117 results found
Reddy UV, Weber DK, Wang S, et al., 2021, A kink in DWORF helical structure controls the activation of the sarcoplasmic reticulum Ca2+-ATPase., Structure
SERCA is a P-type ATPase embedded in the sarcoplasmic reticulum and plays a central role in muscle relaxation. SERCA's function is regulated by single-pass membrane proteins called regulins. Unlike other regulins, dwarf open reading frame (DWORF) expressed in cardiac muscle has a unique activating effect. Here, we determine the structure and topology of DWORF in lipid bilayers using a combination of oriented sample solid-state NMR spectroscopy and replica-averaged orientationally restrained molecular dynamics. We found that DWORF's structural topology consists of a dynamic N-terminal domain, an amphipathic juxtamembrane helix that crosses the lipid groups at an angle of 64°, and a transmembrane C-terminal helix with an angle of 32°. A kink induced by Pro15, unique to DWORF, separates the two helical domains. A single Pro15Ala mutant significantly decreases the kink and eliminates DWORF's activating effect on SERCA. Overall, our findings directly link DWORF's structural topology to its activating effect on SERCA.
Weber DK, Reddy UV, Wang S, et al., 2021, Structural basis for allosteric control of the SERCA-Phospholamban membrane complex by Ca2+ and phosphorylation, ELIFE, Vol: 10, ISSN: 2050-084X
Sanz-Hernandez M, Barritt JD, Sobek J, et al., 2021, Mechanism of misfolding of the human prion protein revealed by a pathological mutation, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 118, ISSN: 0027-8424
Perni M, van der Goot A, Limbocker R, et al., 2021, Comparative studies in the A30P and A53T alpha-Synuclein C. elegans strains to investigate the molecular origins of Parkinson's Disease, Frontiers in Cell and Developmental Biology, Vol: 9, Pages: 1-10, ISSN: 2296-634X
The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two C. elegans models of PD (PDA30P and PDA53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PDWT). PDA30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PDA53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. α-Synuclein was also observed to be less well localized into aggregates in PDA30P worms compared to PDA53T and PDWT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of α-synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of α-synuclein and its associated toxicity in PDA53T and PDWT worms, but had less marked effects in PDA30P. In addition, using an antibody that targets the N-terminal region of α-synuclein, we observed a suppression of toxicity in PDA30P, PDA53T and PDWT worms. These results illustrate the use of these two C. elegans models in fundamental and applied PD research.
Gonzalez-Garcia M, Fusco G, De Simone A, 2021, Membrane interactions and toxicity by misfolded protein oligomers, Frontiers in Cell and Developmental Biology, Vol: 9, Pages: 1-12, ISSN: 2296-634X
The conversion of otherwise soluble proteins into insoluble amyloid aggregates is associated with a range of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, as well as non-neuropathic conditions such as type II diabetes and systemic amyloidoses. It is increasingly evident that the most pernicious species among those forming during protein aggregation are small prefibrillar oligomers. In this review, we describe the recent progress in the characterization of the cellular and molecular interactions by toxic misfolded protein oligomers. A fundamental interaction by these aggregates involves biological membranes, resulting in two major model mechanisms at the onset of the cellular toxicity. These include the membrane disruption model, resulting in calcium imbalance, mitochondrial dysfunction and intracellular reactive oxygen species, and the direct interaction with membrane proteins, leading to the alteration of their native function. A key challenge remains in the characterization of transient interactions involving heterogeneous protein aggregates. Solving this task is crucial in the quest of identifying suitable therapeutic approaches to suppress the cellular toxicity in protein misfolding diseases.
