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1.
Nat Commun ; 12(1): 5460, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34526506

RESUMO

Surgery is an efficient way to treat localized prostate cancer (PCa), however, it is challenging to demarcate rapidly and accurately the tumor boundary intraoperatively, as existing tumor detection methods are seldom performed in real-time. To overcome those limitations, we develop a fluorescent molecular rotor that specifically targets the prostate-specific membrane antigen (PSMA), an established marker for PCa. The probes have picomolar affinity (IC50 = 63-118 pM) for PSMA and generate virtually instantaneous onset of robust fluorescent signal proportional to the concentration of the PSMA-probe complex. In vitro and ex vivo experiments using PCa cell lines and clinical samples, respectively, indicate the utility of the probe for biomedical applications, including real-time monitoring of endocytosis and tumor staging. Experiments performed in a PCa xenograft model reveal suitability of the probe for imaging applications in vivo.


Assuntos
Antígenos de Superfície/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Sondas Moleculares/metabolismo , Imagem Óptica/métodos , Neoplasias da Próstata/metabolismo , Animais , Antígenos de Superfície/química , Sítios de Ligação , Linhagem Celular Tumoral , Endocitose , Glutamato Carboxipeptidase II/química , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Sondas Moleculares/química , Células PC-3 , Neoplasias da Próstata/diagnóstico , Ligação Proteica , Domínios Proteicos , Espectrometria de Fluorescência/métodos , Transplante Heterólogo
2.
Nat Commun ; 12(1): 4693, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344862

RESUMO

Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell-surface receptors. Nucleic acid-based molecular tension probes allow one to visualize the piconewton (pN) forces applied by these receptors. Building on this technology, we recently developed molecular force microscopy (MFM) which uses fluorescence polarization to map receptor force orientation with diffraction-limited resolution (~250 nm). Here, we show that structured illumination microscopy (SIM), a super-resolution technique, can be used to perform super-resolution MFM. Using SIM-MFM, we generate the highest resolution maps of both the magnitude and orientation of the pN traction forces applied by cells. We apply SIM-MFM to map platelet and fibroblast integrin forces, as well as T cell receptor forces. Using SIM-MFM, we show that platelet traction force alignment occurs on a longer timescale than adhesion. Importantly, SIM-MFM can be implemented on any standard SIM microscope without hardware modifications.


Assuntos
Microscopia de Fluorescência/métodos , Receptores de Superfície Celular/metabolismo , Animais , Fenômenos Biomecânicos , Plaquetas/metabolismo , Linfócitos T CD8-Positivos , Corantes Fluorescentes/metabolismo , Humanos , Integrinas/metabolismo , Camundongos , Sondas Moleculares/metabolismo , Células NIH 3T3 , Paxilina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Imagem com Lapso de Tempo
4.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203967

RESUMO

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.


Assuntos
Análise Custo-Benefício , Éxons/genética , Proteínas da Matriz Extracelular/genética , Sondas Moleculares/metabolismo , Sítios de Splice de RNA/genética , Retinite Pigmentosa/genética , Análise de Sequência de DNA , Síndromes de Usher/genética , Sequência de Bases , Variações do Número de Cópias de DNA/genética , Deleção de Genes , Humanos , Polimorfismo de Nucleotídeo Único/genética , Retinite Pigmentosa/economia , Síndromes de Usher/economia
5.
Chem Commun (Camb) ; 57(53): 6507-6510, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34105530

RESUMO

We applied a new in silico approach for using protease-substrate motifs to design a kallikrein 7 (KLK7)-specific phosphonate activity-based probe (ABP) to quantify the active KLK7 in situ. Epidermal application of the ABP-inhibitor on Spink5-/-Klk5-/- mice, a Netherton syndrome model, reversed disease hallmarks, providing preclinical proof-of-concept for using ABPs as theranostics.


Assuntos
Simulação por Computador , Calicreínas/metabolismo , Sondas Moleculares/metabolismo , Dermatopatias/diagnóstico , Dermatopatias/terapia , Dermatopatias/metabolismo
6.
Chem Commun (Camb) ; 57(51): 6249-6252, 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34059853

RESUMO

A hydrophilic probe is employed to enrich exosomes from three kinds of cancer cells by TiO2-phosphate interaction and exosomal glycoproteins by hydrophilic interaction in succession. The probe performs efficiently in both the enrichment processes. And the analytical results confirm that unique exosomal glycoproteins can distinguish parent exosomes from others.


