Protein-Labeling Fluorescent Probe Reveals Ectodomain Shedding of Transmembrane Carbonic Anhydrases.

Ectodomain shedding is a form of limited proteolysis in which a protease cleaves a transmembrane protein, releasing the extracellular domain from the cell surface. Cells use this process to regulate a wide variety of biological events. Typically, immunological detection methods are employed for the analysis of ectodomains secreted into the cultured media. In this paper, we describe a new strategy using an affinity-based protein-labeling fluorescent probe to study ectodomain shedding. We analyzed the ectodomain shedding of cell surface carbonic anhydrases (CAIX and CAXII), which are important biomarkers for tumor hypoxia. Using both chemical and genetic approaches, we identified that the ADAM17 metalloprotease is responsible for the shedding of carbonic anhydrases. Compared to current immunological methods, this protein-labeling approach not only detects ectodomain released into the culture media but also allows real-time living cell tracking and quantitative analysis of remnant proteins on the cell surface, thereby providing a more detailed insight into the mechanism of ectodomain shedding as well as protein lifetime on the cell surface.

Small-Molecule Modulated Affinity-Tunable Semisynthetic Protein Switches.

Engineered protein switches have been widely applied in cell-based protein sensors and point-of-care diagnosis for the rapid and simple analysis of a wide variety of proteins, metabolites, nucleic acids, and enzymatic activities. Currently, these protein switches are based on two main types of switching mechanisms to transduce the target binding event to a quantitative signal, through a change in the optical properties of fluorescent molecules and the activation of enzymatic activities. In this paper, we introduce a new affinity-tunable protein switch strategy in which the binding of a small-molecule target with the protein activates the streptavidin-biotin interaction to generate a readout signal. In the absence of a target, the biotinylated protein switch forms a closed conformation where the biotin is positioned in close proximity to the protein, imposing a large steric hindrance to prevent the effective binding with streptavidin. In the presence of the target molecule, this steric hindrance is removed, thereby exposing the biotin for streptavidin binding to produce strong fluorescent signals. With this modular sensing concept, various sulfonamide, methotrexate, and trimethoprim drugs can be selectively detected on the cell surface of native and genetically engineered cells using different fluorescent dyes and detection techniques.

Cross-Sectional, Short-, Medium-, and Long-Term Effects of Dietary Pattern on Frailty in Taiwan.

This study examined the association between dietary patterns and the development of frailty during 4-, 8-, 12-year follow-up periods in the population-based Taiwan Study. We used the data of an elderly population aged 53 years and over (n = 3486) from four waves of the Taiwan Longitudinal Study on Aging. Frailty was identified by using the modified Fried criteria and the values were summed to derive a frailty score. We applied reduced rank regression to determine dietary patterns, which were divided into tertiles (healthy, general, and unhealthy dietary pattern). We used multinomial logistic regression models to assess the association between dietary patterns and the risk of frailty. The healthy dietary pattern was characterized by a higher intake of antioxidant drinks (tea), energy-rich foods (carbohydrates, e.g., rice, noodles), protein-rich foods (fish, meat, seafood, and eggs), and phytonutrient-rich foods (fruit and dark green vegetables). Compared with the healthy pattern, the unhealthy dietary pattern showed significant cross-sectional, short-term, medium-term, and long-term associations with a higher prevalence of frailty (odds ratios (OR) 2.74; 95% confidence interval (CI) 1.94-3.87, OR 2.55; 95% CI 1.67-3.88, OR 1.66; 95% CI 1.07-2.57, and OR 2.35; 95% CI 1.27-4.34, respectively). Our findings support recommendations to increase the intake of antioxidant drinks, energy-rich foods, protein-rich foods, and phytonutrient-rich foods, which were associated with a non-frail status. This healthy dietary pattern can help prevent frailty over time in elderly people.