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1.
Sci Signal ; 14(675)2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758061

RESUMEN

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.


Asunto(s)
/metabolismo , Señalización del Calcio/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , NADP/análogos & derivados , /fisiología , Marcadores de Afinidad , Animales , Canales de Calcio/metabolismo , Proteínas Portadoras/metabolismo , Química Clic/métodos , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , NADP/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero Secundario/fisiología , Transcriptoma , Internalización del Virus
2.
Methods Mol Biol ; 2266: 3-10, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33759118

RESUMEN

This chapter provides a brief overview of the applications of ZINClick virtual library. In the last years, we have investigated the click-chemical space covered by molecules containing the triazole ring and generated a database of 1,2,3-triazoles called ZINClick, starting from literature reported alkynes and azides synthesizable in no more than three synthetic steps from commercially available products. This combinatorial database contains millions of 1,4-disubstituted 1,2,3-triazoles that are easily synthesizable. The library is regularly updated and can be freely downloaded from http://www.ZINClick.org . This virtual library is a good starting point to explore a new portion of chemical space.


Asunto(s)
Química Clic/métodos , Descubrimiento de Drogas/métodos , Triazoles/química , Alquinos/química , Azidas/química , Química Clic/instrumentación , Bases de Datos de Compuestos Químicos , Diseño de Fármacos , Bibliotecas de Moléculas Pequeñas/química , Programas Informáticos
3.
Molecules ; 26(4)2021 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-33669256

RESUMEN

O-GlcNAcylation is a posttranslational modification that occurs at serine and threonine residues of protein substrates by the addition of O-linked ß-d-N-acetylglucosamine (GlcNAc) moiety. Two enzymes are involved in this modification: O-GlcNac transferase (OGT), which attaches the GlcNAc residue to the protein substrate, and O-GlcNAcase (OGA), which removes it. This biological balance is important for many biological processes, such as protein expression, cell apoptosis, and regulation of enzyme activity. The extent of this modification has sparked interest in the medical community to explore OGA and OGT as therapeutic targets, particularly in degenerative diseases. While some OGA inhibitors are already in phase 1 clinical trials for the treatment of Alzheimer's disease, OGT inhibitors still have a long way to go. Due to complex expression and instability, the discovery of potent OGT inhibitors is challenging. Over the years, the field has grappled with this problem, and scientists have developed a number of techniques and assays. In this review, we aim to highlight assays and techniques for OGT inhibitor discovery, evaluate their strength for the field, and give us direction for future bioassay methods.


Asunto(s)
Bioensayo/métodos , N-Acetilglucosaminiltransferasas/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Fenómenos Biofísicos , Química Clic , Unión Proteica
4.
Molecules ; 26(4)2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33670633

RESUMEN

The development of new and greener approaches to organic synthesis has been a trend in recent years. Continuing the latest publications of our team, in this work, we demonstrate the efficiency of three solvents: eucalyptol (1,8-cineole), cyclopentyl methyl ether (CPME), and 2-methyltetrahydrofuran (2-MeTHF) for the synthesis of O,S,N-heterocyclic compounds.


Asunto(s)
Química Clic/métodos , Tecnología Química Verde , Compuestos Heterocíclicos/síntesis química , Metales/química , Solventes/química , Compuestos Heterocíclicos/química
5.
Carbohydr Polym ; 260: 117812, 2021 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-33712157

RESUMEN

A dual pH-/thermo-responsive hydrogel was designed based on a polyelectrolyte complex of polyacrylic acid (PAA) and norbornene-functionalized chitosan (CsNb), which was synergized with chemical crosslinking using bistetrazine-poly(N-isopropyl acrylamide) (bisTz-PNIPAM). The thermo-responsive polymeric crosslinker, bisTz-PNIPAM, was synthesized via reversible addition-fragmentation transfer polymerization of NIPAM. FTIR, XRD, rheological and morphological analyses demonstrated the successful formation of the polyelectrolyte network. The highly porous structure generated through the in-situ "click" reaction between Tz and Nb resulted in a higher drug loading (29.35 %). The hydrogel (COOH/NH2 mole ratio of 3:1) exhibited limited drug release (8.5 %) of 5-ASA at a pH of 2.2, but it provided an almost complete release (92 %) at pH 7.4 and 37 °C within 48 h due to the pH responsiveness of PAA, hydrogel porosity, and shrinkage behavior of PNIPAM. The hydrogels were biodegradable and non-toxic against human fibroblast cells, suggesting their considerable potential for a colon-targeted drug delivery system.