Assuntos
Exossomos/metabolismo , Glicoproteínas/análise , Sondas Moleculares/metabolismo , Linhagem Celular Tumoral , Óxido Ferroso-Férrico/química , Glutationa/química , Glutationa/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Nanopartículas de Magnetita/química , Sondas Moleculares/química , Proteômica/métodos , Titânio/química
7.
Org Biomol Chem ; 19(20): 4380-4396, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34037044

RESUMO

CK2 is a protein kinase that plays important roles in many physio-pathological cellular processes. As such, the development of chemical probes for CK2 has received increasing attention in the past decade with more than 40 lead compounds developed. In this review, we aim to provide the reader with a comprehensive overview of the chemical probes acting outside the highly-conserved ATP-site developed to date. Such probes belong to different classes of molecules spanning from small molecules to peptides, act with a range of mechanisms of action and some of them present themselves as promising tools to investigate the biology of CK2 and therefore develop therapeutics for many disease areas including cancer and COVID-19.


Assuntos
Caseína Quinase II/química , Caseína Quinase II/metabolismo , Sondas Moleculares/metabolismo , Animais , Biocatálise , Descoberta de Drogas , Humanos
8.
Molecules ; 26(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807474

RESUMO

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase's biology, with wide-reaching implications for drug development.


Assuntos
Caseína Quinase II/metabolismo , Sondas Moleculares/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , COVID-19 , Caseína Quinase II/química , Diclororribofuranosilbenzimidazol/química , Diclororribofuranosilbenzimidazol/farmacologia , Humanos , Sondas Moleculares/metabolismo , Naftiridinas/química , Naftiridinas/farmacologia , Fenazinas/química , Fenazinas/farmacologia , Polifenóis/química , Polifenóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia
9.
J Med Chem ; 64(8): 4396-4409, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33821652

RESUMO

Multiple diseases are at some point associated with altered endothelial function, and endothelial dysfunction (ED) contributes to their pathophysiology. Biochemical changes of the dysfunctional endothelium are linked to various cellular organelles, including the mitochondria, endoplasmic reticulum, and nucleus, so organelle-specific insight is needed for better understanding of endothelial pathobiology. Raman imaging, which combines chemical specificity with microscopic resolution, has proved to be useful in detecting biochemical changes in ED at the cellular level. However, the detection of spectroscopic markers associated with specific cell organelles, while desirable, cannot easily be achieved by Raman imaging without labeling. This critical review summarizes the current advances in Raman-based analysis of ED, with a focus on a new approach involving molecular Raman reporters that could facilitate the study of biochemical changes in cellular organelles. Finally, imaging techniques based on both conventional spontaneous Raman scattering and the emerging technique of stimulated Raman scattering are discussed.


Assuntos
Endotélio/química , Análise Espectral Raman , Vasos Sanguíneos/química , Vasos Sanguíneos/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Endotélio/metabolismo , Endotélio/fisiopatologia , Humanos , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Mitocôndrias/química , Mitocôndrias/metabolismo , Sondas Moleculares/química , Sondas Moleculares/metabolismo
10.
Int J Mol Sci ; 22(6)2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33804798

RESUMO

Sialidase cleaves sialic acid residues from glycans such as glycoproteins and glycolipids. In the brain, desorption of the sialic acid by sialidase is essential for synaptic plasticity, learning and memory and synaptic transmission. BTP3-Neu5Ac has been developed for sensitive imaging of sialidase enzyme activity in mammalian tissues. Sialidase activity in the rat hippocampus detected with BTP3-Neu5Ac increases rapidly by neuronal depolarization. It is presumed that an increased sialidase activity in conjunction with neural excitation is involved in the formation of the neural circuit for memory. Since sialidase inhibits the exocytosis of the excitatory neurotransmitter glutamate, the increased sialidase activity by neural excitation might play a role in the negative feedback mechanism against the glutamate release. Mammalian tissues other than the brain have also been stained with BTP3-Neu5Ac. On the basis of information on the sialidase activity imaging in the pancreas, it was found that sialidase inhibitor can be used as an anti-diabetic drug that can avoid hypoglycemia, a serious side effect of insulin secretagogues. In this review, we discuss the role of sialidase in the brain as well as in the pancreas and skin, as revealed by using a sialidase activity imaging probe. We also present the detection of influenza virus with BTP3-Neu5Ac and modification of BTP3-Neu5Ac.


