Controlling the inhibition of the sarcoplasmic Ca2+-ATPase by tuning phospholamban structural dynamics

Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca2+-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for approximately 70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-of-function (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.     

Seed germination ecology and salt stress response in eight Mediterranean populations of Sarcopoterium spinosum (L.) Spach

This study aims to deepen the analysis of seed germination ecology and salinity tolerance of Sarcopoterium spinosum (Rosaceae). Germination tests were conducted to evaluate the effect of the fruit’s spongy tissue and the intraspecific variability in seed germination among eight populations of the species on responses to light and total darkness, constant and alternating temperatures, salt stress and germination recovery. The effect of the presence of the spongy tissue varied among populations, with significant results for seed germination. For all populations, optimum germination temperatures were observed in the range of 10–20°C, indicating that S. spinosum and its germination in the field might occur preferably in the period between autumn and early spring. The high water availability due to rainfall during this period could be a considerable advantage for the seed germination of this species. Seeds of S. spinosum showed the ability to germinate in up to 250 mM NaCl in the substrate, and their ability to recover after salt exposure may be interpreted as adaptation to the coastal habitats in which they generally grow. These results give this species a halo-tolerant character. Great inter-population variability is detected in this study in several aspects, which indicated that the Mediterranean populations of S. spinosum differ considerably and are adapted to their local conditions. This study provides new information about S. spinosum seed ecology, which could help to preserve and apply effective conservation measures for this species, which in several areas of its distribution range is endangered.     

Exercise-induced and nitroglycerin-induced myocardial preconditioning improves hemodynamics in patients with angina

In humans, regional myocardial dysfunction during ischemia may be improved by ischemic and pharmacological preconditioning. We assessed the possibility that exercise- and nitroglycerin-induced myocardial preconditioning may improve global cardiac performance during subsequent efforts in patients with angina. Ten patients suffering from chronic stable angina and ten healthy volunteers were studied. Through impedance cardiography we assessed hemodynamics during a maximal exercise test, which was used as a baseline (Bas test) and considered as a preconditioning exercise. The Bas test was followed by a sequence of maximal efforts performed during the first (FWOP; 30 min after the Bas test) and second (SWOP; 48 h after the Bas test) windows of protection conferred by ischemic preconditioning. Hemodynamics was further evaluated during maximal exercise performed 48 h later with pharmacologically induced SWOP (PI-SWOP) obtained by transdermal administration of 10 mg of nitroglycerin. In the angina patients, FWOP, SWOP, and PI-SWOP delayed the time to ischemia and allowed them to achieve higher workloads compared with the Bas test. Furthermore, heart rate and cardiac output at peak exercise were enhanced during all the preconditioning phases with respect to the Bas test. However, only SWOP and PI-SWOP increased myocardial contractility and stroke volume. No changes in hemodynamics were detectable in the control subjects. This study demonstrates that in patients with stable angina, although hemodynamics during exercise can be positively improved during both FWOP and SWOP, differences exist between these two phases. Furthermore, the mimicking of exercise-induced SWOP by PI-SWOP with transdermal nitroglycerin may represent an important clinical aspect.     

Design, synthesis and in vitro antitumor activity of new trans 2-[2-(heteroaryl)vinyl]-1,3-dimethylimidazolium iodides

The design, the synthesis, and the in vitro antitumor activities of trans 2-[2-(heteroaryl)vinyl]-1,3-dimethylimidazolium iodides versus MCF7 (human mammary carcinoma) and LNCap (prostate carcinoma) cell lines are reported. The design indicates trans 2-[2-[5-(2-chlorophenyl)furan-2-yl]vinyl]-1, 3-dimethylimidazolium iodide 5 and trans 2-[2-[5-(4-bromophenyl)furan-2-yl]vinyl]-1, 3-dimethylimidazolium iodide 6 as highly active compounds in the series. The synthesis of the above new derivatives and in vitro antitumor tests, confirm their significant antiproliferative and cytotoxic activities.     

Cytochrome c-induced cytosolic nicotinamide adenine dinucleotide oxidation,mitochondrial permeability transition,and apoptosis

A catalytic amount of cytochrome c (cyto-c) added to the incubation medium of isolated mitochondria promotes the transfer of reducing equivalents from extramitochondrial nicotinamide adenine dinucleotide in its reduced state (NADH) to molecular oxygen inside the mitochondria, a process coupled to the generation of a membrane potential. This mimics in many aspects the early stages of those apoptotic pathways characterized by the persistence of mitochondrial membrane potential but with cyto-c already exported into the cytosol. In cyclosporin-sensitive and calcium-induced mitochondrial permeability transition (MPT) a release of cyto-c can also be observed. However, in MPT uncoupled respiration associated with mitochondrial swelling and preceded by the complete dissipation of the membrane potential which cannot be restored with ATP addition or any other source of energy is immediately activated. The results obtained and discussed with regard to intactness of mitochondrial preparations indicate that MPT could be an apoptotic event downstream but not upstream of cyto-c release linked to the energy-requiring processes. In the early stages of apoptosis cytosolic cyto-c participates in the activation of caspases and at the same time can promote the oxidation of cytosolic NADH, making more energy available for the correct execution of the cell death program. This hypothesis is not in contrast with available data in the literature showing that cyto-c is present in the cytosol of both control and apoptosis-induced cultured cell lines.     