Asunto(s)
Quitosano/química , Portadores de Fármacos/química , Hidrogeles/química , Resinas Acrílicas/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Química Clic , Portadores de Fármacos/toxicidad , Liberación de Fármacos , Humanos , Hidrogeles/farmacología , Concentración de Iones de Hidrógeno , Mesalamina/química , Mesalamina/metabolismo , Porosidad , Temperatura
6.
Nat Protoc ; 16(4): 2131-2157, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33686219

RESUMEN

The stable maintenance of DNA methylation patterns during mitotic cell division is crucial for cell identity. Precisely determining the maintenance kinetics and dissecting the exact contributions of relevant regulators requires a method to accurately measure parent and daughter strand DNA methylation at the same time, ideally at the single-molecule level. Recently, we developed a method referred to as Hammer-seq (hairpin-assisted mapping of methylation of replicated DNA) that fulfils the above criteria. This method integrates 5-ethynyl-2'-deoxyuridine (EdU) labeling of replicating DNA, biotin conjugation and streptavidin-based affinity purification, and whole-genome hairpin bisulfite sequencing technologies. Hammer-seq offers the unique advantage of simultaneously measuring the methylation status of parent and daughter strands within a single DNA molecule, which makes it possible to determine maintenance kinetics across various genomic regions without averaging effects from bulk measurements and to assess de novo methylation events that accompany methylation maintenance. Importantly, when combined with mutant cell lines in which mechanisms of interest are disrupted, Hammer-seq can be applied to determine the functional contributions of potential regulators to methylation maintenance, with accurate kinetics information that cannot be acquired with other currently available methods. Hammer-seq library preparation requires ~100 ug EdU-labeled genomic DNA as input (~15 million mammalian cells). The whole protocol, from pulse labeling to library construction, can be completed within 2-3 d, depending on the chasing time.


Asunto(s)
Metilación de ADN/genética , Replicación del ADN/genética , Imagen Individual de Molécula , Biotina/metabolismo , Química Clic , ADN/metabolismo , Células HeLa , Humanos , Ligandos , Sonicación
7.
Molecules ; 26(3)2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33573040

RESUMEN

In an effort to improve and achieve biologically active anticancer agents, a novel series of 1,2,3-triazole-containing hybrids were designed and efficiently synthesized via the Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reaction of substituted-arylazides with alkyne-functionalized pyrazole-[1,2,4]-triazole hybrids. The structure geometry of these new clicked 1,2,3-triazoles was explored by density functional theory (DFT) using the B3LYP/6-311++G(d,p) level; also, the potential activity of the compounds for light absorption was simulated by time-dependent DFT calculations (TD-DFT). The antitumor impacts of the newly synthesized compounds were in vitro estimated to be towards the human liver cancer cell line (HepG-2), the human colon cancer cell line (HCT-116), and human breast adenocarcinoma (MCF-7). Among the tested compounds, conjugate 7 was the most potent cytotoxic candidate towards HepG-2, HCT-116, and MCF-7, with IC50 = 12.22, 14.16, and 14.64 µM, respectively, in comparison to that exhibited by the standard drug doxorubicin (IC50 = 11.21, 12.46, and 13.45 µM). Finally, a molecular docking study was conducted within the epidermal growth factor receptor (EGFR) active site to suggest possible binding modes. Hence, it could conceivably be hypothesized that analogies 7, 6, and 5 could be considered as decent lead candidate compounds for anticancer agents.


Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Triazoles/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Química Clic , Reacción de Cicloadición , Teoría Funcional de la Densidad , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/farmacología
8.
Bioresour Technol ; 324: 124689, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33450627

RESUMEN

A method for specific immobilization of whole-cell with covalent bonds was developed through a click reaction between alkyne and azide groups. In this approach, magnetic nanoparticle Fe3O4@SiO2-NH2-alkyne was synthesized with Fe3O4 core preparation, SiO2 coating, and alkyne functionalization on the surface. The azides were successfully integrated onto the cell surface of the recombinant E. coli harboring glycerol dehydrogenase, which was employed as the model cell. The highest immobilization yield of 83% and activity recovery of 94% were obtained under the conditions of 0.67 mg mg-1 cell-support ratio, pH 6.0, temperature 45 °C, and 20 mM Cu2+ concentration. The immobilized cell showed good reusability, which remained over 50% of initial activity after 10 cycles of utilization. Its activity was 9.7-fold higher than that of the free cell at the condition of pH 8.0 and each optimal temperature. Furthermore, the immobilized cell showed significantly higher activity, operational stability, and reusability.


Asunto(s)
Enzimas Inmovilizadas , Nanopartículas de Magnetita , Azidas , Química Clic , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Polisacáridos , Dióxido de Silicio
9.
Molecules ; 26(1)2021 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-33466477

RESUMEN

Continued expansion of the chemical biology toolbox presents many new and diverse opportunities to interrogate the fundamental molecular mechanisms driving complex plant-microbe interactions. This review will examine metabolic labeling with click chemistry reagents and activity-based probes for investigating the impacts of plant-associated microbes on plant growth, metabolism, and immune responses. While the majority of the studies reviewed here used chemical biology approaches to examine the effects of pathogens on plants, chemical biology will also be invaluable in future efforts to investigate mutualistic associations between beneficial microbes and their plant hosts.


Asunto(s)
Interacciones Huésped-Patógeno , Microbiota , Fenómenos Fisiológicos de las Plantas , Plantas/metabolismo , Plantas/microbiología , Química Clic
10.
ACS Appl Mater Interfaces ; 13(3): 4711-4722, 2021 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-33444000

RESUMEN

Realization of robust and facile surface functionalization processes is critical to biomaterials and biotechnology yet remains a challenge. Here, we report a new chemical approach that enables operationally simple and site-specific surface functionalization. The mechanism involves a catechol-copper redox chemistry, where the oxidative polymerization of an alkynyl catecholamine reduces Cu(II) to Cu(I), which in situ catalyzes a click reaction with azide-containing molecules of interest (MOIs). This process enables drop-coating and grafting of two- and three-dimensional solid surfaces in a single operation using as small as sub-microliter volumes. Generalizability of the method is shown for immobilizing MOIs of diverse structure and chemical or biological activity. Biological applications in anti-biofouling, cellular adhesion, scaffold seeding, and tissue regeneration are demonstrated, in which the activities or fates of cells are site-specifically manipulated. This work advances surface chemistry by integrating simplicity and precision with multipurpose surface functionalization.


Asunto(s)
Azidas/química , Materiales Biocompatibles/química , Catecolaminas/química , Cobre/química , Células 3T3 , Animales , Azidas/síntesis química , Materiales Biocompatibles/síntesis química , Incrustaciones Biológicas/prevención & control , Catálisis , Catecolaminas/síntesis química , Química Clic , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Oxidación-Reducción , Polimerizacion , Propiedades de Superficie
11.
Nat Protoc ; 16(2): 1193-1218, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33442052

RESUMEN

The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.


Asunto(s)
Replicación del ADN/fisiología , ADN/análisis , Química Clic/métodos , ADN/genética , Replicación del ADN/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Estreptavidina
12.
Anal Chem ; 93(4): 2694-2705, 2021 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-33397101