Assuntos
Imagem Molecular , Sondas Moleculares , Neuraminidase/metabolismo , Animais , Meios de Contraste , Ativação Enzimática , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ácido Glutâmico/biossíntese , Humanos , Imagem Molecular/métodos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Neurônios/metabolismo , Imagem Óptica/métodos , Especificidade de Órgãos , Viroses/diagnóstico por imagem , Viroses/metabolismo , Viroses/virologia
11.
Biomolecules ; 11(2)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673160

RESUMO

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by the deficiency of α-galactosidase A (α-GalA) and the consequent accumulation of toxic metabolites such as globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). Early diagnosis and appropriate timely treatment of FD patients are crucial to prevent tissue damage and organ failure which no treatment can reverse. LSDs might profit from four main therapeutic strategies, but hitherto there is no cure. Among the therapeutic possibilities are intravenous administered enzyme replacement therapy (ERT), oral pharmacological chaperone therapy (PCT) or enzyme stabilizers, substrate reduction therapy (SRT) and the more recent gene/RNA therapy. Unfortunately, FD patients can only benefit from ERT and, since 2016, PCT, both always combined with supportive adjunctive and preventive therapies to clinically manage FD-related chronic renal, cardiac and neurological complications. Gene therapy for FD is currently studied and further strategies such as substrate reduction therapy (SRT) and novel PCTs are under investigation. In this review, we discuss the molecular basis of FD, the pathophysiology and diagnostic procedures, together with the current treatments and potential therapeutic avenues that FD patients could benefit from in the future.


Assuntos
Doença de Fabry , Animais , Inibidores Enzimáticos/farmacologia , Terapia de Reposição de Enzimas , Doença de Fabry/diagnóstico , Doença de Fabry/genética , Doença de Fabry/fisiopatologia , Feminino , Humanos , Masculino , Sondas Moleculares/metabolismo , Mutação , alfa-Galactosidase/antagonistas & inibidores , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo
12.
Anal Chem ; 93(7): 3502-3509, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33544570

RESUMO

Visualizing and modulating the mitophagy process is essential for understanding the role of mitophagy in cellular homeostasis, physiology, and pathology. To overcome the sensing limitation of available mitophagy probes to only lysosome fusion or degradation, a molecular logic gate probe showing multiple fluorescence responses to different mitophagy stages was proposed in this study to sense the oxidative stress-induced mitophagy via a dual-channel mode. This new fluorescent molecular logic gate probe, Mito-PN, was composed by integrating a peroxynitrite-responsive 1,8-naphthalimide with an acidity-activatable rhodamine spirolactam and possesses the mitochondria-targeting capability due to its triphenylphosphonium group. This probe is able to sense both the mitophagy initiation triggered by peroxynitrite and lysosome fusion at different fluorescence wavelengths. It can be rapidly activated by mitochondrial peroxynitrite to turn on the green fluorescence of naphthalimide, and subsequent lysosome/mitophagosome fusion activates the probe with protons to generate red fluorescence. Moreover, our preliminary results demonstrate that the fluorescence response of Mito-PN to peroxynitrite-induced mitophagy can be discriminated from the mitophagy stimulated by carbonyl cyanide m-chlorophenyl hydrazone, which further proves the specific mitophagy tracking ability of Mito-PN. Overall, this research offers a potentially powerful tool for studying the role played by peroxynitrite in mitophagy and provides a versatile strategy for monitoring oxidative stress-related pathological processes.


Assuntos
Corantes Fluorescentes , Mitofagia , Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , Sondas Moleculares/metabolismo , Estresse Oxidativo
13.
Nat Chem Biol ; 17(5): 531-539, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33526893

RESUMO

Splitting bioactive proteins into conditionally reconstituting fragments is a powerful strategy for building tools to study and control biological systems. However, split proteins often exhibit a high propensity to reconstitute, even without the conditional trigger, limiting their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific or often ineffective. Here, we report a computational design strategy grounded in fundamental protein biophysics to guide experimental evaluation of a sparse set of mutants to identify an optimal functional window. We hypothesized that testing a limited set of mutants would direct subsequent mutagenesis efforts by predicting desirable mutant combinations from a vast mutational landscape. This strategy varies the degree of interfacial destabilization while preserving stability and catalytic activity. We validate our method by solving two distinct split protein design challenges, generating both design and mechanistic insights. This new technology will streamline the generation and use of split protein systems for diverse applications.


