Crocin Inhibits the Fibrillation of Human α-synuclein and Disassembles Mature Fibrils: Experimental Findings and Mechanistic Insights from Molecular Dynamics Simulation.

The aggregation of human alpha-synuclein (hαS) is pivotally implicated in the development of most types of synucleinopathies. Molecules that can inhibit or reverse the aggregation process of amyloidogenic proteins have potential therapeutic value. The anti-aggregating activity of multiple carotenoid compounds has been reported over the past decades against a growing list of amyloidogenic polypeptides. Here, we aimed to determine whether crocin, the main carotenoid glycoside component of saffron, would inhibit hαS aggregation or could disassemble its preformed fibrils. By employing a series of biochemical and biophysical techniques, crocin was exhibited to inhibit hαS fibrillation in a dose-dependent fashion by stabilizing very early aggregation intermediates in off-pathway non-toxic conformations with little ß-sheet content. We also observed that crocin at high concentrations could efficiently destabilize mature fibrils and disassemble them into seeding-incompetent intermediates by altering their ß-sheet conformation and reshaping their structure. Our atomistic molecular dynamics (MD) simulations demonstrated that crocin molecules bind to both the non amyloid-ß component (NAC) region and C-terminal domain of hαS. These interactions could thereby stabilize the autoinhibitory conformation of the protein and prevent it from adopting aggregation-prone structures. MD simulations further suggested that ligand molecules prefer to reside longitudinally along the fibril axis onto the edges of the inter-protofilament interface where they establish hydrogen and hydrophobic bonds with steric zipper stabilizing residues. These interactions turned out to destabilize hαS fibrils by altering the interstrand twist angles, increasing the rigidity of the fibril core, and elevating its radius of gyration. Our findings suggest the potential pharmaceutical implication of crocin in synucleinopathies.

An efficient method for recombinant production of human alpha synuclein in Escherichia coli using thioredoxin as a fusion partner.

Herein, we describe a simple and efficient approach to produce recombinant human α-synuclein (hAS) with high purity from Escherichia coli (E. coli). The cDNA for hAS was inserted into plasmid pET32a and expressed in E. coli BL21 (DE3) with an N-terminal tag containing E. coli thioredoxin (trx), followed by a histidine hexapeptide, and a tobacco etch virus (TEV) protease cleavage site (trx-6His-TEV). The fusion protein, trx-hAS, was initially released by osmotic shock treatment from the host cells and subsequently purified using a nickel affinity chromatography. A TEV protease cleavage step was performed to liberate the target protein, hAS, from the fusion partner, trx. Finally, an additional nickel affinity chromatography was performed to further purify the digested product. The yield of this method is ∼25 mg of tag-less protein (with ∼99% purity) per liter of culture volume. Reverse phase HPLC (RP-HPLC) and electrospray ionization (ESI) mass spectrometry confirmed the purity and authenticity of the purified protein. Thioflavin T (ThT) fluorescence assay, transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy demonstrated that the purified proteins form fibrils. Our protocol not only provides a convenient procedure for preparing highly pure hAS, but also requires very little specialized laboratory techniques.

An MTHFR variant, plasma homocysteine levels and late-onset coronary artery disease in subjects from southern Iran.

There have been many controversial debates on the role of Hyperhomocysteinaemia (HHcy) as an independent risk factor for Coronary Artery Disease (CAD) during recent years. Furthermore, an alanine/valine (Ala/Val) gene polymorphism at 222nd amino acid of 5,10-methylenetetrahydrofolate reductase (MTHFR) has been considered as a factor that could render this enzyme thermolabile and less active which in turn may yield a subsequent increase in plasma total homocysteine (tHcy) levels. To assess whether this polymorphism is associated with increased risk of CAD and plasma levels of tHcy in a population from southern Iran, a total of 457 patients with angiographically documented multi-vessel CAD were compared with a control group comprised of 371 subjects with <30% stenosis in all major vessels. Nevertheless our results failed to admit a significant difference between CAD individuals and control subjects for Ala/Val polymorphism and plasma Hcy concentrations. However, plasma Hcy concentrations were significantly higher in individuals with Val/Val genotype than subjects with Ala/Ala genotype, but it didn't show a significant association with CAD in our population. Moreover, as the multiple linear regression analysis indicated, smoking habit, folate levels and the MTHFR Val/Val genotype were the only major predictors of tHcy concentrations in the current investigation.

Lack of association between Ser(413)/Cys polymorphism of plasminogen activator inhibitor type 2 (PAI-2) and premature coronary atherosclerotic disease.

Plasminogen activator inhibitor type-2 (PAI-2) is a serine protease inhibitor of the fibrinolytic system produced predominantly by the macrophages and monocytes. It has been demonstrated that fibrinolysis regulation has a great importance in the pathogenesis of atherosclerotic plaques. Thus in the current investigation, we sought to determine whether Ser(413)/Cys polymorphism (rs6104) of PAI-2 gene could be associated with atherosclerosis and cardiovascular risk factors. Ser(413)/Cys polymorphism was determined by PCR-RFLP technique using Mwo I restriction enzyme for 184 men under 50 years of age and 216 women less than 55 years of age who underwent diagnostic coronary angiography. Data on the history of familial myocardial infarction or other heart diseases, hypertension, and smoking habit were collected by a simple questionnaire. Fasting levels of blood sugar, triglycerides, total cholesterol, low-density lipoprotein and high-density lipoprotein cholesterol levels were also measured by enzymatic methods. Frequencies of the Ser(413) and Cys(413) alleles were 0.760 and 0.240 in the whole population, respectively. The PAI-2 gene variant analyzed was not significantly associated with either the prevalence of premature CAD or the classical risk factors of CAD development such as diabetes, serum cholesterol, triglycerides, low-density lipoprotein and high-density lipoprotein cholesterol, body mass index, hypertension, familial history of heart dysfunction or smoking.

Computational analysis of cysteine proteases (Clan CA, Family Cl) of Leishmania major to find potential epitopic regions.

Leishmania is associated with a broad spectrum of diseases, ranging from simple cutaneous to invasive visceral leishmaniasis. Here, the sequences of ten cysteine proteases of types A, B and C of Leishmania major were obtained from GeneDB database. Prediction of MHC class I epitopes of these cysteine proteases was performed by NetCTL program version 1.2. In addition, by using BcePred server, different structural properties of the proteins were predicted to find out their potential B cell epitopes. According to this computational analysis, nine regions were predicted as B cell epitopes. The results provide useful information for designing peptide-based vaccines.