RESUMEN

Glycoproteins secreted by cells play essential roles in the regulation of extracellular activities. Secreted glycoproteins are often reflective of cellular status, and thus glycoproteins from easily accessible bodily fluids can serve as excellent biomarkers for disease detection. Cultured cells have been extensively employed as models in the research fields of biology and biomedicine, and global analysis of glycoproteins secreted from these cells provides insights into cellular activities and glycoprotein functions. However, comprehensive identification and quantification of secreted glycoproteins is a daunting task because of their low abundances compared with the high-abundance serum proteins required for cell growth and proliferation. Several studies employed serum-free media to analyze secreted proteins, but it has been shown that serum starvation, even for a short period of time, can alter protein secretion. To overcome these issues, we developed a method to globally characterize secreted glycoproteins and their N-glycosylation sites from cultured cells by combining selective enrichment of secreted glycoproteins with a boosting approach. The results demonstrated the importance of the boosting sample selection and the boosting-to-sample ratio for improving the coverage of secreted glycoproteins. The method was applied to globally quantify secreted glycoproteins from THP-1 monocytes and macrophages in response to lipopolysaccharides (LPS) and from Hep G2 cells treated with TGF-ß without serum starvation. We found differentially secreted glycoproteins in these model systems that showed the cellular response to the immune activation or the epithelial-to-mesenchymal transition. Benefiting from the selective enrichment and the signal enhancement of low-abundance secreted glycoproteins, this method can be extensively applied to study secreted glycoproteins without serum starvation, which will provide a better understanding of protein secretion and cellular activity.


Asunto(s)
Glicoproteínas/química , Técnicas de Cultivo de Célula , Química Clic , Glicoproteínas/metabolismo , Células Hep G2 , Humanos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Péptidos/química , Factor de Crecimiento Transformador beta/farmacología
13.
Anal Chem ; 93(4): 2610-2618, 2021 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-33470097

RESUMEN

Mass-spectrometry-based chemoproteomics has enabled the rapid and proteome-wide discovery of functional and potentially 'druggable' hotspots in proteins. While numerous transformations are now available, chemoproteomic studies still rely overwhelmingly on copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or 'click' chemistry. The absence of bio-orthogonal chemistries that are functionally equivalent and complementary to CuAAC for chemoproteomic applications has hindered the development of multiplexed chemoproteomic platforms capable of assaying multiple amino acid side chains in parallel. Here, we identify and optimize Suzuki-Miyaura cross-coupling conditions for activity-based protein profiling and mass-spectrometry-based chemoproteomics, including for target deconvolution and labeling site identification. Uniquely enabled by the observed orthogonality of palladium-catalyzed cross-coupling and CuAAC, we combine both reactions to achieve dual labeling. Multiplexed targeted deconvolution identified the protein targets of bifunctional cysteine- and lysine-reactive probes.


Asunto(s)
Alquinos/química , Azidas/química , Cobre/química , Reacción de Cicloadición/métodos , Proteómica/métodos , Catálisis , Química Clic , Células HEK293 , Humanos , Estructura Molecular
14.
Molecules ; 26(2)2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33467746

RESUMEN

A synthesis of a series of mono-T8 and difunctionalized double-decker silsesquioxanes bearing substituted triazole ring(s) has been reported within this work. The catalytic protocol for their formation is based on the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) process. Diverse alkynes were in the scope of our interest-i.e., aryl, hetaryl, alkyl, silyl, or germyl-and the latter was shown to be the first example of terminal germane alkyne which is reactive in the applied process' conditions. From the pallet of 15 compounds, three of them with pyridine-triazole and thiophenyl-triazole moiety attached to T8 or DDSQ core were verified in terms of their coordinating properties towards selected transition metals, i.e., Pd(II), Pt(II), and Rh(I). The studies resulted in the formation of four SQs based coordination compounds that were obtained in high yields up to 93% and their thorough spectroscopic characterization is presented. To our knowledge, this is the first example of the DDSQ-based molecular complex possessing bidentate pyridine-triazole ligand binding two Pd(II) ions.


Asunto(s)
Compuestos de Organosilicio/síntesis química , Paladio/química , Piridinas/química , Siloxanos/química , Triazoles/química , Catálisis , Química Clic , Reacción de Cicloadición
15.
Methods Mol Biol ; 2230: 357-365, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33197025

