron transfer mechanism rather than active species, that is the peroxidase-like catalysts work as electron transfer intermediates and the electron transfer efficiency will increase with the larger electron cloud density of active sites derived from the electronic synergistic effect between Au and Ag, contributing to the enhanced catalytic activity of peroxidase mimics. This finding could provide guidance for the structural design of high-activity peroxidase mimics.


Assuntos
Nanopartículas Metálicas , Peroxidase , Colorimetria , Corantes , Cisteína/análise , Fluorescência , Ouro/química , Peróxido de Hidrogênio/química , Nanopartículas Metálicas/química , Peroxidase/química , Peroxidases/química
19.
Chem Commun (Camb) ; 58(36): 5526-5529, 2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35420608

RESUMO

With the idea of exploiting metal templated C-S bond forming reactions to achieve modification of cysteines in bacterial proteins, a cyclometalated Au(III) compound was explored in a competitive chemoproteomic approach in S. aureus cell extracts. More than 100 ligandable cysteines were identified, of which more than 50% were not engaged by organic α-chloroacetamides in a previous study, indicating that organometallic compounds expand the ligandable space in bacteria. A selected interaction was validated using an enzyme activity assay, and intact protein mass spectrometry showed cysteine arylation of an unprecedented target. The obtained results demonstrate that this family of organogold compounds has potential for therapeutic protein targeting via selective, covalent modification of cysteine residues in bacteria.


Assuntos
Cisteína , Staphylococcus aureus , Cisteína/química , Compostos Organoáuricos/química
20.
J Am Chem Soc ; 144(17): 7911-7918, 2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35451816

RESUMO

Arginylation is an understudied post-translational modification (PTM) involving the transfer of arginine to aspartate or glutamate sidechains in a protein. Among the targets of this PTM is α-synuclein (αS), a neuronal protein involved in regulating synaptic vesicles. The aggregation of αS is implicated in neurodegenerative diseases, particularly in Parkinson's disease, and arginylation has been found to protect against this pathological process. Arginylated αS has been studied through semisynthesis involving multipart native chemical ligation (NCL), but this can be very labor-intensive with low yields. Here, we present a facile way to introduce a mimic of the arginylation modification into a protein of interest, compatible with orthogonal installation of labels such as fluorophores. We synthesize bromoacetyl arginine and react it with recombinant, site-specific cysteine mutants of αS. We validate the mimic by testing the vesicle binding affinity of mimic-arginylated αS, as well as its aggregation kinetics and monomer incorporation into fibrils, and comparing these results to those of authentically arginylated αS produced through NCL. In cultured neurons, we compare the fibril seeding capabilities of preformed fibrils carrying a small percentage of arginylated αS. We find that, consistent with authentically arginylated αS, mimic-arginylated αS does not perturb the protein's native function but alters aggregation kinetics and monomer incorporation. Both mimic and authentically modified αS suppress aggregation in neuronal cells. Our results provide further insight into the neuroprotective effects of αS arginylation, and our alternative strategy to generate arginylated αS enables the study of this PTM in proteins not accessible through NCL.


Assuntos
Fármacos Neuroprotetores , alfa-Sinucleína , Arginina/metabolismo , Cisteína/metabolismo , Fármacos Neuroprotetores/farmacologia , Processamento de Proteína Pós-Traducional , alfa-Sinucleína/metabolismo
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