Nguyen PH, Ramamoorthy A, Sahoo BR, et al., 2021, Amyloid Oligomers: A Joint Experimental/Computational Perspective on Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, and Amyotrophic Lateral Sclerosis, CHEMICAL REVIEWS, Vol: 121, Pages: 2545-2647, ISSN: 0009-2665
Sanz-Hernandez M, De Simone A, 2021, Backbone NMR assignments of the C-terminal domain of the human prion protein and its disease-associated T183A variant, BIOMOLECULAR NMR ASSIGNMENTS, Vol: 15, Pages: 193-196, ISSN: 1874-2718
Man W, Tahirbegi B, Vrettas M, et al., 2021, The docking of synaptic vesicles on the presynaptic membrane induced by α-synuclein is modulated by lipid composition, Nature Communications, Vol: 12, ISSN: 2041-1723
α-Synuclein (αS) is a presynaptic disordered protein whose aberrant aggregation is associated with Parkinson’s disease. The functional role of αS is still debated, although it has been involved in the regulation of neurotransmitter release via the interaction with synaptic vesicles (SVs). We report here a detailed characterisation of the conformational properties of αS bound to the inner and outer leaflets of the presynaptic plasma membrane (PM), using small unilamellar vesicles. Our results suggest that αS preferentially binds the inner PM leaflet. On the basis of these studies we characterise in vitro a mechanism by which αS stabilises, in a concentration-dependent manner, the docking of SVs on the PM by establishing a dynamic link between the two membranes. The study then provides evidence that changes in the lipid composition of the PM, typically associated with neurodegenerative diseases, alter the modes of binding of αS, specifically in a segment of the sequence overlapping with the non-amyloid component region. Taken together, these results reveal how lipid composition modulates the interaction of αS with the PM and underlie its functional and pathological behaviours in vitro.
O'Brien HER, Zhang XF, Sanz-Hernandez M, et al., 2021, Blocking von Willebrand factor free thiols inhibits binding to collagen under high and pathological shear stress, Journal of Thrombosis and Haemostasis, Vol: 19, Pages: 358-369, ISSN: 1538-7836
BackgroundVon Willebrand factor (VWF) contains a number of free thiols, the majority of which are located in its C‐domains, and these have been shown to alter VWF function, However, the impact of free thiols on function following acute exposure of VWF to collagen under high and pathological shear stress has not been determined.MethodsVWF free thiols were blocked with N‐ethylmaleimide and flow assays performed under high and pathological shear rates to determine the impact on platelet capture and collagen binding function. Atomic force microscopy (AFM) was used to probe the interaction of VWF with collagen and molecular simulations conducted to determine the effect of free thiols on the flexibility of the VWF‐C4 domain.ResultsBlockade of VWF free thiols reduced VWF‐mediated platelet capture to collagen in a shear‐dependent manner, with platelet capture virtually abolished above 5000 s−1 and in regions of stenosis in microfluidic channels. Direct visualization of VWF fibers formed under extreme pathological shear rates and analysis of collagen‐bound VWF attributed the effect to altered binding of VWF to collagen. AFM measurements showed that thiol‐blockade reduced the lifetime and strength of the VWF‐collagen bond. Pulling simulations of the VWF‐C4 domain demonstrated that with one or two reduced disulphide bonds the C4 domain has increased flexibility and the propensity to undergo free‐thiol exchange.ConclusionsWe conclude that free thiols in the C‐domains of VWF enhance the flexibility of the molecule and enable it to withstand high shear forces following collagen binding, demonstrating a previously unrecognized role for VWF free thiols.
Boi L, Pisanu A, Fusco G, et al., 2020, Modeling Parkinson’s disease neuropathology and symptoms by intranigral inoculation of preformed human α-synuclein oligomers, International Journal of Molecular Sciences, Vol: 21, ISSN: 1422-0067
The accumulation of aggregated α-synuclein (αSyn) is a hallmark of Parkinson’s disease (PD). Current evidence indicates that small soluble αSyn oligomers (αSynOs) are the most toxic species among the forms of αSyn aggregates, and that size and topological structural properties are crucial factors for αSynOs-mediated toxicity, involving the interaction with either neurons or glial cells. We previously characterized a human αSynO (H-αSynO) with specific structural properties promoting toxicity against neuronal membranes. Here, we tested the neurotoxic potential of these H-αSynOs in vivo, in relation to the neuropathological and symptomatic features of PD. The H-αSynOs were unilaterally infused into the rat substantia nigra pars compacta (SNpc). Phosphorylated αSyn (p129-αSyn), reactive microglia, and cytokine levels were measured at progressive time points. Additionally, a phagocytosis assay in vitro was performed after microglia pre-exposure to αsynOs. Dopaminergic loss, motor, and cognitive performances were assessed. H-αSynOs triggered p129-αSyn deposition in SNpc neurons and microglia and spread to the striatum. Early and persistent neuroinflammatory responses were induced in the SNpc. In vitro, H-αSynOs inhibited the phagocytic function of microglia. H-αsynOs-infused rats displayed early mitochondrial loss and abnormalities in SNpc neurons, followed by a gradual nigrostriatal dopaminergic loss, associated with motor and cognitive impairment. The intracerebral inoculation of structurally characterized H-αSynOs provides a model of progressive PD neuropathology in rats, which will be helpful for testing neuroprotective therapies.
