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1.
Mol Immunol ; 116: 140-150, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31654938

RESUMO

BACKGROUND: Non-specific lipid transfer proteins (LTPs) are important allergens in fruits, pollen, vegetables, nuts and latex. Due to their compact structure, LTPs are highly resistant to heat treatment. Here, Art v 3 from mugwort pollen and Pru p 3 from peach were used as model allergens to in-depth investigate structural and immunological properties upon thermal treatment at different buffer conditions. METHODS: Recombinant Art v 3 and Pru p 3 were purified from E. coli and incubated at 95 °C up to 120 min using sodium phosphate buffer pH 3.4 or 7.3. Physicochemical properties of allergens were analyzed in circular dichroism spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, size exclusion chromatography, and mass spectrometry. The crystal structure of Art v 3.0201 was determined to 1.9 Šresolution. IgG and IgE binding was investigated in ELISA using murine and LTP allergic patients' sera. RESULTS: Highly pure and homogenous recombinant allergens were obtained from bacterial production. The crystal structure of Art v 3.0201 revealed an antiparallel four helix bundle with a C-terminal extension mediating an asymmetric, transient dimer interface and differently sized cavities. Both allergens showed high thermal stability at acidic conditions. In contrast, extensive heat treatment in neutral buffer induced irreversible structural changes due to lanthionine-based cysteine rearrangement. This fostered loss of the typical α-helical structure, increased molecular size and abrogation of IgG and IgE binding epitopes. Pru p 3 lost its structural integrity at shorter heat stress duration than Art v 3, which did however only partially affect the molecule's IgE binding epitopes. CONCLUSION: During thermal treatment, susceptibility to structural changes of the LTP-fold is highly dependent on the surrounding environment but also on intrinsic features of individual LTPs. This is a crucial fact to consider when processing LTP-containing food or food products as this will directly influence their allergenic potential.


Assuntos
Alanina/análogos & derivados , Antígenos de Plantas/metabolismo , Proteínas de Transporte/metabolismo , Cisteína/metabolismo , Proteínas de Plantas/metabolismo , Sulfetos/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Artemisia/metabolismo , Reações Cruzadas/fisiologia , Epitopos/metabolismo , Escherichia coli/metabolismo , Hipersensibilidade Alimentar/metabolismo , Humanos , Imunoglobulina E/metabolismo , Imunoglobulina G/metabolismo , Camundongos , Pólen/metabolismo , Prunus/metabolismo
2.
Chembiochem ; 18(12): 1083-1086, 2017 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-28425643

RESUMO

Chemical (as opposed to light-induced) activation of caged molecules is a rapidly advancing approach to trigger biological processes. We previously introduced the ruthenium-catalyzed release of allyloxycarbonyl (alloc)-protected amines in human cells. A restriction of this and all other methods is the limited lifetime of the catalyst, thus hampering meaningful applications. In this study, we addressed this problem with the development of a new generation of ruthenium complexes for the uncaging of alloc-protected amines with superior catalytic activity. Under biologically relevant conditions, we achieved a turnover number >300, a reaction rate of 580 m-1 s-1 , and we observed high activity in blood serum. Furthermore, alloc-protected doxorubicin, as an anticancer prodrug, could be activated in human cell culture and induced apoptosis with a single low dose (1 µm) of the new catalyst.


Assuntos
Compostos Alílicos/química , Aminas/química , Complexos de Coordenação/síntese química , Doxorrubicina/agonistas , Rutênio/química , Antibióticos Antineoplásicos/sangue , Antibióticos Antineoplásicos/síntese química , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Catálise , Complexos de Coordenação/sangue , Complexos de Coordenação/química , Doxorrubicina/análogos & derivados , Doxorrubicina/sangue , Doxorrubicina/farmacologia , Células HeLa , Humanos , Concentração Inibidora 50 , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Rutênio/sangue
3.
Curr Opin Chem Biol ; 25: 48-54, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25561021

RESUMO

Photolabile protecting groups have been widely used for activation strategies of caged substrates within living cells. However, an alternative uncaging method in which, instead of light, chemical compounds are used as activators (chemical uncaging) is still in its infancy. The recent advances in bioorthogonal reactions mediated by transition metals have shown that bioorthogonal catalysts have the potential to yield such a chemical activator. By now we have seen transition metal compounds that activate caged enzymes, toxigenic prodrugs and other small molecules such as fluorophores within living human cells. In this review we will focus on metal catalysts based on palladium, ruthenium and iron and we will mainly discuss their biocompatibility and catalytic efficiency in uncaging reactions within biological environments.


Assuntos
Elementos de Transição/química , Elementos de Transição/farmacologia , Animais , Sobrevivência Celular , Humanos , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Processos Fotoquímicos
4.
Angew Chem Int Ed Engl ; 53(39): 10536-40, 2014 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-25138780

RESUMO

The catalysis of bioorthogonal transformations inside living organisms is a formidable challenge--yet bears great potential for future applications in chemical biology and medicinal chemistry. We herein disclose highly active organometallic ruthenium complexes for bioorthogonal catalysis under biologically relevant conditions and inside living cells. The catalysts uncage allyl carbamate protected amines with unprecedented high turnover numbers of up to 270 cycles in the presence of water, air, and millimolar concentrations of thiols. By live-cell imaging of HeLa cells and with the aid of a caged fluorescent probe we could reveal a rapid development of intense fluorescence within the cellular cytoplasm and therefore support the proposed bioorthogonality of the catalysts. In addition, to illustrate the manifold applications of bioorthogonal catalysis, we developed a method for catalytic in-cell activation of a caged anticancer drug, which efficiently induced apoptosis in HeLa cells.


Assuntos
Complexos de Coordenação/química , Compostos Organometálicos/química , Pró-Fármacos/química , Aminas/química , Apoptose/efeitos dos fármacos , Carbamatos/química , Catálise , Complexos de Coordenação/síntese química , Complexos de Coordenação/toxicidade , Citoplasma/química , Citoplasma/metabolismo , Corantes Fluorescentes/química , Células HeLa , Humanos , Pró-Fármacos/síntese química , Pró-Fármacos/toxicidade , Rutênio/química
5.
ChemMedChem ; 8(6): 924-7, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23568508

RESUMO

Shedding light on the matter: Rhenium(I) indolato complexes with highly potent visible-light-triggered antiproliferative activity (complex 1: EC50 light=0.1 µM vs EC50 dark=100 µM) in 2D- and 3D-organized cancer cells are reported and can be traced back to an efficient generation of singlet oxygen, causing rapid morphological changes and an induction of apoptosis.


Assuntos
Antineoplásicos/farmacologia , Luz , Compostos Organometálicos/farmacologia , Rênio/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Relação Estrutura-Atividade
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