Structural characterisation of α-synuclein-membrane interactions and the resulting aggregation using small angle scattering.

The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid ß, interact with lipids, and this interaction can strongly favour the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to model membranes formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the model membranes take an active role in the reaction. The binding of α synuclein to the model membranes immediately induces a major structural change in the lipid assembly, which leads to a break-up into small and mostly disc- or rod-like lipid-protein particles. This transition can be reversed by temperature changes or proteolytic protein removal. Incubation of the small lipid-α-synuclein particles for several hours, however, leads to amyloid fibril formation, whereby the lipids are incorporated into the amyloid fibrils.

Factors Contributing to Delayed Return to Work among French Healthcare Professionals Afflicted by COVID-19 at a Hospital in the Rhône-Alpes Region, 2021.

COVID-19 is an emerging disease whose impact on the return to work of hospital staff is not yet known. This study was aimed at evaluating the prevalence of delayed return to work associated with medical, personal, and professional factors in hospital staff who tested positive for COVID-19 during the second epidemic wave. A descriptive, analytical observational study was conducted. The source population consisted of all staff of a French University Hospital Center who had an RT-PCR test or an antigenic test positive for SARS-CoV-2 during the period from 6 September to 30 November 2020. A delayed return to work was defined as a return to work after a period of at least 8 days of eviction, whereas before the eviction period decided by the French government was 14 days. Data collection was carried out through an anonymous online self-questionnaire. The participation rate was 43% (216 participants out of 502 eligible subjects). Moreover, 40% of the staff had a delayed return to work, and 24% of them reported a delayed return to work due to persistent asthenia. Delayed return to work was significantly associated with age, fear of returning to work, and persistent asthenia, but the number of symptoms lasting more than 7 days was the only factor that remained significantly associated after multivariate analysis. From this study, it appears that interest in identifying the number of persistent symptoms as a possible indicator of delayed work emerges. Moreover, persistent asthenia should be given special attention by practitioners to detect a possible long COVID.

Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen's large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.

High-Density Lipoprotein function is modulated by the SARS-CoV-2 spike protein in a lipid-type dependent manner.

There is a close relationship between the SARS-CoV-2 virus and lipoproteins, in particular high-density lipoprotein (HDL). The severity of the coronavirus disease 2019 (COVID-19) is inversely correlated with HDL plasma levels. It is known that the SARS-CoV-2 spike (S) protein binds the HDL particle, probably depleting it of lipids and altering HDL function. Based on neutron reflectometry (NR) and the ability of HDL to efflux cholesterol from macrophages, we confirm these observations and further identify the preference of the S protein for specific lipids and the consequent effects on HDL function on lipid exchange ability. Moreover, the effect of the S protein on HDL function differs depending on the individuals lipid serum profile. Contrasting trends were observed for individuals presenting low triglycerides/high cholesterol serum levels (LTHC) compared to high triglycerides/high cholesterol (HTHC) or low triglycerides/low cholesterol serum levels (LTLC). Collectively, these results suggest that the S protein interacts with the HDL particle and, depending on the lipid profile of the infected individual, it impairs its function during COVID-19 infection, causing an imbalance in lipid metabolism.

Structural Insights into the Catalytic Cycle of a Bacterial Multidrug ABC Efflux Pump.

ABC ("ATP-Binding Cassette") transporters of the type IV subfamily consist of exporters involved in the efflux of many compounds, notably those capable to confer multidrug resistance like the mammalian P-glycoprotein or the bacterial transporter BmrA. They function according to an alternating access mechanism between inward-facing (IF) and outward-facing (OF) conformations, but the extent of physical separation between the two nucleotide-binding domains (NBDs) in different states is still unsettled. Small Angle Neutron Scattering and hydrogen/deuterium exchange coupled to mass spectrometry were used to highlight different conformational states of BmrA during its ATPase cycle. In particular, mutation of the conserved Lysine residue of the Walker-A motif (K380A) captures BmrA in an ATP-bound IF conformation prior to NBD closure. While in the transition-like state induced by vanadate wild-type BmrA is mainly in an OF conformation, the transporter populates only IF conformations in either the apo state or in the presence of ADP/Mg. Importantly, in this post-hydrolytic step, distances between the two NBDs of BmrA seem to be more separated than in the apo state, but they remain shorter than the widest opening found in the related MsbA transporter. Overall, our results highlight the main steps of the catalytic cycle of a homodimeric bacterial multidrug transporter and underline structural and functional commonalities as well as oddities among the type IV subfamily of ABC transporters.

