Semisynthetic, site-specific ubiquitin modification of α-synuclein reveals differential effects on aggregation

The process of neurodegeneration in Parkinson's Disease is intimately associated with the aggregation of the protein α-synuclein into toxic oligomers and fibrils. Interestingly, many of these protein aggregates are found to be post-translationally modified by ubiquitin at several different lysine residues. However, the inability to generate homogeneously ubiquitin modified α-synuclein at each site has prevented the understanding of the specific biochemical consequences. We have used protein semisynthesis to generate nine site-specifically ubiquitin modified α-synuclein derivatives and have demonstrated that different ubiquitination sites have differential effects on α-synuclein aggregation.     

Explaining the structural plasticity of α-synuclein

Given that α-synuclein has been implicated in the pathogenesis of several neurodegenerative disorders, deciphering the structure of this protein is of particular importance. While monomeric α-synuclein is disordered in solution, it can form aggregates rich in cross-β structure, relatively long helical segments when bound to micelles or lipid vesicles, and a relatively ordered helical tetramer within the native cell environment. To understand the physical basis underlying this structural plasticity, we generated an ensemble for monomeric α-synuclein using a Bayesian formalism that combines data from NMR chemical shifts, RDCs, and SAXS with molecular simulations. An analysis of the resulting ensemble suggests that a non-negligible fraction of the ensemble (0.08, 95% confidence interval 0.03-0.12) places the minimal toxic aggregation-prone segment in α-synuclein, NAC(8-18), in a solvent exposed and extended conformation that can form cross-β structure. Our data also suggest that a sizable fraction of structures in the ensemble (0.14, 95% confidence interval 0.04-0.23) contains long-range contacts between the N- and C-termini. Moreover, a significant fraction of structures that contain these long-range contacts also place the NAC(8-18) segment in a solvent exposed orientation, a finding in contrast to the theory that such long-range contacts help to prevent aggregation. Lastly, our data suggest that α-synuclein samples structures with amphipathic helices that can self-associate via hydrophobic contacts to form tetrameric structures. Overall, these observations represent a comprehensive view of the unfolded ensemble of monomeric α-synuclein and explain how different conformations can arise from the monomeric protein.     

Structural modification of silica-containing α-zirconium phosphate

Silica-containing -zirconium phosphate (-ZrSiP) was prepared by the precipitation method. X-ray diffractometry was used to characterize -ZrSiP and to compare it with other -zirconium phosphates (ZrP). A monoclinic cell with parameters of a=0.9038 nm, b=0.529 nm, c=1.4183 nm and =91.76° was identified for -ZrSiP, i.e. silica modifies the original -ZrP structure. The specific surface area of -ZrSiP was found to be two orders of magnitude higher then that of -ZrP.     

Mechanism of membrane interaction and disruption by α-synuclein

Parkinson's disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein α-synuclein. There is increasing evidence suggesting that the aggregation of α-synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface-sensitive imaging technique supercritical angle fluorescence microscopy and F?rster resonance energy transfer, we report the direct observation of α-synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of α-synuclein studied, namely, the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing α-synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross-β-sheet aggregates and damaged the membrane at submicromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble part of the yet poorly understood cellular toxicity phenomenon in vivo.     

A photoelectrochemical immunosensor based on Au-doped TiO2 nanotube arrays for the detection of α-synuclein

α‐Synuclein (α‐SYN) is a very important neuronal protein that is associated with Parkinson’s disease. In this paper, we utilized Au‐doped TiO₂ nanotube arrays to design a photoelectrochemical immunosensor for the detection of α‐SYN. The highly ordered TiO₂ nanotubes were fabricated by using an electrochemical anodization technique on pure Ti foil. After that, a photoelectrochemical deposition method was exploited to modify the resulting nanotubes with Au nanoparticles, which have been demonstrated to facilitate the improvement of photocurrent responses. Moreover, the Au‐doped TiO₂ nanotubes formed effective antibody immobilization arrays and immobilized primary antibodies (Ab₁) with high stability and bioactivity to bind target α‐SYN. The enhanced sensitivity was obtained by using {Ab₂‐Au‐GOx} bioconjugates, which featured secondary antibody (Ab₂) and glucose oxidase (GOx) labels linked to Au nanoparticles for signal amplification. The GOx enzyme immobilized on the prepared immunosensor could catalyze glucose in the detection solution to produce H₂O₂, which acted as a sacrificial electron donor to scavenge the photogenerated holes in the valence band of TiO₂ nanotubes upon irradiation of the other side of the Ti foil and led to a prompt photocurrent. The photocurrents were proportional to the α‐SYN concentrations, and the linear range of the developed immunosensor was from 50 pg mL^?1 to 100 ng mL^?1 with a detection limit of 34 pg mL^?1. The proposed method showed high sensitivity, stability, reproducibility, and could become a promising technique for protein detection.     

