Effects of macromolecular crowding and osmolyte on human Tau fibrillation.

Tau fibrillation is reported to be involved in neurodegenerative disorders, such as Alzheimer's disease, in which the natural environment is very crowded in the cells. Understanding the role of crowding environments in regulating Tau fibrillation is of great importance for elucidating the etiology of these diseases. In this experiment, the effects of macromolecular crowding and osmolyte reagents in the crowding environment on Tau fibrillation were studied by thioflavin T binding, SDS-PAGE and TEM assays. Ficoll 70 and Dextran 70 of different concentrations were used as macromolecular crowding reagents inside the cells and showed a strong enhancing effect on the fibrillation of normal and hyperphosphorylated Tau. The enhancing effect of Dextran is stronger than that of Ficoll 70 at the same concentration. In addition, the cellular osmolyte sucrose was found to protect Tau against fibrillation, and inhibit the enhancing effect of macromolecular crowding on Tau fibrillation. A possible model for the fibrillation process of Tau and the effect of macromolecular crowding and osmolyte on this process was proposed based on these experimental results. The information obtained from our study can enhance the understanding of how proteins aggregate and avoid aggregation in crowded physiological environments and might lead to a better understanding of the molecular mechanisms of Alzheimer's disease in vivo.

Chaperone-like effects of a scFv antibody on the folding of human muscle creatine kinase.

Molecular chaperones play an essential role in assisting the folding of a myriad of nascent peptides to form different biologically active proteins. Therefore, their low substrate specificity is important for the functions of these housekeeping proteins. However, discovering chaperones which assist the folding of a particular protein can shed new light on the folding pathway of the protein, offering an interesting approach for developing specific therapeutic agents to treat protein-misfolding diseases. Screening of antibodies with chaperone-like function represents a novel strategy to meet the challenges. In this study, some single-chain variable fragment (scFv) antibodies were selected from a high-capacity phage antibody library using human muscle creatine kinase (HCK) as antigen. A scFv antibody (scFv A4) was determined to inhibit aggregation and favor recovery of the native conformation of HCK during its refolding. This antibody also increased the stability of HCK during its heat-induced unfolding process. Our findings demonstrate that scFv A4 has dual-chaperone-like activities: assisting in correct protein folding as well as protecting the native protein from unfolding. A molecular mechanism by which scFv A4 exhibits chaperone-like effects on HCK was proposed. This study demonstrates that phage antibody libraries can lead to chaperone-like proteins, and the specificity of the resulting antibody toward its antigen could provide new molecular details regarding how the chaperone interacts with the protein's unfolding and folding pathways.