Cryo-EM structure of MsbA in saposin-lipid nanoparticles (Salipro) provides insights into nucleotide coordination.

The ATP-binding cassette transporter MsbA is a lipid flippase, translocating lipid A, glycolipids, and lipopolysaccharides from the inner to the outer leaflet of the inner membrane of Gram-negative bacteria. It has been used as a model system for time-resolved structural studies as several MsbA structures in different states and reconstitution systems (detergent/nanodiscs/peptidiscs) are available. However, due to the limited resolution of the available structures, detailed structural information on the bound nucleotides has remained elusive. Here, we have reconstituted MsbA in saposin A-lipoprotein nanoparticles (Salipro) and determined the structure of ADP-vanadate-bound MsbA by single-particle cryo-electron microscopy to 3.5 Å resolution. This procedure has resulted in significantly improved resolution and enabled us to model all side chains and visualise detailed ADP-vanadate interactions in the nucleotide-binding domains. The approach may be applicable to other dynamic membrane proteins.

Telework and Psychological Health in Hospital Staff during the First Wave of the COVID-19 Epidemic in France.

BACKGROUND: The COVID-19 pandemic led to a change in work organization with the development of telework. The purpose of this study was to assess the prevalence of anxiety and depressive symptoms in teleworking staff in a university hospital center in France during the first lockdown, and to identify personal, medical and occupational factors associated with anxiety disorder. METHODS: A cross-sectional observational study was conducted in 474 hospital staff working from home during the first lockdown. The sociodemographic, occupational and medical information (anxiety and depressive disorders measured on the Hospital Anxiety and Depression (HAD) scale) was collected by an anonymous online self-administered questionnaire. The variables associated with anxiety disorder were investigated by a univariate analysis (chi² and Fisher tests) and a multivariate analysis (logistic regression model). RESULTS: Three hundred and forty hospital staff participated in the study (72% response rate). Of the participants, 106 subjects (32.1%) showed signs of an anxiety disorder and 26 (7.65%) of a depressive disorder. An anxiety disorder was significantly associated with mental workload, changes in working hours, difficulties in teleworking due to issues of internet connection or due to noise, difficulties in combining family and occupational life, sleep disturbance, worry about media information and worry about the health of a loved one. An anxiety disorder remained associated with occupational stress and personal stress during lockdown after a multivariate logistic regression. CONCLUSIONS: This study highlighted the association between an anxiety disorder and perceived occupational and personal stress levels in hospital staff teleworking during the first lockdown. Stress management workshops could be proposed to hospital staff. Prevention of anxiety requires reinforced medical monitoring and reduced stress.

Elongation rate and average length of amyloid fibrils in solution using isotope-labelled small-angle neutron scattering.

We demonstrate a solution method that allows both elongation rate and average fibril length of assembling amyloid fibrils to be estimated. The approach involves acquisition of real-time neutron scattering data during the initial stages of seeded growth, using contrast matched buffer to make the seeds effectively invisible to neutrons. As deuterated monomers add on to the seeds, the labelled growing ends give rise to scattering patterns that we model as cylinders whose increase in length with time gives an elongation rate. In addition, the absolute intensity of the signal can be used to determine the number of growing ends per unit volume, which in turn provides an estimate of seed length. The number of ends did not change significantly during elongation, demonstrating that any spontaneous or secondary nucleation was not significant compared with growth on the ends of pre-existing fibrils, and in addition providing a method of internal validation for the technique. Our experiments on initial growth of alpha synuclein fibrils using 1.2 mg ml-1 seeds in 2.5 mg ml-1 deuterated monomer at room temperature gave an elongation rate of 6.3 ± 0.5 Å min-1, and an average seed length estimate of 4.2 ± 1.3 µm.

