Structural and Folding Dynamic Properties of the T70N Variant of Human Lysozyme

Definition of the transition mechanism from the native globular protein into fibrillar polymer was greatly improved by the biochemical and biophysical studies carried out on the two amyloidogenic variants of human lysozyme, I56T and D67H. Here we report thermodynamic and kinetic data on folding as well as structural features of a naturally occurring variant of human lysozyme, T70N, which is present in the British population at an allele frequency of 5% and, according to clinical and histopathological data, is not amyloidogenic. This variant is less stable than the wild-type protein by 3.7 kcal/mol, but more stable than the pathological, amyloidogenic variants. Unfolding kinetics in guanidine are six times faster than in the wild-type, but three and twenty times slower than in the amyloidogenic variants. Enzyme catalytic parameters, such as maximal velocity and affinity, are reduced in comparison to the wild-type. The solution structure, determined by 1H NMR and modeling calculations, exhibits a more compact arrangement at the interface between the beta-sheet domain and the subsequent loop on one side and part of the alpha domain on the other side, compared with the wild-type protein. This is the opposite of the conformational variation shown by the amyloidogenic variant D67H, but it accounts for the reduced stability and catalytic performance of T70N

Protein Aggregation

Protein aggregation occurs in vivo as a result of improper folding or misfolding. Diverse diseases arise from protein misfolding and are now grouped under the term "protein conformational diseases", including most of the neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, the prion encephalopathies and Huntington's disease, as well as cystic fibrosis, sickle cell anemia and other less common conditions. The hallmark event in these diseases is a change in the secondary and/or tertiary structure of a normal, functional protein, leading to the formation of protein aggregates with various supramolecular organizations. In most cases the aggregates are organized in structurally well-defined fibrils forming amyloid deposits. The crucial feature of the amyloidogenic proteins is their structural instability induced either by mutations, post-translational modifications, or local conditions, such as pH, temperature, and co-solutes. The conformational change may promote the disease either by gain of a toxic activity or by the lack of biological function of the natively folded protein. As different molecular mechanisms are involved in the formation of the various forms of protein aggregates, the laboratory diagnostic approach remains frequently elusive

Amyloid fibrils derived from the apolipoprotein A1 Leu174Ser variant contain elements of ordered helical structure

We recently described a new apolipoprotein A1 variant presenting a Leu174Ser replacement mutation that is associated with a familial form of systemic amyloidosis displaying predominant heart involvement. We have now identified a second unrelated patient with very similar clinical presentation and carrying the identical apolipoprotein A1 mutation. In this new patient the main protein constituent of the amyloid fibrils is the polypeptide derived from the first 93 residues of the protein, the identical fragment to that found in the patient previously described to carry this mutation. The X-ray fiber diffraction pattern obtained from preparations of partially aligned fibrils displays the cross-β reflections characteristic of all amyloid fibrils. In addition to these cross-β reflections, other reflections suggest the presence of well-defined coiled-coil helical structure arranged with a defined orientation within the fibrils. In both cases the fibrils contain a trace amount of full-length apolipoprotein A1 with an apparent prevalence of the wild-type species over the variant protein. We have found a ratio of full-length wild-type to mutant protein in plasma HDL of three to one. The polypeptide 1–93 purified from natural fibrils can be solubilized in aqueous solutions containing denaturants, and after removal of denaturants it acquires a monomeric state that, based on CD and NMR studies, has a predominantly random coil structure. The addition of phospholipids to the monomeric form induces the formation of some helical structure, thought most likely to occur at the C-terminal end of the polypeptide

In vitro evaluation of the wound healing activity of Drypetes klainei stem bark extracts

Ethnopharmacological relevance Drypetes klainei Pierre ex Pax is used in Cameroon by Baka Pygmies in the wound healing process and for the treatment of burns. Aim of the study To validate the traditional use of D. klainei Pierre ex Pax stem bark extracts through the evaluation of their antimicrobial properties and their ability to improve wound healing process in fibroblast cell cultures. Materials and methods The antimicrobial properties of D. klainei extracts were evaluated against Staphylococcus aureus ATCC 6538, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 10536, Candida albicans ATCC 10231, on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal–fungicidal concentration (MBC–MFC) by the macrodilution method. The extracts abilities to accelerate wound healing were studied on murine and human fibroblasts in terms of cell viability and migration (scratch wound-healing assay). Results All the extracts were non-toxic against the selected microorganisms at the tested concentrations, and significantly improve wound healing process in vitro, compared to untreated controls. However, the defatted methanol extract was active at lower concentrations, compared to the water extract. Conclusions The ability of both water and defatted methanol extracts to accelerate scratch wound closure in fibroblast cultures may support the traditional use of D. klainei stem bark in the treatment of skin lesions (such as burns) even if no antimicrobial activity was evidenced

Structural and folding dynamics properties of T70N variant of human lysozyme.

Definition of the transition mechanism from the native globular protein into fibrillar polymer was greatly improved by the biochemical and biophysical studies carried out on the two amyloidogenic variants of human lysozyme, I56T and D67H. Here we report thermodynamic and kinetic data on folding as well as structural features of a naturally occurring variant of human lysozyme, T70N, which is present in the British population at an allele frequency of 5% and, according to clinical and histopathological data, is not amyloidogenic. This variant is less stable than the wild-type protein by 3.7 kcal/mol, but more stable than the pathological, amyloidogenic variants. Unfolding kinetics in guanidine are six times faster than in the wild-type, but three and twenty times slower than in the amyloidogenic variants. Enzyme catalytic parameters, such as maximal velocity and affinity, are reduced in comparison to the wild-type. The solution structure, determined by 1H NMR and modeling calculations, exhibits a more compact arrangement at the interface between the beta-sheet domain and the subsequent loop on one side and part of the alpha domain on the other side, compared with the wild-type protein. This is the opposite of the conformational variation shown by the amyloidogenic variant D67H, but it accounts for the reduced stability and catalytic performance of T70N

Search for Bc+→π+μ+μ−B_c^+\to\pi^+\mu^+\mu^- decays and measurement of the branching fraction ratio B(Bc+→ψ(2S)π+)/B(Bc+→J/ψπ+){\cal B}(B_c^+\to\psi(2S)\pi^+)/{\cal B}(B_c^+\to J/\psi \pi^+)

International audienceThe first search for nonresonant $B_c^+\to\pi^+\mu^+\mu^-$ decays is reported. The analysis uses proton-proton collision data collected with the LHCb detector between 2011 and 2018, corresponding to an integrated luminosity of 9 fb$^{-1}$. No evidence for an excess of signal events over background is observed and an upper limit is set on the branching fraction ratio ${\cal B}(B_c^+\to\pi^+\mu^+\mu^-)/{\cal B}(B_c^+\to J/\psi \pi^+) < 2.1\times 10^{-4}$ at $90\%$ confidence level. Additionally, an updated measurement of the ratio of the $B_c^+\to\psi(2S)\pi^+$ and $B_c^+\to J/\psi \pi^+$ branching fractions is reported. The ratio ${\cal B}(B_c^+\to\psi(2S)\pi^+)/{\cal B}(B_c^+\to J/\psi \pi^+)$ is measured to be $0.254\pm 0.018 \pm 0.003 \pm 0.005$, where the first uncertainty is statistical, the second systematic, and the third is due to the uncertainties on the branching fractions of the leptonic $J/\psi$ and $\psi(2S)$ decays. This measurement is the most precise to date and is consistent with previous LHCb results