Search results for "TLE"
showing 10 items of 2417 documents
Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C
2014
Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad ran…
Cryo-negative staining
1998
Abstract A procedure is presented for the preparation of thin layers of vitrified biological suspensions in the presence of ammonium molybdate, which we termcryo-negative staining. The direct blotting of sample plus stain solution on holey carbon supports produces thin aqueous films across the holes, which are routinely thiner than the aqueous film produced by conventional negative staining on a continuous carbon layer. Because of this, a higher than usual concentration of negative stain (ca. 16% rather than 2%) is required for cryo-negative staining in order to produce an optimal image contrast. The maintenance of the hydrated state, the absence of adsorption to a carbon film and associate…
Bortezomib Partially Improves Laminin α2 Chain–Deficient Muscular Dystrophy
2014
Congenital muscular dystrophy, caused by mutations in LAMA2 (the gene encoding laminin α2 chain), is a severe and incapacitating disease for which no therapy is yet available. We have recently demonstrated that proteasome activity is increased in laminin α2 chain-deficient muscle and that treatment with the nonpharmaceutical proteasome inhibitor MG-132 reduces muscle pathology in laminin α2 chain-deficient dy(3K)/dy(3K) mice. Here, we explore the use of the selective and therapeutic proteasome inhibitor bortezomib (currently used for treatment of relapsed multiple myeloma and mantle cell lymphoma) in dy(3K)/dy(3K) mice and in congenital muscular dystrophy type 1A muscle cells. Outcome measu…
Optimization of peptidomimetic boronates bearing a P3 bicyclic scaffold as proteasome inhibitors
2014
Abstract A new series of pseudopeptide boronate proteasome inhibitors (2–3) was developed, through optimization of our previously described analogs of bortezomib, bearing a bicyclic 1,6-naphthyridin-5(6H)-one scaffold as P3 fragment (1). The biological evaluation on human 20S proteasome displayed a promising inhibition profile, especially for compounds bearing a P2 ethylene fragment, which exhibited Ki values in the nanomolar range for the ChT-L activity (e.g. 2a, Ki = 0.057 μM) and considerable selectivity for proteasome over bovine pancreatic α-chymotrypsin. Docking experiments into the yeast 20S proteasome revealed that the ligands are accommodated predominantly into the ChT-L site and t…
Aggregation Kinetics of Bovine Serum Albumin Studied by FTIR Spectroscopy and Light Scattering
2003
To investigate which type of structural and conformational changes is involved in the aggregation processes of bovine serum albumin (BSA), we have performed thermal aggregation kinetics in D(2)O solutions of this protein. The tertiary conformational changes are followed by Amide II band, the secondary structural changes and the formation of beta-aggregates by the Amide I' band and, finally, the hydrodynamic radius of aggregates by dynamic light scattering. The results show, as a function of pD, that: tertiary conformational changes are more rapid as pD increases; the aggregation proceeds through formation of ordered aggregates (oligomers) at pD far from the isoelectric point of the protein;…
Conformational changes involved in thermal aggregation processes of bovine serum albumin
2003
We report a kinetic study on thermal aggregation process of the model protein bovine serum albumin (BSA) in low concentration regime. Aim of this study is to provide information on relationship between conformational changes and initial step of aggregation. The experimental approach is based on steady-state fluorescence spectra of the two tryptophans located in two different domains, in way to study conformational changes in the surrounding of these residues. We also follow emission spectra of Fluorescein-5-Maleimide dye bound to the single free cysteine of BSA. Complementary information on the extent of aggregation and on the structural changes is obtained by Rayleigh scattering and circul…
Thermal aggregation of beta-lactoglobulin in presence of metal ions.
2007
In this work, we report a study of the effects of zinc and copper ions on the heat-induced aggregation of beta-lactoglobulin (BLG). Kinetics investigations on aggregates growth by light scattering measurements and on secondary structure changes by FTIR absorption measurements show the different role played by the two metals during the whole process. In particular, the presence of zinc in solution promotes the formation of aggregates of BLG at a lower temperature than copper. Then, at fixed temperature, formation of a large amount of aggregates, of large dimension, is observed for Zn-BLG in shorter time; on the contrary, the presence of copper in solution does not affect the aggregation proc…
Protein Thermal Denaturation and Matrix Glass Transition in Different Protein−Trehalose−Water Systems
2011
Biopreservation by saccharides is a widely studied issue due to its scientific and technological importance; in particular, ternary amorphous protein-saccharide-water systems are extensively exploited to model the characteristics of the in vivo biopreservation process. We present here a differential scanning calorimetry (DSC) study on amorphous trehalose-water systems with embedded different proteins (myoglobin, lysozyme, BSA, hemoglobin), which differ for charge, surface, and volume properties. In our study, the protein/trehalose molar ratio is kept constant at 1/40, while the water/sugar molar ratio is varied between 2 and 300; results are compared with those obtained for binary trehalose…
Identification of disulphide bonds in the refolding of bovine pancreatic RNase A
1996
Background: Comprehension of the rules that govern the folding process is still far from satisfactory, though it is nevertheless clear that all the information required to define the folding is encoded in the amino acid sequence. In proteins that contain disulphide bonds, folding is associated with disulphide bond formation. Protein species with different numbers of disulphides tend to accumulate during the process; these species can be trapped in a stable form, by quenching any remaining free SH groups, and then characterized in order to identify the disulphide bonds formed. Results The refolding pathway of reduced and denatured RNase A has been studied using mass spectrometric strategies …
Self-assembly of biopolymeric structures below the threshold of random cross-link percolation
1996
Self-assembly of extended structures via cross-linking of individual biomolecules often occurs in solutions at concentrations well below the estimated threshold for random cross-link percolation. This requires solute-solute correlations. Here we study bovine serum albumin. Its unfolding causes the appearance of an instability region of the sol, not observed for native bovine serum albumin. As a consequence, spinodal demixing of the sol is observed. The thermodynamic phase transition corresponding to this demixing is the determinative symmetry-breaking step allowing the subsequent occurrence of (correlated) cross-linking and its progress up to the topological phase transition of gelation. Th…