Membrane protein dynamics: limited lipid control

Correlation of lipid disorder with membrane protein dynamics has been studied with infrared spectroscopy, by combining data characterizing lipid phase, protein structure and, via hydrogen-deuterium (H/D) exchange, protein dynamics. The key element was a new measuring scheme, by which the combined effects of time and temperature on the H/D exchange could be separated. Cyanobacterial and plant thylakoid membranes, mammalian mitochondria membranes, and for comparison, lysozyme were investigated. In dissolved lysozyme, as a function of temperature, H/D exchange involved only reversible movements (the secondary structure did not change considerably); heat-denaturing was a separate event at much higher temperature. Around the low-temperature functioning limit of the biomembranes, lipids affected protein dynamics since changes in fatty acyl chain disorders and H/D exchange exhibited certain correlation. H/D exchange remained low in all membranes over physiological temperatures. Around the high-temperature functioning limit of the membranes, the exchange rates became higher. When temperature was further increased, H/D exchange rates went over a maximum and afterwards decreased (due to full H/D exchange and/or protein denaturing). Maximal H/D exchange rate temperatures correlated neither with the disorder nor with the unsaturation of lipids. In membrane proteins, in contrast to lysozyme, the onsets of sizable H/D exchange rates were the onsets of irreversible denaturing as well. Seemingly, at temperatures where protein self-dynamics allows large-scale H/D exchange, lipid-protein coupling is so weak that proteins prefer aggregating to limit the exposure of their hydrophobic surface regions to water. In all membranes studied, dynamics seemed to be governed by lipids around the low-temperature limit, and by proteins around the high-temperature limit of membrane functionality

Calcium Ions Promote Formation of Amyloid β-Peptide (1–40) Oligomers Causally Implicated in Neuronal Toxicity of Alzheimer's Disease

Amyloid β-peptide (Aβ) is directly linked to Alzheimer's disease (AD). In its monomeric form, Aβ aggregates to produce fibrils and a range of oligomers, the latter being the most neurotoxic. Dysregulation of Ca2+ homeostasis in aging brains and in neurodegenerative disorders plays a crucial role in numerous processes and contributes to cell dysfunction and death. Here we postulated that calcium may enable or accelerate the aggregation of Aβ. We compared the aggregation pattern of Aβ(1–40) and that of Aβ(1–40)E22G, an amyloid peptide carrying the Arctic mutation that causes early onset of the disease. We found that in the presence of Ca2+, Aβ(1–40) preferentially formed oligomers similar to those formed by Aβ(1–40)E22G with or without added Ca2+, whereas in the absence of added Ca2+ the Aβ(1–40) aggregated to form fibrils. Morphological similarities of the oligomers were confirmed by contact mode atomic force microscopy imaging. The distribution of oligomeric and fibrillar species in different samples was detected by gel electrophoresis and Western blot analysis, the results of which were further supported by thioflavin T fluorescence experiments. In the samples without Ca2+, Fourier transform infrared spectroscopy revealed conversion of oligomers from an anti-parallel β-sheet to the parallel β-sheet conformation characteristic of fibrils. Overall, these results led us to conclude that calcium ions stimulate the formation of oligomers of Aβ(1–40), that have been implicated in the pathogenesis of AD

The Disulfide Bonds in Glycoprotein E2 of Hepatitis C Virus Reveal the Tertiary Organization of the Molecule

Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% β-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change

Doxorubicin-induced chronic dilated cardiomyopathy—the apoptosis hypothesis revisited