Assuntos
Sondas Moleculares/química , Engenharia de Proteínas/métodos , Fatores de Transcrição/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Genes Reporter , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Sondas Moleculares/genética , Sondas Moleculares/metabolismo , Mutação , Multimerização Proteica , Proteólise , Sirolimo/metabolismo , Sirolimo/farmacologia , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional
14.
J Am Chem Soc ; 143(8): 3037-3042, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33596067

RESUMO

Post-translational modification of proteins with poly(ADP-ribose) (PAR) is an important component of the DNA damage response. Four PAR synthesis inhibitors have recently been approved for the treatment of breast, ovarian, and prostate cancers. Despite the clinical significance of PAR, a molecular understanding of its function, including its binding partners, remains incomplete. In this work, we synthesized a PAR photoaffinity probe that captures and isolates endogenous PAR binders. Our method identified dozens of known PAR-binding proteins and hundreds of novel candidates involved in DNA repair, RNA processing, and metabolism. PAR binding by eight candidates was confirmed using pull-down and/or electrophoretic mobility shift assays. Using PAR probes of defined lengths, we detected proteins that preferentially bind to 40-mer versus 8-mer PAR, indicating that polymer length may regulate the outcome and timing of PAR signaling pathways. This investigation produces the first census of PAR-binding proteins, provides a proteomics analysis of length-selective PAR binding, and associates PAR binding with RNA metabolism and the formation of biomolecular condensates.


Assuntos
Luz , Sondas Moleculares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteômica/métodos , Sondas Moleculares/síntese química , Sondas Moleculares/química , Transdução de Sinais
15.
Proc Natl Acad Sci U S A ; 118(9)2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33622793

RESUMO

Reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are transient species that have broad actions in signaling and stress, but spatioanatomical understanding of their biology remains insufficient. Here, we report a tandem activity-based sensing and labeling strategy for H2O2 imaging that enables capture and permanent recording of localized H2O2 fluxes. Peroxy Green-1 Fluoromethyl (PG1-FM) is a diffusible small-molecule probe that senses H2O2 by a boronate oxidation reaction to trigger dual release and covalent labeling of a fluorescent product, thus preserving spatial information on local H2O2 changes. This unique reagent enables visualization of transcellular redox signaling in a microglia-neuron coculture cell model, where selective activation of microglia for ROS production increases H2O2 in nearby neurons. In addition to identifying ROS-mediated cell-to-cell communication, this work provides a starting point for the design of chemical probes that can achieve high spatial fidelity by combining activity-based sensing and labeling strategies.


Assuntos
Corantes Fluorescentes/metabolismo , Peróxido de Hidrogênio/metabolismo , Microglia/metabolismo , Sondas Moleculares/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Animais , Ácidos Borônicos/química , Comunicação Celular , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Embrião de Mamíferos , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Sondas Moleculares/síntese química , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oxirredução , Paraquat/farmacologia , Células RAW 264.7 , Coloração e Rotulagem/métodos
16.
Nat Commun ; 12(1): 109, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397937

RESUMO

Zn2+ plays important roles in metabolism and signaling regulation. Subcellular Zn2+ compartmentalization is essential for organelle functions and cell biology, but there is currently no method to determine Zn2+ signaling relationships among more than two different organelles with one probe. Here, we report simultaneous Zn2+ tracking in multiple organelles (Zn-STIMO), a method that uses structured illumination microscopy (SIM) and a single Zn2+ fluorescent probe, allowing super-resolution morphology-correlated organelle identification in living cells. To guarantee SIM imaging quality for organelle identification, we develop a new turn-on Zn2+ fluorescent probe, NapBu-BPEA, by regulating the lipophilicity of naphthalimide-derived Zn2+ probes to make it accumulate in multiple organelles except the nucleus. Zn-STIMO with this probe shows that CCCP-induced mitophagy in HeLa cells is associated with labile Zn2+ enhancement. Therefore, direct organelle identification supported by SIM imaging makes Zn-STIMO a reliable method to determine labile Zn2+ dynamics in various organelles with one probe. Finally, SIM imaging of pluripotent stem cell-derived organoids with NapBu-BPEA demonstrates the potential of super-resolution morphology-correlated organelle identification to track biospecies and events in specific organelles within organoids.