Heller GT, Aprile FA, Michaels TCT, et al., 2020, Small-molecule sequestration of amyloid-beta as a drug discovery strategy for Alzheimer's disease, SCIENCE ADVANCES, Vol: 6, ISSN: 2375-2548
Stephens AD, Zacharopoulou M, Moons R, et al., 2020, Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity, NATURE COMMUNICATIONS, Vol: 11, ISSN: 2041-1723
Navarro-Paya C, Sanz-Hernandez M, De Simone A, 2020, In Silico Study of the Mechanism of Binding of the N-Terminal Region of alpha Synuclein to Synaptic-Like Membranes, LIFE-BASEL, Vol: 10
Carta AR, Boi L, Pisanu A, et al., 2020, Advances in modelling alpha-synuclein-induced Parkinson's diseases in rodents: Virus-based models versus inoculation of exogenous preformed toxic species, JOURNAL OF NEUROSCIENCE METHODS, Vol: 338, ISSN: 0165-0270
Visconti L, Toto A, Jarvis J, et al., 2020, Demonstration of binding induced structural plasticity in a SH2 domain, Frontiers in Molecular Biosciences, Vol: 7, ISSN: 2296-889X
SH2 domains are common protein interaction domains able to recognize short aminoacidic sequences presenting a phosphorylated tyrosine (pY). In spite of their fundamental importance for cell physiology there is a lack of information about the mechanism by which these domains recognize and bind their natural ligands. The N-terminal SH2 (N-SH2) domain of PI3K mediates the interaction with different scaffolding proteins and is known to recognize a specific pY-X-X-M consensus sequence. These interactions are at the cross roads of different molecular pathways and play a key role for cell development and division. By combining mutagenesis, chemical kinetics and NMR, here we provide a complete characterization of the interaction between N-SH2 and a peptide mimicking the scaffolding protein Gab2. Our results highlight that N-SH2 is characterized by a remarkable structural plasticity, with the binding reaction being mediated by a diffused structural region and not solely by the residues located in the binding pocket. Furthermore, the analysis of kinetic data allow us to pinpoint an allosteric network involving residues far from the binding pocket involved in specificity. Results are discussed on the light of previous works on the binding properties of SH2 domains.
Man WK, De Simone A, Barritt JD, et al., 2020, A Role of Cholesterol in Modulating the Binding of alpha-Synuclein to Synaptic-Like Vesicles, FRONTIERS IN NEUROSCIENCE, Vol: 14
Runfola M, De Simone A, Vendruscolo M, et al., 2020, The N-terminal Acetylation of alpha-Synuclein Changes the Affinity for Lipid Membranes but not the Structural Properties of the Bound State, SCIENTIFIC REPORTS, Vol: 10, ISSN: 2045-2322
Qu F, ElOmari K, Wagner A, et al., 2019, Desolvation of the substrate binding protein TauA dictates ligand specificity for the alkanesulfonate ABC importer TauABC, Biochemical Journal, Vol: 476, Pages: 3649-3660, ISSN: 0264-6021
Under limiting sulfur availability, bacteria can assimilate sulfur from alkanesulfonates. Bacteria utilize ATP-binding cassette (ABC) transporters to internalise them for further processing to release sulfur. In gram-negative bacteria the TauABC and SsuABC ensure internalization, although, these two systems have common substrates, the former has been characterised as a taurine specific system. TauA and SsuA are substrate binding proteins (SBPs) that bind and bring the alkanesulfonates to the ABC importer for transport. Here, we have determined the crystal structure of TauA and have characterised its thermodynamic binding parameters by isothermal titration calorimetry in complex with taurine and different alkanesulfonates. Our structures revealed that the coordination of the alkanesulfonates is conserved, with the exception of Asp205 that is absent in SsuA, but the thermodynamic parameters revealed a very high enthalpic penalty cost for binding of the other alkanesulfonates relative to taurine. Our molecular dynamic simulations indicated that the different levels of hydration of the binding site contributed to the selectivity for taurine over the other alkanesulfonates. Such selectivity mechanism is very likely to be employed by other SBPs of ABC transporters.
