Harnessing polyhydroxylated pyrrolidines as a stabilizer of acid alpha-glucosidase (GAA) to enhance the efficacy of enzyme replacement therapy in Pompe disease.

To discover small molecules as acid alpha-glucosidase (GAA) stabilizers for potential benefits of the exogenous enzyme treatment toward Pompe disease cells, we started from the initial screening of the unique chemical space, consisting of sixteen stereoisomers of 2-aminomethyl polyhydroxylated pyrrolidines (ADMDPs) to find out two primary stabilizers 17 and 18. Further external or internal structural modifications of 17 and 18 were performed to increase structural diversity, followed by the protein thermal shift study to evaluate the GAA stabilizing ability. Fortunately, pyrrolidine 21, possessing an l-arabino-typed configuration pattern, was identified as a specific potent rh-GAA stabilizer, enabling the suppression of rh-GAA protein denaturation. In a cell-based Pompe model, co-administration of 21 with rh-GAA protein significantly improved enzymatic activity (up to 5-fold) compared to administration of enzyme alone. Potentially, pyrrolidine 21 enables the direct increase of ERT (enzyme replacement therapy) efficacy in cellulo and in vivo.

Polyethylene glycol perturbs the unfolding of CRABP I: A correlation between experimental and theoretical approach.

The importance of macromolecules paves the way towards a detailed molecular level investigation as all most all cellular processes occurring at the interior of cells in the form of proteins, enzymes, and other biological molecules are significantly affected because of their crowding. Thus, exploring the role of crowding environment on the denaturation and renaturation kinetics of protein molecules is of great importance. Here, CRABP I (cellular retinoic acid binding protein I) is employed as a model protein along with different molecular weights of Polyethylene glycol (PEG) as molecular crowders. The experimental evaluations are done by accessing the protein secondary structure analysis using circular dichroism (CD) spectroscopy and unfolding kinetics using intrinsic fluorescence of CRABP I at 37 °C to mimic the in vivo crowding environment. The unfolding kinetics results indicated that both PEG 2000 and PEG 4000 act as stabilizers by retarding the unfolding kinetic rates. Both kinetic and stability outcomes presented the importance of crowding environment on stability and kinetics of CRABP I. The molecular dynamics (MD) studies revealed that thirteen PEG 2000 molecules assembled during the 500 ns simulation, which increases the stability and percentage of ß-sheet. The experimental findings are well supported by the molecular dynamics simulation results.

Enhancement of Wound Healing Efficacy by Increasing the Stability and Skin-Penetrating Property of bFGF Using 30Kc19α-Based Fusion Protein.

The instability of recombinant basic fibroblast growth factor (bFGF) is a major disadvantage for its therapeutic use and means frequent applications to cells or tissues are required for sustained effects. Originating from silkworm hemolymph, 30Kc19α is a cell-penetrating protein that also has protein stabilization properties. Herein, it is investigated whether fusing 30Kc19α to bFGF can enhance the stability and skin penetration properties of bFGF, which may consequently increase its therapeutic efficacy. The fusion of 30Kc19α to bFGF protein increases protein stability, as confirmed by ELISA. 30Kc19α-bFGF also retains the biological activity of bFGF as it facilitates the migration and proliferation of fibroblasts and angiogenesis of endothelial cells. It is discovered that 30Kc19α can improve the transdermal delivery of a small molecular fluorophore through the skin of hairless mice. Importantly, it increases the accumulation of bFGF and further facilitates its translocation into the skin through follicular routes. Finally, when applied to a skin wound model in vivo, 30Kc19α-bFGF penetrates the dermis layer effectively, which promotes cell proliferation, tissue granulation, angiogenesis, and tissue remodeling. Consequently, the findings suggest that 30Kc19α improves the therapeutic functionalities of bFGF, and would be useful as a protein stabilizer and/or a delivery vehicle in therapeutic applications.

Development of a simple assay system for protein-stabilizing efficiency based on hemoglobin protection against denaturation and measurement of the cooperative effect of mixing protein stabilizers.

We have elucidated the cooperative stabilization of proteins by sugars, amino acids, and other protein-stabilizing agents using a new and simple assay system. Our system determines the protein-stabilizing ability of various compounds by measuring their ability to protect hemoglobin from denaturation. Hemoglobin denaturation was readily measured by quantitative changes in its ultraviolet-visible absorption spectrum. The efficiency of our assay was confirmed using various sugars such as trehalose and sucrose that are known to be good protein stabilizers. We have also found that mixtures of two different types of protein stabilizers resulted in a cooperative stabilizing effect on protein.

Effect of urea and trimethylamine N-oxide on the binding between actin molecules.

Urea and trimethylamine N-oxide (TMAO) are known to denature and stabilize proteins, respectively. We examined two actin-binding processes, namely, end-to-end annealing of actin filaments (F-form) and the polymerization of actin monomers (G-form) into filaments, in the presence of urea, TMAO, and both solutes. Fluorescence microscopy for direct observation of actin filaments bound by fluorescent phalloidin revealed that the annealing rate constant decreased as the concentrations of urea or TMAO increased. Fluorescence spectroscopy with pyrene-labeled actin monomers showed that urea decreased the polymerization rate, whereas TMAO enhanced the rate. The decrease in the polymerization rate constant and thermal stability induced by 0.6M urea was almost completely ameliorated by the addition of 0.3M TMAO. These results suggest that TMAO-dependent stabilization of actin structure facilitates the binding of G-form actin to the ends of F-form actin filaments. Conversely, the binding between ends of mature filaments was impaired by TMAO.