Direct Observation of Defects and Increased Ion Permeability of a Membrane Induced by Structurally Disordered Cu/Zn-Superoxide Dismutase Aggregates

Interactions between protein aggregates and a cellular membrane have been strongly implicated in many protein conformational diseases. However, such interactions for the case of Cu/Zn superoxide dismutase (SOD1) protein, which is related to fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS), have not been explored yet. For the first time, we report the direct observation of defect formation and increased ion permeability of a membrane induced by SOD1 aggregates using a supported lipid bilayer and membrane patches of human embryonic kidney cells as model membranes. We observed that aggregated SOD1 significantly induced the formation of defects within lipid membranes and caused the perturbation of membrane permeability, based on surface plasmon resonance spectroscopy, atomic force microscopy and electrophysiology. In the case of apo SOD1 with an unfolded structure, we found that it bound to the lipid membrane surface and slightly perturbed membrane permeability, compared to other folded proteins (holo SOD1 and bovine serum albumin). The changes in membrane integrity and permeability were found to be strongly dependent on the type of proteins and the amount of aggregates present. We expect that the findings presented herein will advance our understanding of the pathway by which structurally disordered SOD1 aggregates exert toxicity in vivo

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Expression and Characterization of a Recombinant Psychrophilic Cu/Zn Superoxide Dismutase from Deschampsia antarctica E. Desv. [Poaceae]

"We present here the structural modeling and biochemical characterization of a recombinant superoxide dismutase (SOD) from Deschampsia antarctica E. Desv. [Poaceae] produced in Escherichia coli. The recombinant protein was purified by affinity chromatography nickel-nitrilotriacetic acid (Ni-NTA), and its identity was demonstrated by immunoblotting and inhibition by H2O2 and KCN. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis confirmed the presence of Cu and Zn. Modeling of the D. antarctica Cu/Zn-SOD (DaSOD) amino acid sequence using the SWISS-MODEL and 2Q2L_B monomer of the psychrophilic Cu/Zu-SOD from Potentilla atrosanguinea (PaSOD) as template produced a structure similar to that of the typical eukaryotic Cu/Zn-SODs. Activity assays using the p-nitro blue tetrazolium chloride (NBT) solution method showed that the purified DaSOD had a specific activity of 5818 U/mg at 25 °C and pH 7.2 and that it was active in a pH interval of 5–8 and a temperature interval of 0–40 °C. Furthermore, DaSOD was still active at −20 °C as observed by a zymogram assay. We found 100 % activity when it was heated at 80 °C for 60 min, indicating a high thermostability. DaSOD properties suggest that this enzyme could be useful for preventing the oxidation of refrigerated or frozen foods, as well as in the preparation of cosmetic and pharmaceutical products.

Insights into SOD1-linked amyotrophic lateral sclerosis from NMR studies of Ni2+- and other metal-ion-substituted wild-type copper–zinc superoxide dismutases

The dimeric copper-zinc superoxide dismutase Cu(2)Zn(2)SOD1 is a particularly interesting system for biological inorganic chemical studies because substitutions of the native Cu and/or Zn ions by a non-native metal ion cause minimal structural changes and result in high enzymatic activity for those derivatives with Cu remained in the Cu site. The pioneering NMR studies by Ivano Bertini and coworkers of the magnetically coupled derivative Cu(2)Co(2)SOD1 are of particular importance in this regard. In addition to Co(2+), Ni(2+) is also a versatile metal ion for substitution into Cu(2)Zn(2)SOD1, showing very little disturbance of the structure in Cu(2)Ni(2)SOD1 and a very good mimic of the native Cu ion in Ni(2)Zn(2)SOD1. The NMR studies presented here were inspired by and indebted to Professor Bertini's paramagnetic NMR pursuits of metalloproteins. In the current study, we report Ni(2+) binding to apo-wild type SOD1 and a time-dependent Ni(2+) ion migration from the Zn site to the Cu site and preparation and characterization of Ni(2)Ni(2)SOD1, which shows similar coordination properties to those of Cu(2)Cu(2)SOD1, namely a different anion binding property from the wild type and a possibly broken bridging His. Mutations in the human SOD1 gene can cause familial amyotrophic lateral sclerosis (ALS), and mutant SOD1 proteins with significantly altered metal binding behaviors are implicated in causing the disease. We therefore conclude by discussing the effects of the ALS mutations on the remarkable stabilities and metal-binding properties of wild type SOD1 proteins and the implications concerning the causes of SOD1-linked ALS

Genetic factors affecting EBV copy number in lymphoblastoid cell lines derived from the 1000 Genome Project samples

Epstein-Barr virus (EBV), human herpes virus 4, has been classically associated with infectious mononucleosis, multiple sclerosis and several types of cancers. Many of these diseases show marked geographical differences in prevalence, which points to underlying genetic and/or environmental factors. Those factors may include a different susceptibility to EBV infection and viral copy number among human populations. Since EBV is commonly used to transform B-cells into lymphoblastoid cell lines (LCLs) we hypothesize that differences in EBV copy number among individual LCLs may reflect differential susceptibility to EBV infection. To test this hypothesis, we retrieved whole-genome sequenced EBV-mapping reads from 1,753 LCL samples derived from 19 populations worldwide that were sequenced within the context of the 1000 Genomes Project. An in silico methodology was developed to estimate the number of EBV copy number in LCLs and validated these estimations by real-time PCR. After experimentally confirming that EBV relative copy number remains stable over cell passages, we performed a genome wide association analysis (GWAS) to try detecting genetic variants of the host that may be associated with EBV copy number. Our GWAS has yielded several genomic regions suggestively associated with the number of EBV genomes per cell in LCLs, unraveling promising candidate genes such as CAND1, a known inhibitor of EBV replication. While this GWAS does not unequivocally establish the degree to which genetic makeup of individuals determine viral levels within their derived LCLs, for which a larger sample size will be needed, it potentially highlighted human genes affecting EBV-related processes, which constitute interesting candidates to follow up in the context of EBV related pathologiesThis work was supported by Instituto de Salud Carlos III (ES) (RD07/0060); Spanish Government Grants (BFU2012-38236); Departament d'Innovació, Universitats I Empresa, Generalitat de Catalunya (2014SGR1311); Instituto de Salud Carlos III (PT13/0001/0026); FEDER (Fondo Europeo de Desarrollo Regional)/FSE (Fondo Social Europeo)