Conformational preferences of the amylin nucleation site in SDS micelles: an NMR study

Human islet amyloid polypeptide (hIAPP), or amylin, is a 37 amino acid hormone secreted by pancreatic beta-cells. hIAPP constitutes approximately 90% of the amyloid deposits found in type II diabetic patients. It has been shown that the central region of the peptide (hIAPP(20-29)) constitutes the nucleation site for the amyloidogenic process with F23 playing a key role in the formation of the beta-pleated structures. In addition, it has been proposed that an important stage in the cytotoxicity of hIAPP is its interaction with the beta-cell membranes. As a first step toward the characterization of the interaction of hIAPP with cell membranes, we determined conformational preferences of hIAPP(20-29) in membrane-mimicking environments. We found that upon interacting with negatively charged micelles, the dominant conformation of hIAPP(20-29) is a distorted type I beta-turn centered on residues F23 and G24, with F23, A25, and I26 forming a small hydrophobic cluster that may facilitate the interaction of this peptide with the membrane bilayer. Moreover, we were able to elucidate the topological orientation of the peptide that is absorbed on the micelle surface, with the hydrophobic cluster oriented toward the hydrocarbon region of the micelles and both N- and C-termini exposed to the solvent.     

ErbB2 is essential in the prevention of dilated cardiomyopathy

Amplification of the gene encoding the ErbB2 (Her2/neu) receptor tyrosine kinase is critical for the progression of several forms of breast cancer. In a large-scale clinical trial, treatment with Herceptin (trastuzumab), a humanized blocking antibody against ErbB2, led to marked improvement in survival. However, cardiomyopathy was uncovered as a mitigating side effect, thereby suggesting an important role for ErbB2 signaling as a modifier of human heart failure. To investigate the physiological role of ErbB2 signaling in the adult heart, we generated mice with a ventricular-restricted deletion of Erbb2. These ErbB2-deficient conditional mutant mice were viable and displayed no overt phenotype. However, physiological analysis revealed the onset of multiple independent parameters of dilated cardiomyopathy, including chamber dilation, wall thinning and decreased contractility. Additionally, cardiomyocytes isolated from these conditional mutants were more susceptible to anthracycline toxicity. ErbB2 signaling in cardiomyocytes is therefore essential for the prevention of dilated cardiomyopathy.     

Selectivity in heavy metal- binding to peptides and proteins

The metal-binding affinities and three-dimensional structures of three synthetic 18-residue peptides with sequences derived from that of the highly conserved metal-binding motif MXCXXC found in many heavy metal-binding proteins were determined. A change in register of the cysteines and alanines of the sequence from the periplasmic mercury-binding protein, MerP, i.e., CAAC, CACA, and CCAA, affects the specificity of metal binding, in particular, the peptide with vicinal cysteines binds only mercury. The three-dimensional structures of the mercury-bound forms of the three peptides determined in solution by NMR spectroscopy peptides differ considerably, even though they are all linear bicoordinate complexes. The three-dimensional structure of the peptide with CAAC bound to Cd(II) demonstrates that the metal-binding loop is malleable enough to accommodate modes of coordination other than linear bicoordinate.     

Variability of HXT2 at the protein and gene level among the Saccharomyces sensu stricto group

Variability of HXT2 at the protein and gene level was investigated among Saccharomyces sensu stricto and other yeast species. Results showed that the HXT2 gene is probably present in yeast genera other than Saccharomyces, suggesting that this gene is widely distributed in the yeast world. Chromosomal analyses indicated the stable location of HXT2 on the same chromosome and with the same copy number throughout the entire sensu stricto group. Results of the immunoblotting assay demonstrated that all strains tested (with the exception of S. cerevisiae DBVPG 6042) exhibited a lower level of Hxt2p expression than that shown by laboratory wild-type. Moreover, Hxt2p expression seems to reinforce the taxonomical differences between the two pairs of species (S. cerevisiae and S. paradoxus vs. S. pastorianus and S. bayanus) within the sensu stricto group of the genus of Saccharomyces that also reflect their different ecological niche.