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Interaction Between alpha-Synuclein and Metal Ions, Still Looking for a Role in the Pathogenesis of Parkinson's Disease
No full textThe most recent literature on the interaction between α-synuclein in its several aggregation states and metal ions is discussed. This analysis shows two major types of interactions. Binding sites are present in the C-terminal region, and similar, low affinity (in the millimolar range) is exhibited toward many different metal ions, including copper and iron. A more complex scenario emerges for these latter metal ions, which are also able to coordinate with high affinity (in the micromolar range) to the N-terminal region of α-synuclein. Moreover, these redox-active metal ions may induce chemical modifications on the protein in vitro and in the reducing intracellular environment, and these modifications might be relevant for the aggregation properties of α-synuclein. Finally, an attempt is made to contextualize the interaction between α-synuclein and these metal ions in the framework of the elusive and multifactorial pathogenesis of Parkinson’s disease
Observing the osmophobic effect in action at the single molecule level
No full textProtecting osmolytes are widespread small organic molecules able to stabilize the folded state of most proteins against various denaturing stresses in vivo. The osmophobic model explains thermodynamically their action through a preferential exclusion of the osmolyte molecules from the protein surface, thus favoring the formation of intrapeptide hydrogen bonds. Few works addressed the influence of protecting osmolytes on the protein unfolding transition state and kinetics. Among those, previous single molecule force spectroscopy experiments evidenced a complexation of the protecting osmolyte molecules at the unfolding transition state of the protein, in apparent contradiction with the osmophobic nature of the protein backbone. We present single-molecule evidence that glycerol, which is a ubiquitous protecting osmolyte, stabilizes a globular protein against mechanical unfolding without binding into its unfolding transition state structure. We show experimentally that glycerol does not change the position of the unfolding transition state as projected onto the mechanical reaction coordinate. Moreover, we compute theoretically the projection of the unfolding transition state onto two other common reaction coordinates, that is, the number of native peptide bonds and the weighted number of native contacts. To that end, we augment an analytic Ising-like protein model with support for group-transfer free energies. Using this model, we find again that the position of the unfolding transition state does not change in the presence of glycerol, giving further support to the conclusions based on the single-molecule experiments
Single-molecule force spectroscopy of chimeric polyprotein constructs containing intrinsically disordered domains
No full textHere, we describe the single molecule force spectroscopy (SMFS)-based experimental protocol we have recently used to single out different classes of conformations in a chimeric multimodular protein containing an intrinsically disordered (human Alpha Synuclein) domain. Details are provided regarding cloning, expression and purification of the chimeric polyprotein constructs, optimal surface preparation, SMFS data collection and filtering. Although the specificity of the issue and the ensemble of nonstandard techniques needed to perform the described procedures render this a rather unorthodox protocol, it is relatively straightforward to adapt it to the study of other protein domains
Worm-like ising model for protein mechanical unfolding under the effect of osmolytes.
No full textWe show via single-molecule mechanical unfolding experiments that the osmolyte glycerol stabilizes the native state of the human cardiac I27 titin module against unfolding without shifting its unfolding transition state on the mechanical reaction coordinate. Taken together with similar findings on the immunoglobulin-binding domain of streptococcal protein G (GB1), these experimental results suggest that osmolytes act on proteins through a common mechanism that does not entail a shift of their unfolding transition state. We investigate the above common mechanism via an Ising-like model for protein mechanical unfolding that adds worm-like-chain behavior to a recent generalization of the Wako-Saitô-Muñoz-Eaton model with support for group-transfer free energies. The thermodynamics of the model are exactly solvable, while protein kinetics under mechanical tension can be simulated via Monte Carlo algorithms. Notably, our force-clamp and velocity-clamp simulations exhibit no shift in the position of the unfolding transition state of GB1 and I27 under the effect of various osmolytes. The excellent agreement between experiment and simulation strongly suggests that osmolytes do not assume a structural role at the mechanical unfolding transition state of proteins, acting instead by adjusting the solvent quality for the protein chain analyte
Cloning, expression, purification, and spectroscopic analysis of the fragment 57-102 of human alpha-synuclein
No full textThe protein alpha-synuclein plays an important role in many neurodegenerative disorders.. referred to as alpha-synucleinopathies, that include, among others, Parkinson's and Alzheimer's diseases. The central region of the wild type protein, known as the non-Abeta component of amyloid plaques (NAC, amino acids 61-95), seems to be responsible for its aggregation process. To structurally characterize this fragment by nuclear magnetic resonance, we produced it by DNA recombinant technology. This technique, unlike chemical synthesis, allows the production of labeled samples (C-13, N-15) required for NMR studies. Because the NAC region is very sparingly soluble in aqueous buffer, we cloned a slightly larger portion of alpha-synuclein, alphasyn57-102, with the presence of several charged residues in both extremities of the NAC region. The conformational preferences of purified alphasyn57-102, in solution and bound to SDS micelles, was studied. Our results indicate that the protein is largely unfolded in solution but exhibits a helical conformation in the lipid-associated state. The methodology that we have used in this work for the cloning, expression, and purification of alphasyn57-102 can be easily applied to most small proteins, thus representing a powerful tool for structural NMR analysis of labeled peptides
DJ-1 is a copper chaperone acting on SOD1 activation.