Wang X, Kirkpatrick JP, Launay HMM, et al., 2019, Probing the dynamic stalk region of the ribosome using solution NMR, SCIENTIFIC REPORTS, Vol: 9, ISSN: 2045-2322
Achour A, Broggini L, Han X, et al., 2019, Biochemical and biophysical comparison of human and mouse beta-2 microglobulin reveals the molecular determinants of low amyloid propensity, FEBS JOURNAL, Vol: 287, Pages: 546-560, ISSN: 1742-464X
Vivoli Vega M, Cascella R, Chen SW, et al., 2019, The Toxicity of Misfolded Protein Oligomers Is Independent of Their Secondary Structure., ACS Chem Biol, Vol: 14, Pages: 1593-1600
The self-assembly of proteins into structured fibrillar aggregates is associated with a range of neurodegenerative diseases, including Alzheimer's and Parkinson's diseases, in which an important cytotoxic role is thought to be played by small soluble oligomers accumulating during the aggregation process or released by mature fibrils. As the structural characteristics of such species and their links with toxicity are still not fully defined, we have compared six examples of preformed misfolded protein oligomers with different β-sheet content, as determined using Fourier transform infrared spectroscopy, and with different toxicity, as determined by three cellular readouts of cell viability. The results show the absence of any measurable correlation between the nature of their secondary structure and their cellular toxicity, both when comparing the six types of oligomers as a group and when comparing species in subgroups characterized by either the same size or the same exposure of hydrophobic moieties.
Cascella R, Perni M, Chen SW, et al., 2019, Probing the Origin of the Toxicity of Oligomeric Aggregates of alpha-Synuclein with Antibodies, ACS CHEMICAL BIOLOGY, Vol: 14, Pages: 1352-1362, ISSN: 1554-8929
He S, Biedermann F, Vankova N, et al., 2018, Cavitation energies can outperform dispersion interactions, Nature Chemistry, Vol: 10, Pages: 1252-1257, ISSN: 1755-4330
The accurate dissection of binding energies into their microscopic components is challenging, especially in solution. Here we study the binding of noble gases (He-Xe) with the macrocyclic receptor cucurbituril in water by displacement of methane and ethane as 1H NMR probes. We dissect the hydration free energies of the noble gases into an attractive dispersive component and a repulsive one for formation of a cavity in water. This allows us to identify the contributions to host-guest binding and to conclude that the binding process is driven by differential cavitation energies rather than dispersion interactions. The free energy required to create a cavity to accept the noble gas inside the cucurbituril is much lower than that to create a similarly sized cavity in bulk water. The recovery of the latter cavitation energy drives the overall process, which has implications for the refinement of gas-storage materials and the understanding of biological receptors.
Balasco N, Smaldone G, Ruggiero A, et al., 2018, Local structural motifs in proteins: Detection and characterization of fragments inserted in helices, INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, Vol: 118, Pages: 1924-1930, ISSN: 0141-8130
Patel JR, Xu Y, Capitini C, et al., 2018, Backbone NMR assignments of HypF-N under conditions generating toxic and non-toxic oligomers, BIOMOLECULAR NMR ASSIGNMENTS, Vol: 12, Pages: 273-277, ISSN: 1874-2718
Gianni S, McCully ME, Malagrino F, et al., 2018, A Carboxylate to Amide Substitution That Switches Protein Folds, ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, Vol: 57, Pages: 12795-12798, ISSN: 1433-7851
Balasco N, Barone D, Iaccarino E, et al., 2018, Intrinsic structural versatility of the highly conserved 412-423 epitope of the Hepatitis C Virus E2 protein, INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, Vol: 116, Pages: 620-632, ISSN: 0141-8130
Capitini C, Patel JR, Natalello A, et al., 2018, Structural differences between toxic and nontoxic HypF-N oligomers, CHEMICAL COMMUNICATIONS, Vol: 54, Pages: 8637-8640, ISSN: 1359-7345
Fusco G, Sanz-Hernandez M, Ruggeri FS, et al., 2018, Molecular determinants of the interaction of EGCG with ordered and disordered proteins, BIOPOLYMERS, Vol: 109, ISSN: 0006-3525
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