RESUMEN

Identifying and tracking proliferating and quiescent cells in situ is an important phenotyping component of skeletal tissues in development, physiology and disease. Among all the methods that exist, which include immunostaining for cell cycle-specific proteins, the gold standards use thymidine analogs. These compounds label proliferating cells by being incorporated into de novo-synthesized genomic DNA. 5-bromo-2'-deoxyuridine (BrdU) has traditionally been used for this purpose, but its detection is lengthy and requires harsh treatment of tissue sections to give access of anti-BrdU antibody to DNA. An alternative, more recently developed, uses 5-ethynyl-2'-deoxyuridine (EdU). This thymidine analog is detected by click chemistry, that is, covalent cross-linking of its ethynyl group with a fluorescent azide that is small enough to easily penetrate native tissues and reach DNA. In addition to being simple and quick, this EdU-based assay is compatible with other protocols, such as immunostaining, on the same tissue sections. We here describe an EdU-based protocol optimized to label and functionally assess actively proliferating cells as well as slowly dividing cells, including stem cells, in mouse skeletal tissues.


Asunto(s)
Desarrollo Óseo/efectos de los fármacos , Huesos/ultraestructura , Proliferación Celular/efectos de los fármacos , Coloración y Etiquetado/métodos , Animales , Huesos/efectos de los fármacos , Química Clic/métodos , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacología , Citometría de Flujo/métodos , Ratones
16.
Methods Mol Biol ; 2213: 147-161, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33270200

RESUMEN

Interdisciplinary chemical proteomics approaches have been widely applied to the identification of specific targets of bioactive small molecules or drugs. In this chapter, we describe the application of a cell-permeable activity-based curcumin probe (Cur-P) with an alkyne moiety to detect and identify specific binding targets of curcumin in HCT116 colon cancer cells. Through click chemistry, a fluorescent tag or a biotin tag is attached to the probe-modified curcumin targets for visualization or affinity purification followed by mass spectrometric identification. A quantitative proteomics approach of isobaric tags for relative and absolute quantification (iTRAQ)™ is applied to distinguish specific curcumin targets from nonspecific binding proteins.


Asunto(s)
Antineoplásicos/farmacología , Curcumina/farmacología , Proteómica/métodos , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Química Clic , Electroforesis en Gel de Poliacrilamida , Fluorescencia , Células HCT116 , Humanos , Marcaje Isotópico , Nanotecnología , Péptidos/metabolismo , Rodaminas , Estreptavidina/química , Espectrometría de Masas en Tándem , Tripsina/metabolismo
17.
Methods Mol Biol ; 2219: 163-180, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33074540

RESUMEN

Many species of aquatic worms, including members of the phyla Nemertea, Annelida, Platyhelminthes, and Xenacoelomorpha, can regenerate large parts of their body after amputation. In most species, cell proliferation plays key roles in the reconstruction of lost tissues. For example, in annelids and flatworms, inhibition of cell proliferation by irradiation or chemicals prevents regeneration. Cell proliferation also plays crucial roles in growth, body patterning (e.g., segmentation) and asexual reproduction in many groups of aquatic worms. Cell proliferation dynamics in these organisms can be studied using immunohistochemical detection of proteins expressed during proliferation-associated processes or by incorporation and labeling of thymidine analogues during DNA replication. In this chapter, we present protocols for labeling and quantifying cell proliferation by (a) antibody-based detection of either phosphorylated histone H3 during mitosis or proliferating cell nuclear antigen (PCNA) during S-phase, and (b) incorporation of two thymidine analogues, 5'-bromo-2'-deoxyuridine (BrdU) and 5'-ethynyl-2'-deoxyuridine (EdU), detected by immunohistochemistry or inorganic "click" chemistry, respectively. Although these protocols have been developed for whole mounts of small (<2 cm) marine and freshwater worms, they can also be adapted for use in larger specimens or tissue sections.


Asunto(s)
Anélidos/fisiología , Platelmintos/fisiología , Animales , Anélidos/citología , Ciclo Celular , Proliferación Celular , Química Clic/métodos , Inmunohistoquímica/métodos , Platelmintos/citología , Regeneración , Fijación del Tejido/métodos
18.
Int J Biol Macromol ; 169: 51-59, 2021 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-33333092