The chemotherapeutic agent doxorubicin (DOX) has significantly increased survival rates of pediatric and adult cancer patients. However, 10% of pediatric cancer survivors will 10–20 years later develop severe dilated cardiomyopathy (DCM), whereby the exact molecular mechanisms of disease progression after this long latency time remain puzzling. We here revisit the hypothesis that elevated apoptosis signaling or its increased likelihood after DOX exposure can lead to an impairment of cardiac function and cause a cardiac dilation. Based on recent literature evidence, we first argue why a dilated phenotype can occur when little apoptosis is detected. We then review findings suggesting that mature cardiomyocytes are protected against DOX-induced apoptosis downstream, but not upstream of mitochondrial outer membrane permeabilisation (MOMP). This lack of MOMP induction is proposed to alter the metabolic phenotype, induce hypertrophic remodeling, and lead to functional cardiac impairment even in the absence of cardiomyocyte apoptosis. We discuss findings that DOX exposure can lead to increased sensitivity to further cardiomyocyte apoptosis, which may cause a gradual loss in cardiomyocytes over time and a compensatory hypertrophic remodeling after treatment, potentially explaining the long lag time in disease onset. We finally note similarities between DOX-exposed cardiomyocytes and apoptosis-primed cancer cells and propose computational system biology as a tool to predict patient individual DOX doses. In conclusion, combining recent findings in rodent hearts and cardiomyocytes exposed to DOX with insights from apoptosis signal transduction allowed us to obtain a molecularly deeper insight in this delayed and still enigmatic pathology of DC

On improvement in ejection fraction with iron chelation in thalassemia major and the risk of future heart failure

<p>Abstract</p> <p>Background</p> <p>Trials of iron chelator regimens have increased the treatment options for cardiac siderosis in beta-thalassemia major (TM) patients. Treatment effects with improved left ventricular (LV) ejection fraction (EF) have been observed in patients without overt heart failure, but it is unclear whether these changes are clinically meaningful.</p> <p>Methods</p> <p>This retrospective study of a UK database of TM patients modelled the change in EF between serial scans measured by cardiovascular magnetic resonance (CMR) to the relative risk (RR) of future development of heart failure over 1 year. Patients were divided into 2 strata by baseline LVEF of 56-62% (below normal for TM) and 63-70% (lower half of the normal range for TM).</p> <p>Results</p> <p>A total of 315 patients with 754 CMR scans were analyzed. A 1% absolute increase in EF from baseline was associated with a statistically significant reduction in the risk of future development of heart failure for both the lower EF stratum (EF 56-62%, RR 0.818, p < 0.001) and the higher EF stratum (EF 63-70%, RR 0.893 p = 0.001).</p> <p>Conclusion</p> <p>These data show that during treatment with iron chelators for cardiac siderosis, small increases in LVEF in TM patients are associated with a significantly reduced risk of the development of heart failure. Thus the iron chelator induced improvements in LVEF of 2.6% to 3.1% that have been observed in randomized controlled trials, are associated with risk reductions of 25.5% to 46.4% for the development of heart failure over 12 months, which is clinically meaningful. In cardiac iron overload, heart mitochondrial dysfunction and its relief by iron chelation may underlie the changes in LV function.</p

The prion-like RNA-processing protein HNRPDL forms inherently toxic amyloid-like inclusion bodies in bacteria

BACKGROUND: The formation of protein inclusions is connected to the onset of many human diseases. Human RNA binding proteins containing intrinsically disordered regions with an amino acid composition resembling those of yeast prion domains, like TDP-43 or FUS, are being found to aggregate in different neurodegenerative disorders. The structure of the intracellular inclusions formed by these proteins is still unclear and whether these deposits have an amyloid nature or not is a matter of debate. Recently, the aggregation of TDP-43 has been modelled in bacteria, showing that TDP-43 inclusion bodies (IBs) are amorphous but intrinsically neurotoxic. This observation raises the question of whether it is indeed the lack of an ordered structure in these human prion-like protein aggregates the underlying cause of their toxicity in different pathological states. RESULTS: Here we characterize the IBs formed by the human prion-like RNA-processing protein HNRPDL. HNRPDL is linked to the development of limb-girdle muscular dystrophy 1G and shares domain architecture with TDP-43. We show that HNRPDL IBs display characteristic amyloid hallmarks, since these aggregates bind to amyloid dyes in vitro and inside the cell, they are enriched in intermolecular β-sheet conformation and contain inner amyloid-like fibrillar structure. In addition, despite their ordered structure, HNRPDL IBs are highly neurotoxic. CONCLUSIONS: Our results suggest that at least some of the disorders caused by the aggregation of human prion-like proteins would rely on the formation of classical amyloid assemblies rather than being caused by amorphous aggregates. They also illustrate the power of microbial cell factories to model amyloid aggregation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0284-7) contains supplementary material, which is available to authorized users