SARS-CoV-2 spike protein removes lipids from model membranes and interferes with the capacity of high density lipoprotein to exchange lipids.

Cholesterol has been shown to affect the extent of coronavirus binding and fusion to cellular membranes. The severity of Covid-19 infection is also known to be correlated with lipid disorders. Furthermore, the levels of both serum cholesterol and high-density lipoprotein (HDL) decrease with Covid-19 severity, with normal levels resuming once the infection has passed. Here we demonstrate that the SARS-CoV-2 spike (S) protein interferes with the function of lipoproteins, and that this is dependent on cholesterol. In particular, the ability of HDL to exchange lipids from model cellular membranes is altered when co-incubated with the spike protein. Additionally, the S protein removes lipids and cholesterol from model membranes. We propose that the S protein affects HDL function by removing lipids from it and remodelling its composition/structure.

ApoE and ApoE Nascent-Like HDL Particles at Model Cellular Membranes: Effect of Protein Isoform and Membrane Composition.

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body.

Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis.

The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.

Apolipoprotein E Binding Drives Structural and Compositional Rearrangement of mRNA-Containing Lipid Nanoparticles.

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP's plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis.

Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to de-hydro-isourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hy-droxy-isourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the 'peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O-H⋯π interaction with 8AZA. The active site Lys10-Thr57 dyad features a charged Lys10-NH3 + side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1-HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis.

Observing Protein Degradation by the PAN-20S Proteasome by Time-Resolved Neutron Scattering.

The proteasome is a key player of regulated protein degradation in all kingdoms of life. Although recent atomic structures have provided snapshots on a number of conformations, data on substrate states and populations during the active degradation process in solution remain scarce. Here, we use time-resolved small-angle neutron scattering of a deuterium-labeled GFPssrA substrate and an unlabeled archaeal PAN-20S system to obtain direct structural information on substrate states during ATP-driven unfolding and subsequent proteolysis in solution. We find that native GFPssrA structures are degraded in a biexponential process, which correlates strongly with ATP hydrolysis, the loss of fluorescence, and the buildup of small oligopeptide products. Our solution structural data support a model in which the substrate is directly translocated from PAN into the 20S proteolytic chamber, after a first, to our knowledge, successful unfolding process that represents a point of no return and thus prevents dissociation of the complex and the release of harmful, aggregation-prone products.

Lipoprotein ability to exchange and remove lipids from model membranes as a function of fatty acid saturation and presence of cholesterol.

Lipoproteins play a central role in the development of atherosclerosis. High and low-density lipoproteins (HDL and LDL), known as 'good' and 'bad' cholesterol, respectively, remove and/or deposit lipids into the artery wall. Hence, insight into lipid exchange processes between lipoproteins and cell membranes is of particular importance in understanding the onset and development of cardiovascular disease. In order to elucidate the impact of phospholipid tail saturation and the presence of cholesterol in cell membranes on these processes, neutron reflection was employed in the present investigation to follow lipid exchange with both HDL and LDL against model membranes. Mirroring clinical risk factors for the development of atherosclerosis, lower exchange was observed in the presence of cholesterol, as well as for an unsaturated phospholipid, compared to faster exchange when using a fully saturated phospholipid. These results highlight the importance of membrane composition on the interaction with lipoproteins, chiefly the saturation level of the lipids and presence of cholesterol, and provide novel insight into factors of importance for build-up and reversibility of atherosclerotic plaque. In addition, the correlation between the results and well-established clinical risk factors suggests that the approach taken can be employed also for understanding a broader set of risk factors including, e.g., effects of triglycerides and oxidative stress, as well as local effects of drugs on atherosclerotic plaque formation.

Interdomain Flexibility within NADPH Oxidase Suggested by SANS Using LMNG Stealth Carrier.

