Substitution of Met-38 to Ile in γ-synuclein found in two patients with amyotrophic lateral sclerosis induces aggregation into amyloid.

α-, ß-, and γ-Synuclein are intrinsically disordered proteins implicated in physiological processes in the nervous system of vertebrates. α-synuclein (αSyn) is the amyloidogenic protein associated with Parkinson's disease and certain other neurodegenerative disorders. Intensive research has focused on the mechanisms that cause αSyn to form amyloid structures, identifying its NAC region as being necessary and sufficient for amyloid assembly. Recent work has shown that a 7-residue sequence (P1) is necessary for αSyn amyloid formation. Although γ-synuclein (γSyn) is 55% identical in sequence to αSyn and its pathological deposits are also observed in association with neurodegenerative conditions, γSyn is resilient to amyloid formation in vitro. Here, we report a rare single nucleotide polymorphism (SNP) in the SNCG gene encoding γSyn, found in two patients with amyotrophic lateral sclerosis (ALS). The SNP results in the substitution of Met38 with Ile in the P1 region of the protein. These individuals also had a second, common and nonpathological, SNP in SNCG resulting in the substitution of Glu110 with Val. In vitro studies demonstrate that the Ile38 variant accelerates amyloid fibril assembly. Contrastingly, Val110 retards fibril assembly and mitigates the effect of Ile38. Substitution of residue 38 with Leu had little effect, while Val retards, and Ala increases the rate of amyloid formation. Ile38 γSyn also results in the formation of γSyn-containing inclusions in cells. The results show how a single point substitution can enhance amyloid formation of γSyn and highlight the P1 region in driving amyloid formation in another synuclein family member.

Single residue modulators of amyloid formation in the N-terminal P1-region of α-synuclein.

Alpha-synuclein (αSyn) is a protein involved in neurodegenerative disorders including Parkinson's disease. Amyloid formation of αSyn can be modulated by the 'P1 region' (residues 36-42). Here, mutational studies of P1 reveal that Y39A and S42A extend the lag-phase of αSyn amyloid formation in vitro and rescue amyloid-associated cytotoxicity in C. elegans. Additionally, L38I αSyn forms amyloid fibrils more rapidly than WT, L38A has no effect, but L38M does not form amyloid fibrils in vitro and protects from proteotoxicity. Swapping the sequence of the two residues that differ in the P1 region of the paralogue γSyn to those of αSyn did not enhance fibril formation for γSyn. Peptide binding experiments using NMR showed that P1 synergises with residues in the NAC and C-terminal regions to initiate aggregation. The remarkable specificity of the interactions that control αSyn amyloid formation, identifies this region as a potential target for therapeutics, despite their weak and transient nature.

Looking Beyond the Core: The Role of Flanking Regions in the Aggregation of Amyloidogenic Peptides and Proteins.

Amyloid proteins are involved in many neurodegenerative disorders such as Alzheimer's disease [Tau, Amyloid ß (Aß)], Parkinson's disease [alpha-synuclein (αSyn)], and amyotrophic lateral sclerosis (TDP-43). Driven by the early observation of the presence of ordered structure within amyloid fibrils and the potential to develop inhibitors of their formation, a major goal of the amyloid field has been to elucidate the structure of the amyloid fold at atomic resolution. This has now been achieved for a wide variety of sequences using solid-state NMR, microcrystallography, X-ray fiber diffraction and cryo-electron microscopy. These studies, together with in silico methods able to predict aggregation-prone regions (APRs) in protein sequences, have provided a wealth of information about the ordered fibril cores that comprise the amyloid fold. Structural and kinetic analyses have also shown that amyloidogenic proteins often contain less well-ordered sequences outside of the amyloid core (termed here as flanking regions) that modulate function, toxicity and/or aggregation rates. These flanking regions, which often form a dynamically disordered "fuzzy coat" around the fibril core, have been shown to play key parts in the physiological roles of functional amyloids, including the binding of RNA and in phase separation. They are also the mediators of chaperone binding and membrane binding/disruption in toxic amyloid assemblies. Here, we review the role of flanking regions in different proteins spanning both functional amyloid and amyloid in disease, in the context of their role in aggregation, toxicity and cellular (dys)function. Understanding the properties of these regions could provide new opportunities to target disease-related aggregation without disturbing critical biological functions.

Spot the Difference: Function versus Toxicity in Amyloid Fibrils.

In a recent study, Hervas et al. extracted Orb2 fibrils, that are involved in long-term memory formation, from Drosophila brains, characterised their function, and determined their structure using cryo-electron microscopy (cryo-EM). The fibrils show a remarkable resemblance to amyloid ß (Aß) fibrils associated with Alzheimer's disease, highlighting the subtle difference between functional and dysfunctional amyloid.

A short motif in the N-terminal region of α-synuclein is critical for both aggregation and function.

Aggregation of human α-synuclein (αSyn) is linked to Parkinson's disease (PD) pathology. The central region of the αSyn sequence contains the non-amyloid ß-component (NAC) crucial for aggregation. However, how NAC flanking regions modulate αSyn aggregation remains unclear. Using bioinformatics, mutation and NMR, we identify a 7-residue sequence, named P1 (residues 36-42), that controls αSyn aggregation. Deletion or substitution of this 'master controller' prevents aggregation at pH 7.5 in vitro. At lower pH, P1 synergises with a sequence containing the preNAC region (P2, residues 45-57) to prevent aggregation. Deleting P1 (ΔP1) or both P1 and P2 (ΔΔ) also prevents age-dependent αSyn aggregation and toxicity in C. elegans models and prevents αSyn-mediated vesicle fusion by altering the conformational properties of the protein when lipid bound. The results highlight the importance of a master-controller sequence motif that controls both αSyn aggregation and function-a region that could be targeted to prevent aggregation in disease.

Photoinduced Electron Transfer between Psoralens and DNA: Influence of DNA Sequence and Substitution.

Psoralens are heterocyclic compounds which are, among other uses, used to treat skin deseases in the framework of PUVA therapy. In the dark, they intercalate into DNA and can form photoadducts with thymines upon UV-A excitation, which harms the affected cells. We have recently discovered that after excitation of intercalated psoralens, an efficient photoinduced electron transfer (PET) from DNA occurs. Here, the PET is studied in detail by means of femtosecond transient absorption spectroscopy. Using DNA samples that contain either only GC or AT base pairs, we show that only guanine donates the electrons. Additionally, the substituent effects on PET are studied relying on three different psoralen derivatives. The substitution alters spectroscopic and electrochemical properties of the psoralens, which are determined by cyclic voltammetry and steady state spectroscopy. These experiments allow us to estimate the PET energetics, which are in line with the measured kinetics. Implications for the applications of psoralens are discussed.