Full text linkLack of oxidative stress control is a common and often prime feature observed in many neurodegenerative diseases. Both DJ-1 and SOD1, proteins involved in familial Parkinson disease and amyotrophic lateral sclerosis, respectively, play a protective role against oxidative stress. Impaired activity and modified expression of both proteins have been observed in different neurodegenerative diseases. A potential cooperative action of DJ-1 and SOD1 in the same oxidative stress response pathway may be suggested basedonacopper- mediatedinteractionbetweenthetwo proteins reported here. To investigate the mechanisms underlying the antioxidative function of DJ-1 in relation to SOD1 activity, we investigated the ability of DJ-1 to bind copper ions.Westructurally characterized a novel copper binding site involving Cys-106, and we investigated, using different techniques, the kinetics of DJ-1 binding to copper ions. The copper transfer between the two proteins was also examined using both fluorescence spectroscopy and specific biochemical assays for SOD1 activity. The structural and functional analysis of the novel DJ-1 copper binding site led us to identify a putative role for DJ-1 as a copper chaperone. Alteration of the coordination geometry of the copper ion in DJ-1 may be correlated to the physiological role of the protein, to a potential failure in metal transfer to SOD1, and to successive implications in neurodegenerative etiopathogenesis
Single-molecule-level evidence for the osmophobic effect.
No full textProtecting osmolytes play a crucial role in preventing protein denaturation in harsh environmental conditions of living organisms. Experimental evidence is provided for a mechanism of protein-fold stabilization by these molecules that is in accord with the hypothesis of a backbone-based osmophobic effect. (In picture: ΔG=free energy, [O]=osmolyte concentration, χ=unfolding reaction coordinate.
Interaction of Curcumin with α-Synuclein and its Relationship to Curcumin's Ability to Inhibit Fibril Deposit.
No full textLewy bodies and Lewy neurites in the brain constitute the main histopathological features of Parkinson’s disease (PD), and are comprised of amyloid-like fibrils composed of a small protein named α-synuclein (AS). As the aggregation of AS in the brain has been implicated as a critical step in the development of the diseases, the current search for disease-modifying drugs is focused on molecules that can act on the process of AS deposition in the brain. Recently, curcumin, a constituent of the Indian spice Turmeric, structurally similar to Congo Red, has been demonstrated to bind Aβ amyloid and prevent further oligomerization of Aβ monomers onto growing amyloid β-sheets. Reasoning that oligomerization kinetics and mechanism of amyloid formation may be similar in Parkinson’s disease and Alzheimer’s disease, some authors suggested the use of curcumin to prevent AS aggregation and/or to reduce AS fibrils. Although the mechanisms by which this molecule inhibits Aβ fibril formation and destabilizes preformed fibrils are still unclear, it could be an effective starting template for the development of preventive and therapeutic drugs for PD.
With the aim to unravel the mechanism of action of curcumin, we investigated the interactions of curcumin with wild-type AS by NMR, CD and fluorescence spectroscopies. The chiroptical properties of curcumin make this molecule an ideal compound to perform interaction CD studies both in the far-UV and in the visible region of the spectrum. On the other hand, the presence of four tyrosine residues in the AS sequence provide a useful in-site fluorescence probe for quenching studies
Dopamine-derived Quinones Affect the Structure of the Redox Sensor DJ-1 through Modifications at Cys-106 andCys-53
No full textThe physiological role of DJ-1, a protein involved in familial Parkinson disease is still controversial. One of the hypotheses proposed indicates a sensor role for oxidative stress, through oxidation of a conserved cysteine residue (Cys-106). The association of DJ-1 mutations with Parkinson disease suggests a loss of function, specific to dopaminergic neurons. Under oxidative conditions, highly reactive dopamine quinones (DAQs) can be produced, which can modify cysteine residues. In cellular models, DJ-1 was found covalently modified by dopamine. We analyzed the structural modifications induced on human DJ-1 by DAQs in vitro. We described the structural perturbations induced byDAQadduct formation on each of the three cysteine residues of DJ-1 using specific mutants. Cys-53 is the most reactive residue and forms a covalent dimer also in SH-SY5Y DJ-1- transfected cells, but modification of Cys-106 induces the most severe structural perturbations; Cys-46 is not reactive. The relevance of these covalent modifications to the several functions ascribed to DJ-1 is discussed in the context of the cell response to a dopamine-derived oxidative insult
CD and Fluorescence Screening of α-Synuclein-Peptide Interactions.
No full textα-Synuclein (AS), a natively unfolded protein, is the major components of the intracellular protein-aggregates, the Lewy bodies, found in the dopaminergic neurons of Parkinson's disease patients. The aggregates called "protofibrils," an intermediate in the fibrillogenesis process, are more cytotoxic than the amyloid-like fibrils in most of the proteins which generate fibrils. On one hand, aggregation inhibitors are expected to reduce AS cytotoxicity by preventing protofibril formation; on the other, an aggregation accelerator has recently been reported to reduce AS cytotoxicity, likely by causing protofibril precipitation. Therefore, amyloid aggregation modulating ligands are expected to serve as therapeutic medicines.
In the present study, we evaluated the interaction of peptide ligands with AS by CD and fluorescence spectroscopies. For this purpose, we synthesized two peptides, H-RKVFYTW-NH2 and H-RGAVVTGR-NH2, named BB1 and BB2, respectively, and their all-D amino acid analogues. In addition, a rotamer-scan of the phenylalanine residue into the BB1 peptide was performed with the aim to evaluate the influence of the topography of this residue in the binding process. To this end, the Phe residue was replaced by L- or D-NMePhe, L- or D-Tic and Ala residues.
Far-UV CD studies showed that AS conformation was strongly influenced by the interaction with these peptides. While the interaction with BB1 and BB2 induced an increase of the negative band at 198 nm, suggesting a corresponding increase of the unordered conformation of AS, other peptides caused a decrease of the same band. Surprisingly, L-NMePhe and L-Tic BB1 analogues did not interact with AS. The binding properties of the BB1 analogues was also confirmed by near-UV CD and fluorescence spectroscopies
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