Mutations of Profilin‑1 Associated with Amyotrophic Lateral Sclerosis Promote Aggregation Due to Structural Changes of Its Native State

The PFN1 gene, coding for profilin-1, has recently been associated with familial amyotrophic lateral sclerosis (fALS), as three mutations, namely C71G, M114T, and G118V, have been found in patients with familial forms of the disease and another, E117G, has been proposed to be a moderate risk factor for disease onset. In this work, we have purified the four profilin-1 variants along with the wild-type protein. The resulting aggregates appear to be fibrillar, to have a weak binding to ThT, and to possess a significant amount of intermolecular β-sheet structure. Using ThT fluorescence assays, far-UV circular dichroism, and dynamic light scattering, we found that all four variants have an aggregation propensity higher than that of the wild-type counterpart. In particular, the C71G mutation was found to induce the most dramatic change in aggregation, followed by the G118V and M114T substitutions and then the E117G mutation. Such a propensity was found not to strictly correlate with the conformational stability in this group of profilin-1 variants, determined using both urea-induced denaturation at equilibrium and folding/unfolding kinetics. However, it correlated with structural changes of the folded states, as monitored with far-UV circular dichroism, intrinsic fluorescence spectroscopy, ANS binding, acrylamide quenching, and dynamic light scattering. Overall, the results suggest that all four mutations increase the tendency of profilin-1 to aggregate and that such aggregation behavior is largely determined by the mutation-induced structural changes occurring in the folded state of the protein

A single amino acid mutation affects elicitor and expansins-like activities of cerato-platanin, a non-catalytic fungal protein

<div><p>Cerato-platanin (CP) is a non-catalytic, cysteine-rich protein, the first member of the cerato-platanin family. It is a single-domain protein with a double Ψ/β barrel domain resembling the D1 domain of plant and bacterial expansins. Similarly to expansins, CP shows a cell wall-loosening activity on cellulose and can be defined as an expanisin-like protein, in spite of the missing D2 domain, normally present in plant expansins. The weakening activity shown on cellulose may facilitate the CP-host interaction, corroborating the role of CP in eliciting plant defence response. Indeed, CP is an elicitor of primary defences acting as a Pathogen-Associated Molecular Patterns (PAMP). So far, structure-function relationship study has been mainly performed on the bacterial BsEXLX1 expansin, probably due to difficulties in expressing plant expansins in heterologous systems. Here, we report a subcloning and purification method of CP in the engineered <i>E</i>. <i>coli</i> SHuffle cells, which proved to be suitable to obtain the properly folded and biologically active protein. The method also enabled the production of the mutant D77A, rationally designed to be inactive. The wild-type and the mutated CP were characterized for cellulose weakening activity and for PAMP activity (i.e. induction of Reactive Oxygen Species synthesis and phytoalexins production). Our analysis reveals that the carboxyl group of D77 is crucial for expansin-like and PAMP activities, thus permitting to establish a correlation between the ability to weaken cellulose and the capacity to induce defence responses in plants. Our results enable the structural and functional characterization of a mono-domain eukaryotic expansin and identify the essential role of a specific aspartic residue in cellulose weakening.</p></div

3D model of CP structure.

<p>A) Surface representation. Coloured residues are located in the putative oligosaccharide binding region. Colours shown conserved amino acid along all CPF members. Red for acid residues, blue for basic residues and yellow for-polar residues. B) H bonds distances in wtCP. The bonds between D77-Y9, D77-S78 and D77-A19 are indicated. Yellow rectangle indicates the zoomed region in C. C) H bonds in mut CP. Lack of H bonds in mutCP with A77 instead D77.</p

MALDI–TOF mass spectrometry.

<p>Twenty picomoles of proteins were dissolved in 50% acetonitrile containing 0.05% TFA, diluted 1:1 in saturated sinapinic acid matrix and analysed on a MALDI–TOF mass spectrometer. A) nCP. B) wtCP. C) mutCP.</p

Structural characterisation of CP.

<p>A) Far-UV CD spectra of 6.25 μM nCP, wtCP and mutCP. B) Near-UV CD spectra of 1.5 mg/mL wtCP and mutCP. All samples were dissolved in 10 mM Na-phosphate buffer.C) equilibrium denaturation experiment of 0.04 mg/ml wtCP and mutCP in the presence of GndHCl concentrations ranging from 0 to 5.6 M.</p

Weakening activity of CP on filter paper.

<p>A) Releasing of paper fragments from the paper disc Each tube contains a filter paper disc in 0.5 mL of 50 mM sodium acetate buffer and 30 μM of wtCP or mutCP. Buffer only or buffer containing 30μM BSA at the same concentration were used as negative controls. Weakening activity was visible as paper fragments released in suspension from the paper disc after 48h, at 38°C, with shaking at 700 rpm. B) Quantification of the paper fragments produced. Absorbance at 500 nm was measured on a Ultraspec 2000 (Pharmacia biotech) spectrophotometer. Error bars indicate the standard deviation of measurements from three separated experiments. Values marked with different letters are significantly different at p< 0.05 according to the <i>t-test</i>.</p

Purification yield of wild type and mutated CP starting from 1L of induced culture.

<p>Values are the means of data from four independent experiments ± s.d.</p

A Complex Equilibrium among Partially Unfolded Conformations in Monomeric Transthyretin

Aggregation of transthyretin (TTR) is known to be linked to the development of systemic and localized amyloidoses. It also appears that TTR exerts a protective role against aggregation of the Aβ peptide, a process linked to Alzheimer’s disease. <i>In vitro</i>, both processes correlate with the ability of TTR to populate a monomeric state, yet a complete description of the possible conformational states populated by monomeric TTR <i>in vitro</i> at physiological pH is missing. Using an array of biophysical methods and kinetic tests, we show that once monomers of transthyretin are released from the tetramer, equilibrium is established between a set of conformational states possessing different degrees of disorder. A molten globular state appears in equilibrium with the fully folded monomer, whereas an off-pathway species accumulates transiently during refolding of TTR. These two conformational ensembles are distinct in terms of structure, kinetics, and their pathways of formation. Further subpopulations of the protein fold differently because of the occurrence of proline isomerism. The identification of conformational states unrevealed in previous studies opens the way for further characterization of the amyloidogenicity of TTR and its protective role in Alzheimer’s disease