Small angle neutron scattering (SANS) provides a method to obtain important low-resolution information for integral membrane proteins (IMPs), challenging targets for structural determination. Specific deuteration furnishes a "stealth" carrier for the solubilized IMP. We used SANS to determine a structural envelope of SpNOX, the Streptococcus pneumoniae NADPH oxidase (NOX), a prokaryotic model system for exploring structure and function of eukaryotic NOXes. SpNOX was solubilized in the detergent lauryl maltose neopentyl glycol, which provides optimal SpNOX stability and activity. Using deuterated solvent and protein, the lauryl maltose neopentyl glycol was experimentally undetected in SANS. This affords a cost-effective SANS approach for obtaining novel structural information on IMPs. Combining SANS data with molecular modeling provided a first, to our knowledge, structural characterization of an entire NOX enzyme. It revealed a distinctly less compact structure than that predicted from the docking of homologous crystal structures of the separate transmembrane and dehydrogenase domains, consistent with a flexible linker connecting the two domains.

A Dimerization Site at SCR-17/18 in Factor H Clarifies a New Mechanism for Complement Regulatory Control.

Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.

In Vivo Water Dynamics in Shewanella oneidensis Bacteria at High Pressure.

Following observations of survival of microbes and other life forms in deep subsurface environments it is necessary to understand their biological functioning under high pressure conditions. Key aspects of biochemical reactions and transport processes within cells are determined by the intracellular water dynamics. We studied water diffusion and rotational relaxation in live Shewanella oneidensis bacteria at pressures up to 500 MPa using quasi-elastic neutron scattering (QENS). The intracellular diffusion exhibits a significantly greater slowdown (by -10-30%) and an increase in rotational relaxation times (+10-40%) compared with water dynamics in the aqueous solutions used to resuspend the bacterial samples. Those results indicate both a pressure-induced viscosity increase and slowdown in ionic/macromolecular transport properties within the cells affecting the rates of metabolic and other biological processes. Our new data support emerging models for intracellular organisation with nanoscale water channels threading between macromolecular regions within a dynamically organized structure rather than a homogenous gel-like cytoplasm.

Time-resolved small-angle neutron scattering as a probe for the dynamics of lipid exchange between human lipoproteins and naturally derived membranes.

Atherosclerosis is the main killer in the western world. Today's clinical markers include the total level of cholesterol and high-/low-density lipoproteins, which often fails to accurately predict the disease. The relationship between the lipid exchange capacity and lipoprotein structure should explain the extent by which they release or accept lipid cargo and should relate to the risk for developing atherosclerosis. Here, small-angle neutron scattering and tailored deuteration have been used to follow the molecular lipid exchange between human lipoprotein particles and cellular membrane mimics made of natural, "neutron invisible" phosphatidylcholines. We show that lipid exchange occurs via two different processes that include lipid transfer via collision and upon direct particle tethering to the membrane, and that high-density lipoprotein excels at exchanging the human-like unsaturated phosphatidylcholine. By mapping the specific lipid content and level of glycation/oxidation, the mode of action of specific lipoproteins can now be deciphered. This information can prove important for the development of improved diagnostic tools and in the treatment of atherosclerosis.

Protein Short-Time Diffusion in a Naturally Crowded Environment.

The interior of living cells is a dense and polydisperse suspension of macromolecules. Such a complex system challenges an understanding in terms of colloidal suspensions. As a fundamental test we employ neutron spectroscopy to measure the diffusion of tracer proteins (immunoglobulins) in a cell-like environment (cell lysate) with explicit control over crowding conditions. In combination with Stokesian dynamics simulation, we address protein diffusion on nanosecond time scales where hydrodynamic interactions dominate over negligible protein collisions. We successfully link the experimental results on these complex, flexible molecules with coarse-grained simulations providing a consistent understanding by colloid theories. Both experiments and simulations show that tracers in polydisperse solutions close to the effective particle radius Reff = ⟨ Ri3⟩1/3 diffuse approximately as if the suspension was monodisperse. The simulations further show that macromolecules of sizes R > Reff ( R < Reff) are slowed more (less) effectively even at nanosecond time scales, which is highly relevant for a quantitative understanding of cellular processes.