ATR-FTIR studies of phospholipid vesicle interactions with α-FeOOH and α-Fe2O3 surfaces

Prior infrared spectroscopic studies of extracellular polymeric substances (EPS) and live bacterial cells have indicated that organic phosphate groups mediate cell adhesion to iron oxides via inner-sphere P–OFe surface complexation. Since cell membrane phospholipids are a potential source of organic phosphate groups, we investigated the adhesion of phospholipidic vesicles to the surfaces of the iron (oxyhydr)oxides goethite (α-FeOOH) and hematite (α-Fe₂O₃) using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. l-α-Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidic acid (PA) were used because they are vesicle forming phospholipids representative of prokaryotic and eukaryotic cell surface membranes. Phospholipid vesicles, formed in aqueous suspension, were characterized by transmission electron microscopy (TEM), multi-angle laser light scattering (MALS) and quasi-elastic light scattering (QELS). Their adhesion to goethite and hematite surfaces was studied with ATR-FTIR at pH 5. Results indicate that PC and PE adsorption is affected by electrostatic interaction and H-bonding (PE). Conversely, adsorption of PA involves phosphate inner-sphere complexes, for both goethite and hematite, via P–OFe bond formation. Biomolecule adsorption at the interface was observed to occur on the scale of minutes to hours. Exponential and linear increases in peak intensity were observed for goethite and hematite, respectively. Our ATR-FTIR results on the PA terminal phosphate are in good agreement with those on EPS reacted with goethite and on bacterial cell adhesion to hematite. These findings suggest that the plasma membrane, and the PA terminal phosphate in particular, may play a role in mediating the interaction between bacteria and iron oxide surfaces during initial stages of biofilm formation.     

Autophagic failure promotes the exocytosis and intercellular transfer of α-synuclein

The accumulation of abnormal protein aggregates is a major characteristic of many neurodegenerative disorders, including Parkinson''s disease (PD). The intracytoplasmic deposition of α-synuclein aggregates and Lewy bodies, often found in PD and other α-synucleinopathies, is thought to be linked to inefficient cellular clearance mechanisms, such as the proteasome and autophagy/lysosome pathways. The accumulation of α-synuclein aggregates in neuronal cytoplasm causes numerous autonomous changes in neurons. However, it can also affect the neighboring cells through transcellular transmission of the aggregates. Indeed, a progressive spreading of Lewy pathology among brain regions has been hypothesized from autopsy studies. We tested whether inhibition of the autophagy/lysosome pathway in α-synuclein-expressing cells would increase the secretion of α-synuclein, subsequently affecting the α-synuclein deposition in and viability of neighboring cells. Our results demonstrated that autophagic inhibition, via both pharmacological and genetic methods, led to increased exocytosis of α-synuclein. In a mixed culture of α-synuclein-expressing donor cells with recipient cells, autophagic inhibition resulted in elevated transcellular α-synuclein transmission. This increase in protein transmission coincided with elevated apoptotic cell death in the recipient cells. These results suggest that the inefficient clearance of α-synuclein aggregates, which can be caused by reduced autophagic activity, leads to elevated α-synuclein exocytosis, thereby promoting α-synuclein deposition and cell death in neighboring neurons. This finding provides a potential link between autophagic dysfunction and the progressive spread of Lewy pathology.     

Structural characterizations of oligopyridyl foldamers, α-helix mimetics

Protein-protein interactions are central to many biological processes, from intracellular communication to cytoskeleton assembly, and therefore represent an important class of targets for new therapeutics. The most common secondary structure in natural proteins is an α-helix. Small molecules seem to be attractive candidates for stabilizing or disrupting protein-protein interactions based on α-helices. In our study, we assessed the ability of oligopyridyl scaffolds to mimic the α-helical twist. The theoretical as well as experimental studies (X-ray diffraction and NMR) on conformations of bipyridines in the function of substituent and pyridine nitrogen positions were carried out. Furthermore, the experimental techniques showed that the conformations observed in bipyridines are maintained within a longer oligopyridyl scaffold (quaterpyridines). The alignment of the synthesized quaterpyridine with two methyl substituents showed that it is an α-helix foldamer; their methyl groups overlap very well with side chain positions, i and i + 3, of an ideal α-helix.     

Cleavage of hindered α-methylbenzylamides,intermediates in the resolution of carboxylic acids

α-Methylbenzylamides, useful intermediates in the resolution of carboxylic acids, are cleaved with a two step procedure; enolizable amides are not racemized.     