Assuntos
Rastreamento de Células , Organelas/metabolismo , Zinco/metabolismo , Autofagossomos/metabolismo , Autofagia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Sobrevivência Celular , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Espaço Intracelular/metabolismo , Lisossomos/metabolismo , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Naftalimidas/metabolismo , Organoides/metabolismo , Espectrometria de Fluorescência
17.
Nat Commun ; 12(1): 717, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514717

RESUMO

The Neisseria meningitidis protein FrpC contains a self-processing module (SPM) undergoing autoproteolysis via an aspartic anhydride. Herein, we establish NeissLock, using a binding protein genetically fused to SPM. Upon calcium triggering of SPM, the anhydride at the C-terminus of the binding protein allows nucleophilic attack by its target protein, ligating the complex. We establish a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We optimize NeissLock using the Ornithine Decarboxylase/Antizyme complex. Various sites on the target (α-amine or ε-amines) react with the anhydride, but reaction is blocked if the partner does not dock. Ligation is efficient at pH 7.0, with half-time less than 2 min. We arm Transforming Growth Factor-α with SPM, enabling specific covalent coupling to Epidermal Growth Factor Receptor at the cell-surface. NeissLock harnesses distinctive protein chemistry for high-yield covalent targeting of endogenous proteins, advancing the possibilities for molecular engineering.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Sondas Moleculares/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos , Anidridos/metabolismo , Animais , Imagem Molecular/métodos , Sondas Moleculares/química , Sondas Moleculares/genética , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
18.
J Am Chem Soc ; 143(3): 1341-1347, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33433199

RESUMO

We have developed a novel bioorthogonal reaction that can selectively displace fluorine substitutions alpha to amide bonds. This fluorine-thiol displacement reaction (FTDR) allows for fluorinated cofactors or precursors to be utilized as chemical reporters, hijacking acetyltransferase-mediated acetylation both in vitro and in live cells, which cannot be achieved with azide- or alkyne-based chemical reporters. The fluoroacetamide labels can be further converted to biotin or fluorophore tags using FTDR, enabling the general detection and imaging of acetyl substrates. This strategy may lead to a steric-free labeling platform for substrate proteins, expanding our chemical toolbox for functional annotation of post-translational modifications in a systematic manner.


Assuntos
Acetilcoenzima A/metabolismo , Acetiltransferases/metabolismo , Sondas Moleculares/metabolismo , Compostos de Sulfidrila/química , Acetilcoenzima A/química , Acetilação , Biotina/análogos & derivados , Corantes Fluorescentes/química , Células HEK293 , Humanos , Sondas Moleculares/química , Estrutura Molecular , Estudo de Prova de Conceito , Rodaminas/química
19.
J Appl Microbiol ; 130(2): 493-503, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32738017

RESUMO

AIMS: Diagnosis of Staphylococcus aureus is important in various diseases from hospital-acquired infections to foodborne diseases. This work reports two new luminescent affiprobes for specific detection of S. aureus. METHODS AND RESULTS: To develop advanced luminescent affiprobes, enhanced green fluorescent protein (EGFP) was flanked by single and double repeats of ZpA963 affibody using molecular biology studies. The recombinant proteins including fluorescent monomeric affibody (fA1 ) and fluorescent dimeric affibody (fA2 ) were expressed in the bacterial expression system, purified and used to identify the S. aureus. Fluorescence microscope and flow cytometry results demonstrated that the treated samples with fA1 and fA2 had relatively high fluorescent mean intensities in comparison to the untreated S. aureus cells. Moreover, it was revealed that 'fA2 ' affiprobe had lower dissociation constant value (about 25-fold) and was more effective for detection of S. aureus than the 'fA1 ' affiprobe. In addition, the binding of the affiprobes for some other pathogenic bacteria i.e. Escherichia coli, Bacillus cereus, Enterococcus faecalis and Staphylococcus saprophyticus was examined. Expectedly, no cross-reaction was observed for binding the constructed affiprobes to these bacteria, eliminating possibilities for false positive results. CONCLUSIONS: The results show that 'fA1 ' affiprobe and 'fA2 ' affiprobe are two new efficient luminescent affiprobes for detecting S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a new approach for detection of Staphylococcus aureus in a simple one-step process and in low concentrations of probes. In the best of our knowledge, this is the first study to direct detection of bacterial cells by affiprobes and may be used to develop new diagnostic kits.


Assuntos
Técnicas Bacteriológicas/métodos , Citometria de Fluxo/métodos , Sondas Moleculares/metabolismo , Staphylococcus aureus/isolamento & purificação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/isolamento & purificação , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luminescência , Sondas Moleculares/genética , Sondas Moleculares/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/citologia , Staphylococcus aureus/metabolismo
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