Ambroxol increases glucocerebrosidase (GCase) activity and restores GCase translocation in primary patient-derived macrophages in Gaucher disease and Parkinsonism.

Mutations in the glucocerebrosidase gene (GBA) encoding the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease (GD) and are the most commonly known genetic risk factor for Parkinson disease (PD). Ambroxol is one of the most effective pharmacological chaperones of GCase. Fourteen GD patients, six PD patients with mutations in the GBA gene (GBA-PD), and thirty controls were enrolled. GCase activity and hexosylsphingosine (HexSph) concentration were measured in dried blood and macrophage spots using liquid chromatography coupled with tandem mass spectrometry. The effect of ambroxol on GCase translocation to lysosomes was assessed using confocal microscopy. The results showed that ambroxol treatment significantly increased GCase activity in cultured macrophages derived from patient blood monocytic cell (PBMC) of GD (by 3.3-fold) and GBA-PD patients (by 3.5-fold) compared to untreated cells (p < 0.0001 and p < 0.0001, respectively) four days after cultivation. Ambroxol treatment significantly reduced HexSph concentration in GD (by 2.1-fold) and GBA-PD patients (by 1.6-fold) (p < 0.0001 and p < 0.0001, respectively). GD macrophage treatment resulted in increased GCase level and increased enzyme colocalization with the lysosomal marker LAMP2. The possible binding modes of ambroxol to mutant GCase carrying N370S amino acid substitution at pH 4.7 were examined using molecular docking and molecular dynamics simulations. The ambroxol position characterized by minimal binding free energy was observed in close vicinity to the residue, at position 370. Taken together, these data showed that PBMC-derived macrophages could be used for assessing ambroxol therapy response for GD patients and also for GBA-PD patients.

Thioflavin T Interaction with Acetylcholinesterase: New Evidence of 1:1 Binding Stoichiometry Obtained with Samples Prepared by Equilibrium Microdialysis.

The aim of the present work was investigation of the fluorescent dye thioflavin T (ThT) binding to acetylcholinesterase (AChE). ThT is an effective test for protease activity, as well as a probe for amyloid fibril formation. Despite the extended and active investigation of ThT-AChE binding, there is still no common view on the stoichiometry of this interaction. In particular, there is a hypothesis explaining the spectral properties of bound to AChE dye and high quantum yield of its fluorescence by formation of dimers or excimers of ThT. In order to confirm or deny this hypothesis, we proposed a new experimental approach for examination of ThT-AChE interaction based on spectroscopic investigation of samples prepared by equilibrium microdialysis. This approach allowed us to prove 1/1 ThT/AChE binding stoichiometry. The increase of ThT fluorescence quantum yield and lifetime accompanying its binding to AChE can be explained by the molecular rotor nature of this dye. Together with the coincidence of the positions of free and AChE-bound ThT fluorescence spectra, the obtained results prove the groundlessness of the hypotheses about ThT aggregation while binding to AChE. The model of ThT localization in the active site of AChE was proposed by using molecular docking simulations. These results also allowed us to suggest the key role of aromatic residues in ThT-AChE interaction, as observed for some amyloid fibrils.

Interaction with serum albumin as a factor of the photodynamic efficacy of novel bacteriopurpurinimide derivatives.

Optimization of the chemical structure of antitumor photosensitizers (PSs) is aimed at increasing their affinity to a transport protein, albumin and irreversible light-induced tumor cell damage. Bacteriopurpurinimide derivatives are promising PSs thanks to their ability to absorb light in the near infrared spectral region. Using spectrophotometry, we show that two new bacteriopurpurinimide derivatives with different substituents at the N atoms of the imide exocycle and the pyrrole ring A are capable of forming non-covalent complexes with human serum albumin (HSA). The association constant (calculated with the Benesi-Hildebrand equation) for N-ethoxybacteriopurpurinimide ethyloxime (compound 1) is higher than that for the methyl ether of methoxybacteriopurpurinimide (compound 2) (1.18×10(5) M-1 vs. 1.26×10(4) M(-1), respectively). Molecular modeling provides details of the atomic interactions between 1 and 2 and amino acid residues in the FA1 binding site of HSA. The ethoxy group stabilizes the position of 1 within this site due to hydrophobic interaction with the protein. The higher affinity of 1 for HSA makes this compound more potent than 2 in photodynamic therapy for cultured human colon carcinoma cells. Photoactivation of 1 and 2 in cells induces rapid (within a few minutes of irradiation) necrosis. This mechanism of cell death may be efficient for eliminating tumors resistant to other therapies.

[Knotted proteins].

For a long time the presence of knots in a protein structure was not admitted. However, the existence of proteins with various types of knots has now been proven. The functional significance of knotted topology remains unclear despite numerous assumptions. Studing the structure of knots in proteins and their impact on the acquisition of native structure of proteins is important for the understanding of protein folding as a whole. We review the types of knots in the proteins discovered to date, including trefoil knot, figure-of-eight knot, and more complex knots with 5 and 6 crossings of polypeptide chain. We survey the folding of knotted proteins as well.

Improvement of the efficiency of transglycosylation catalyzed by α-galactosidase from Thermotoga maritima by protein engineering.

At high concentrations of p-nitrophenyl-α-D-galactopyranoside (pNPGal) as a substrate, its hydrolysis catalyzed by α-galactosidase from Thermotoga maritima (TmGalA) is accompanied by transglycosylation resulting in production of a mixture of (α1,2)-, (α1,3)-, and (α1,6)-p-nitrophenyl (pNP)-digalactosides. Molecular modeling of the reaction stage preceding the formation of the pNP-digalactosides within the active site of the enzyme revealed amino acid residues which modification was expected to increase the efficiency of transglycosylation. Upon the site-directed mutagenesis to the predicted substitutions of the amino acid residues, genes encoding the wild type TmGalA and its mutants were expressed in E. coli, and the corresponding enzymes were isolated and tested for the presence of the transglycosylating activity in synthesis of different pNP-digalactosides. Three mutants, F328A, P402D, and G385L, were shown to markedly increase the total transglycosylation as compared to the wild type enzyme. Moreover, the F328A mutant displayed an ability to produce a regio-isomer with the (α1,2)-bond at yield 16-times higher than the wild type TmGalA.