RESUMEN
Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin-streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells.
Asunto(s)
Metaloendopeptidasas/metabolismo , Rutenio/metabolismo , Triyodotironina/metabolismo , Biotina/química , Biotina/metabolismo , Biotinilación , Catálisis , Células HEK293 , Humanos , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Estructura Molecular , Mutación Puntual , Rutenio/química , Estereoisomerismo , Estreptavidina/química , Estreptavidina/metabolismo , Triyodotironina/genéticaRESUMEN
The growth of the dietary supplement in Japanese market suggests that the patient's need for assistance with self-care will also continue to grow. Patients' burgeoning acceptance and use of alternative therapies is another indication that patients seeking more from the health care system. The questionnaire showed that the most of them are expecting the pharmacists to provide their knowledge of dietary supplements. However, only limited amount of information is available. We founded the group Alterna in 2003, that compose the pharmacists working in community pharmacies accompanied with those in pharmaceutical universities. We have published the journal named "Alterna" that includes the information of dietary supplements, and it attained Vol. 9 in 2008. In the past studies, we evaluated the content and solubility of coenzyme Q10 dietary supplements in Japanese markets, some of which showed poor solubility. In others, we had taken up the information about vitamin and minerals, tea catechins, DHA and EPA, cooking oils to reduce body fat, collagen, etc. Findings in these studies present the opportunities for the pharmacists to provide the significant positive impact on health care outcomes and costs to patients.