TTR (transthyretin) amyloidoses are diseases characterized by the aggregation and extracellular deposition of the normally soluble plasma protein TTR. incubation of the human cardiomyocytes with V122I TTR but not with T119M TTR, generates superoxide species and activates caspase 3/7. In summary, our results show that the interaction of the amyloidogenic V122I TTR is distinct from that of a non-amyloidogenic TTR variant and is characterized by its retention at the cell membrane, where it initiates the cytotoxic cascade. expression system as described elsewhere . The last step of purification consisted in gel filtration chromatography on a Superdex 75 column (GE Biosciences) to obtain tetrameric TTR free of aggregates. When the recombinant TTR was intended to be used for biophysical studies, the gel filtration purification was performed Rabbit polyclonal to Aquaporin3 in 10?mM phosphate buffer (sodium) pH?7.6/100?mM KCl/1?mM EDTA buffer (GF buffer); when the TTR was intended for cell culture experiments, HBSS (Hank’s balanced salt solution; Mediatech) buffer was used instead. The plasmids to obtain the TTR variants C10A/V122I/P125C and C10A/V122I/E127C were produced by PCR-assisted site directed mutagenesis using the V122I TTR plasmids as template. The new plasmids were sequenced to ensure that the desired mutations had been introduced. All the purified recombinant proteins were stored at ?80C at concentrations lower than 2.5?mg/ml, conditions under which the proteins are stable and do not aggregate. LCCESICMS (liquid chromatographyCelectrospray ionization mass spectrometry) was used to confirm the molecular mass of the recombinant proteins: V122I TTR, 13905.4 (expected, 13906.6), T119M, 13921.6 (expected 13922.6), C10A/V122I/P125C, 13878.9 (expected 13880.5), C10A/V122I/E127C, 13847.5 (expected 13848.5). Labelling of V122I TTR variants with fluorescent probes The cysteine residues of Exo1 V122I TTR, C10A/V122I/E127C TTR and C10A/V122I/P125C TTR variants were labelled with Oregon Green 488 maleimide (O-6034, Molecular Probes) using thiol chemistry. The cysteine residues of C10A/V122I/E127C TTR and C10A/V122I/P125C TTR variants were also derivatized with Alexa Fluor 488 C5-maleimide (A-10254, Molecular Probes) following the manufacturer’s instructions. Briefly, TTR solutions (~2?mg/ml) were dialysed against 50?mM of sodium phosphate buffer pH?7.2 with 100?M TCEP Exo1 [tris(2-carboxyethyl) phosphine-hydrochloride, Biosynth], at room temperature for 2?h. TCEP was required Exo1 to maintain the cysteine residues in reduced form and available for derivatization. Stock solutions of the fluorophores were prepared at 5?mM (in DMSO) and added dropwise to TTR solutions with vigorous agitation. We used 5 and 8 molar excess dye:TTR for Alexa Fluor 488 and Oregon Green 488, respectively. The conjugation reactions were allowed to proceed at 4C overnight in the dark, under mild agitation. In all the subsequent steps the labelled proteins were protected from the light. The crude reaction mixtures were dialysed against GF buffer at room temperature for 2?h and the proteins re-purified by gel filtration at 4C on a Superdex 75 column (GE Biosciences) in GF buffer to remove aggregates that could have formed through Exo1 the labelling procedure. LCCESICMS was utilized to confirm the type from the derivatized protein and the effectiveness of the task. The molecular mass from the labelled proteins had been: C10A/V122I/P125C-Oregon Green 488, 14343.8 (expected, 14343.5), C10A/V122I/E127C-Oregon Green 488, 14311.1 (expected, 14311.5), C10A/V122I/P125C-Alexa Fluor 488, 14577.9 (expected, 14577.5), C10A/V122I/E127C-Alexa Fluor 488, 14545.8 (expected, 14545.5). The amount of labelling was 2.5C2.8 TTR subunits per TTR tetramer for the Oregon Green 488-labelled proteins and four TTR subunits per TTR tetramer for the Alexa Fluor 488-labelled proteins. Covalent V122I kinetic stabilization having a resveratrol analogue V122I TTR was kinetically stabilized having a resveratrol analogue (SM) that binds covalently to Lys15 of TTR within the T4-binding pocket (substance.