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Journal ArticleDOI

Impact of the Acidic C-Terminal Region Comprising Amino Acids 109−140 on α-Synuclein Aggregation in Vitro†

26 Nov 2004-Biochemistry (American Chemical Society)-Vol. 43, Iss: 51, pp 16233-16242
TL;DR: The effects of the C-terminus on aggregation cannot be rationalized merely by a contribution to the protein net charge, but rather suggest a specific role of aa109-140 in the regulation of aggregation, presumably involving formation of intramolecular contacts.
Abstract: The aggregation of alpha-synuclein, involved in the pathogenesis of several neurodegenerative disorders such as Parkinson's disease, is enhanced in vitro by biogenic polyamines binding to the highly charged C-terminal region aa109-140. In this study, we investigated the influence of this region on the aggregation kinetics, monitored by thioflavin T binding and static light scattering, and morphology, assessed by electron microscopy, fluorescence microscopy, and turbidity, by comparing the effect of various solution conditions on the wild-type protein, the disease related mutants A53T and A30P, and two truncated variants, syn(1-108) and syn(1-124), lacking the complete or the C-terminal half of the polyamine binding site. In the presence of the intact C-terminus, aggregation was strongly retarded in physiological buffer. This inhibition of aggregation was overridden by (i) addition of spermine or MgCl(2) or lowering of pH, leading to strong charge shielding in the C-terminus or (ii) by truncation of aa125-140 or aa109-140. Addition of MgCl(2) or spermine or acidification were not effective in promoting aggregation of syn(1-108). The impact of the disease-related mutations on the aggregation kinetics was dependent on the solution conditions, with the aggregation propensity order A53T approximately wt > A30P at low ionic strength, but A53T > wt approximately A30P at high ionic strength, with exceedingly potent promotion of aggregation by the A53T mutation in the presence of spermine. In contrast to full-length alpha-synuclein aggregates, those formed from syn(1-108) did not exhibit a pronounced polymorphism. The effects of the C-terminus on aggregation cannot be rationalized merely by a contribution to the protein net charge, but rather suggest a specific role of aa109-140 in the regulation of aggregation, presumably involving formation of intramolecular contacts.
Citations
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Journal ArticleDOI
TL;DR: The relative importance of the common main-chain and side-chain interactions in determining the propensities of proteins to aggregate is discussed and some of the evidence that the oligomeric fibril precursors are the primary origins of pathological behavior is described.
Abstract: Peptides or proteins convert under some conditions from their soluble forms into highly ordered fibrillar aggregates. Such transitions can give rise to pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. In this review, we identify the diseases known to be associated with formation of fibrillar aggregates and the specific peptides and proteins involved in each case. We describe, in addition, that living organisms can take advantage of the inherent ability of proteins to form such structures to generate novel and diverse biological functions. We review recent advances toward the elucidation of the structures of amyloid fibrils and the mechanisms of their formation at a molecular level. Finally, we discuss the relative importance of the common main-chain and side-chain interactions in determining the propensities of proteins to aggregate and describe some of the evidence that the oligomeric fibril precursors are the primary origins of pathological behavior.

5,897 citations

Journal ArticleDOI
TL;DR: It is shown that a small percentage of newly synthesized α-syn is rapidly secreted from cells via unconventional, endoplasmic reticulum/Golgi-independent exocytosis and this finding proves that intravesicular localization and secretion are part of normal life cycle of α- syn and might also contribute to pathological function of this protein.
Abstract: α-Synuclein (α-syn), particularly in its aggregated forms, is implicated in the pathogenesis of Parkinson's disease and other related neurological disorders. However, the normal biology of α-syn and how it relates to the aggregation of the protein are not clearly understood. Because of the lack of the signal sequence and its predominant localization in the cytosol, α-syn is generally considered exclusively an intracellular protein. Contrary to this assumption, here, we show that a small percentage of newly synthesized α-syn is rapidly secreted from cells via unconventional, endoplasmic reticulum/Golgi-independent exocytosis. Consistent with this finding, we also demonstrate that a portion of cellular α-syn is present in the lumen of vesicles. Importantly, the intravesicular α-syn is more prone to aggregation than the cytosolic protein, and aggregated forms of α-syn are also secreted from cells. Furthermore, secretion of both monomeric and aggregated α-syn is elevated in response to proteasomal and mitochondrial dysfunction, cellular defects that are associated with Parkinson's pathogenesis. Thus, intravesicular localization and secretion are part of normal life cycle of α-syn and might also contribute to pathological function of this protein.

740 citations


Cites background from "Impact of the Acidic C-Terminal Reg..."

