In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ_1–42) is the most important pathophysiological event associated with Alzheimer''s disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α-helix to a β-sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu²⁺ and various drugs used for AD treatment, such as galanthamine (Reminyl®), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu²⁺ and galanthamine prevent the formation of Aβ_1–42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ_1–42 acquires a characteristic U-shape before the α-helix to β-sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ₁₋₄₂ in the presence of Cu²⁺ or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu²⁺) or to Lys 28 (galanthamine), which prevents Aβ₁₋₄₂ from adopting the U-characteristic conformation that allows the amino acids to transition to a β-sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu²⁺ and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.     

Inhibition of proinsulin biosynthesis and conversion by a putative insulin mediator

The phospho-oligosaccharide (POS), presumed to act at the postreceptor level as the insulin second messenger, was recently reported to inhibit glucose-stimulated insulin release from rat pancreatic islets. In the present study, POS was also found to inhibit glucose-stimulated proinsulin biosynthesis and conversion in rat islets. By comparison with prior findings on the effects of both exogenous insulin and anti-insulin serum upon proinsulin synthesis, these results argue against the view that insulin would normally exert a negative feedback control upon the biosynthetic and secretory activities of islet B-cells.     

Wheat Stripe Rust Resistance Protein WKS1 Reduces the Ability of the Thylakoid-Associated Ascorbate Peroxidase to Detoxify Reactive Oxygen Species

Stripe rust is a devastating fungal disease of wheat caused by Puccinia striiformis f. sp tritici (Pst). The WHEAT KINASE START1 (WKS1) resistance gene has an unusual combination of serine/threonine kinase and START lipid binding domains and confers partial resistance to Pst. Here, we show that wheat (Triticum aestivum) plants transformed with the complete WKS1 (variant WKS1.1) are resistant to Pst, whereas those transformed with an alternative splice variant with a truncated START domain (WKS1.2) are susceptible. WKS1.1 and WKS1.2 preferentially bind to the same lipids (phosphatidic acid and phosphatidylinositol phosphates) but differ in their protein-protein interactions. WKS1.1 is targeted to the chloroplast where it phosphorylates the thylakoid-associated ascorbate peroxidase (tAPX) and reduces its ability to detoxify peroxides. Increased expression of WKS1.1 in transgenic wheat accelerates leaf senescence in the absence of Pst. Based on these results, we propose that the phosphorylation of tAPX by WKS1.1 reduces the ability of the cells to detoxify reactive oxygen species and contributes to cell death. This response takes several days longer than typical hypersensitive cell death responses, thus allowing the limited pathogen growth and restricted sporulation that is characteristic of the WKS1 partial resistance response to Pst.     

Identification and characterization of associated with lipid droplet protein 1: A novel membrane-associated protein that resides on hepatic lipid droplets

Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix-assisted laser desorption ionization-time of flight and LC-MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation-related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD-associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.     

Stimulus-secretion coupling of arginine-induced insulin release. Metabolism of L-arginine and L-ornithine in pancreatic islets

Exogenous L-arginine and L-ornithine rapidly accumulate in rat pancreatic islets. L-Arginine is converted to L-ornithine and urea. Endogenous or exogenous L-ornithine generates di- and polyamines, the putrescine turnover being faster than that of spermidine and spermine. However, the major pathway for L-ornithine metabolism consists of its transamination to L-glutamaldehyde and further conversion to L-glutamate. The amines and L-glutamate derived from exogenous L-ornithine are incorporated into islet proteins at the intervention of transglutaminase and cycloheximide-sensitive biosynthetic processes, respectively. These findings suggest the hypothesis that the insulinotropic action of L-arginine and L-ornithine could somehow be related to the metabolism of these cationic amino acids in islet cells.     

Quantitative genetic analysis of salicylic acid perception in Arabidopsis

Salicylic acid (SA) is a phytohormone required for a full resistance against some pathogens in Arabidopsis, and NPR1 (Non-Expressor of Pathogenesis Related Genes 1) is the only gene with a strong effect on resistance induced by SA which has been described. There can be additional components of SA perception that escape the traditional approach of mutagenesis. An alternative to that approach is searching in the natural variation of Arabidopsis. Different methods of analyzing the variation between ecotypes have been tried and it has been found that measuring the growth of a virulent isolate of Pseudomonas syringae after the exogenous application of SA is the most effective one. Two ecotypes, Edi-0 and Stw-0, have been crossed, and their F2 has been studied. There are two significant quantitative trait loci (QTLs) in this population, and there is one QTL in each one of the existing mapping populations Col-4 × Laer-0 and Laer-0 × No-0. They have different characteristics: while one QTL is only detectable at low concentrations of SA, the other acts after the point of crosstalk with methyl jasmonate signalling. Three of the QTLs have candidates described in SA perception as NPR1, its interactors, and a calmodulin binding protein.