RESUMEN

The essential human O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme responsible for modifying thousands of intracellular proteins with the monosaccharide O-GlcNAc. This unique modification plays crucial roles in human health and disease, but the substrate recognition of OGT remains poorly understood. Intriguingly, the only human enzyme reported to remove this modification, O-GlcNAcase (OGA), is O-GlcNAc modified. Here, we exploited a GlcNAc electrophilic probe (GEP1A) to rapidly screen OGT mutants in a fluorescence assay that can discriminate between altered OGT-sugar and -protein substrate binding to help elucidate the binding mode of OGT toward OGA protein substrate. Since OGT tetratricopeptide repeat (TPR) domain plays a key role in OGT-OGA binding, we screened 30 OGT TPR mutants, which revealed 15 "ladder like" asparagine or aspartate residues spanning TPRs 3-7 and 10-13.5 that affect OGA O-GlcNAcylation. By applying a truncated OGA construct, we found that OGA's N-terminal region or pseudo histone acetyltransferase domain is not required for its O-GlcNAcylation, suggesting OGT functionally interacts with OGA through its catalytic and/or stalk domains. This work represents the first effort to systemically investigate each OGT TPR and our findings will facilitate the development of new strategies to investigate the role of substrate-specific O-GlcNAcylation.


Asunto(s)
N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/metabolismo , beta-N-Acetilhexosaminidasas/química , Acetilglucosamina/metabolismo , Química Clic/métodos , Humanos , N-Acetilglucosaminiltransferasas/ultraestructura , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , beta-N-Acetilhexosaminidasas/metabolismo , beta-N-Acetilhexosaminidasas/ultraestructura
19.
Carbohydr Polym ; 251: 117101, 2021 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-33142639

RESUMEN

Numbers of UV crosslinkable chitosan hydrogels through chemical modification had drawn increasing attention, however most of these chitosan hydrogels lost the pH-responsive performance because plenty of amino groups (‒NH2) in chitosan were consumed by reacting with other functional groups. To construct a pH-responsive UV-crosslinkable chitosan hydrogel for active modulating drug release with desired behavior, C6-OH selectively modified chitosan via protection/deprotection strategy to amino groups was synthesized, the allyl groups on C6 site and amino groups on C2 site endowed chitosan with UV crosslinking capability and pH responsiveness, respectively. Rapid UV crosslinking gelation (30 s) with low-dose UV irradiation (4 mW/cm2) via "thiol-ene" click chemistry were demonstrated for the patterned microgel and in-situ formed hydrogel in vivo. The swelling and shrinkage of hydrogel could active modulate the opposite release behaviors of doxorubicin (DOX) and bovine serum albumin (BSA) in different pH medium. The smart UV-crosslinkable chitosan hydrogel via click chemistry might provide a new drug carrier for active modulating opposite drug release behaviors.


Asunto(s)
Quitosano/química , Química Clic/métodos , Hidrogeles/química , Compuestos de Sulfhidrilo/química , Animales , Supervivencia Celular , Reactivos de Enlaces Cruzados/química , Doxorrubicina/química , Sistemas de Liberación de Medicamentos/métodos , Liberación de Fármacos , Fibroblastos/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Albúmina Sérica Bovina/metabolismo , Inhibidores de Topoisomerasa II/metabolismo , Rayos Ultravioleta
20.
Methods Mol Biol ; 2192: 159-181, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33230773

RESUMEN

Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial factories, whether these align with incoming polypeptides, and if there is evidence for co-translational translation that is regulated and limited by the interaction of the incoming proteins with synthesis of their mtDNA-encoded partners. Two methods are described in this chapter to visualize the distribution of mitochondrial ribosomal RNAs in conjunction with newly synthesized mitochondrial proteins. The first combines RNA Fluorescent In Situ Hybridization (FISH) and super-resolution immunocytochemistry to pinpoint mitochondrial ribosomal RNA. The second localizes nascent translation within the mitochondrial network through non-canonical amino acid labeling, click chemistry and fluorescent microscopy.


Asunto(s)
Química Clic/métodos , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Proteínas Mitocondriales/metabolismo , Ribosomas Mitocondriales/metabolismo , ARN Mitocondrial/metabolismo , ARN Ribosómico/metabolismo , Aminoácidos/química , Línea Celular Tumoral , ADN Mitocondrial/genética , Humanos , Microscopía Fluorescente/métodos , Fosforilación Oxidativa , Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo
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