  • ...Some of the known characteristics of certain vesicular environment, such as high calcium concentration (Lowe et al., 2004) and low pH (Hoyer et al., 2004), are known to increase the rate of -syn aggregation....

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Journal ArticleDOI
TL;DR: Stabilization of the native, autoinhibitory structure of alphaS constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.
Abstract: In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein α-synuclein (αS) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, αS to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric αS assumes conformations that are stabilized by long-range interactions and act to inhibit oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to micro-seconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and/or temperature increase, conditions that induce aggregation in vitro, release this inherent tertiary structure, leading to a completely unfolded conformation that associates readily. Stabilization of the native, autoinhibitory structure of αS constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.

711 citations

Journal ArticleDOI
18 Sep 2013-Neuron
TL;DR: The role of synuclein at the nerve terminal and in membrane remodeling is reviewed and the prion-like propagation of misfoldedsynuclein is considered as a mechanism for the spread of degeneration through the neuraxis.

616 citations


Cites background from "Impact of the Acidic C-Terminal Reg..."

  • ...In addition, truncation of the C terminus, which promotes aggregation of synuclein in vitro and in vivo (Crowther et al., 1998; Hoyer et al., 2004; Li et al., 2005; Wakamatsu et al., 2008), had no effect on the inhibition of release, supporting a specific effect of synuclein independent of toxicity....

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Journal ArticleDOI
TL;DR: The structure and dynamics of full-length AS fibrils by high-resolution solid-state NMR spectroscopy are investigated to provide insight into the amyloid fibrilructure and dynamics with residue-specific resolution.
Abstract: The 140-residue protein α-synuclein (AS) is able to form amyloid fibrils and as such is the main component of protein inclusions involved in Parkinson's disease. We have investigated the structure and dynamics of full-length AS fibrils by high-resolution solid-state NMR spectroscopy. Homonuclear and heteronuclear 2D and 3D spectra of fibrils grown from uniformly 13C/15N-labeled AS and AS reverse-labeled for two of the most abundant amino acids, K and V, were analyzed. 13C and 15N signals exhibited linewidths of <0.7 ppm. Sequential assignments were obtained for 48 residues in the hydrophobic core region. We identified two different types of fibrils displaying chemical-shift differences of up to 13 ppm in the 15N dimension and up to 5 ppm for backbone and side-chain 13C chemical shifts. EM studies suggested that molecular structure is correlated with fibril morphology. Investigation of the secondary structure revealed that most amino acids of the core region belong to β-strands with similar torsion angles in both conformations. Selection of regions with different mobility indicated the existence of monomers in the sample and allowed the identification of mobile segments of the protein within the fibril in the presence of monomeric protein. At least 35 C-terminal residues were mobile and lacked a defined secondary structure, whereas the N terminus was rigid starting from residue 22. Our findings agree well with the overall picture obtained with other methods and provide insight into the amyloid fibril structure and dynamics with residue-specific resolution. EM protein structure amyloid Parkinson's disease protein aggregation

598 citations

References
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Journal ArticleDOI
TL;DR: It is demonstrated that at higher concentrations, Lewy body-like fibrils and discrete spherical assemblies are formed; most rapidly by A53T, suggesting mutation-induced acceleration of α-synuclein fibril formation may contribute to the early onset of familial PD.
Abstract: Accelerated in vitro fibril formation by a mutant α-synuclein linked to early-onset Parkinson disease

1,488 citations

Journal ArticleDOI
TL;DR: Fibrils generated in vitro from alpha-synuclein, wild-type and both mutant forms, are shown to possess very similar features that are characteristic of amyloid fibrils, including a wound and predominantly unbranched morphology, distinctive dye-binding properties, and antiparallel beta-sheet structure.
Abstract: Two missense mutations in the gene encoding α-synuclein have been linked to rare, early-onset forms of Parkinson's disease (PD). These forms of PD, as well as the common idiopathic form, are characterized by the presence of cytoplasmic neuronal deposits, called Lewy bodies, in the affected region of the brain. Lewy bodies contain α-synuclein in a form that resembles fibrillar Aβ derived from Alzheimer's disease (AD) amyloid plaques. One of the mutant forms of α-synuclein (A53T) fibrillizes more rapidly in vitro than does the wild-type protein, suggesting that a correlation may exist between the rate of in vitro fibrillization and/or oligomerization and the progression of PD, analogous to the relationship between Aβ fibrillization in vitro and familial AD. In this paper, fibrils generated in vitro from α-synuclein, wild-type and both mutant forms, are shown to possess very similar features that are characteristic of amyloid fibrils, including a wound and predominantly unbranched morphology (demonstrated by...

793 citations

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