β1-integrin-dependent migration of microglia in response to neuron-released α-synuclein

Chronic neuroinflammation is an integral pathological feature of major neurodegenerative diseases. The recruitment of microglia to affected brain regions and the activation of these cells are the major events leading to disease-associated neuroinflammation. In a previous study, we showed that neuron-released α-synuclein can activate microglia through activating the Toll-like receptor 2 (TLR2) pathway, resulting in proinflammatory responses. However, it is not clear whether other signaling pathways are involved in the migration and activation of microglia in response to neuron-released α-synuclein. In the current study, we demonstrated that TLR2 activation is not sufficient for all of the changes manifested by microglia in response to neuron-released α-synuclein. Specifically, the migration of and morphological changes in microglia, triggered by neuron-released α-synuclein, did not require the activation of TLR2, whereas increased proliferation and production of cytokines were strictly under the control of TLR2. Construction of a hypothetical signaling network using computational tools and experimental validation with various peptide inhibitors showed that β1-integrin was necessary for both the morphological changes and the migration. However, neither proliferation nor cytokine production by microglia was dependent on the activation of β1-integrin. These results suggest that β1-integrin signaling is specifically responsible for the recruitment of microglia to the disease-affected brain regions, where neurons most likely release relatively high levels of α-synuclein.     

Loss of glucocerebrosidase 1 activity causes lysosomal dysfunction and α-synuclein aggregation

Lysosomal dysfunction is a common pathological feature of neurodegenerative diseases. GTP-binding protein type A1 (GBA1) encodes β-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase. Homozygous mutations in GBA1 cause Gaucher disease, the most common lysosomal storage disease, while heterozygous mutations are strong risk factors for Parkinson''s disease. However, whether loss of GCase 1 activity is sufficient for lysosomal dysfunction has not been clearly determined. Here, we generated human neuroblastoma cell lines with nonsense mutations in the GBA1 gene using zinc-finger nucleases. Depending on the site of mutation, GCase 1 activity was lost or maintained. The cell line with GCase 1 deficiency showed indications of lysosomal dysfunction, such as accumulation of lysosomal substrates, reduced dextran degradation and accumulation of enlarged vacuolar structures. In contrast, the cell line with C-terminal truncation of GCase 1 but with intact GCase 1 activity showed normal lysosomal function. When α-synuclein was overexpressed, accumulation and secretion of insoluble aggregates increased in cells with GCase 1 deficiency but did not change in mutant cells with normal GCase 1 activity. These results demonstrate that loss of GCase 1 activity is sufficient to cause lysosomal dysfunction and accumulation of α-synuclein aggregates.     

The Effect of α-Branched Side Chains on the Structural and Opto-Electronic Properties of Poly(Diketopyrrolopyrrole-alt-Terthiophene)

Introducing solubilizing α-branched alkyl chains on a poly(diketopyrrolopyrrole-alt-terthiophene) results in a dramatic change of the structural, optical, and electronic properties compared to the isomeric polymer carrying β-branched alkyl side chains. When branched at the α-position the alkyl substituent creates a steric hindrance that reduces the tendency of the polymer to π–π stack and endows the material with a much higher solubility in common organic solvents. The wider π–π stacking and reduced tendency to crystallize, evidenced from grazing-incidence wide-angle X-ray scattering, result in a wider optical band gap in the solid state. In solar cells with a fullerene acceptor, the α-branched isomer affords a higher open-circuit voltage, but an overall lower power conversion efficiency as a result of a too well-mixed nanomorphology. Due its reduced π–π stacking, the α-branched isomer fluoresces and affords near-infrared light-emitting diodes emitting at 820 nm.     

Synthesis and Structural Analysis of Aspergillus fumigatus Galactosaminogalactans Featuring α-Galactose, α-Galactosamine and α-N-Acetyl Galactosamine Linkages

Galactosaminogalactan (GAG) is a prominent cell wall component of the opportunistic fungal pathogen Aspergillus fumigatus. GAG is a heteropolysaccharide composed of α-1,4-linked galactose, galactosamine and N-acetylgalactosamine residues. To enable biochemical studies, a library of GAG-fragments was constructed featuring specimens containing α-galactose-, α-galactosamine and α-N-acetyl galactosamine linkages. Key features of the synthetic strategy include the use of di-tert-butylsilylidene directed α-galactosylation methodology and regioselective benzoylation reactions using benzoyl-hydroxybenzotriazole (Bz-OBt). Structural analysis of the Gal, GalN and GalNAc oligomers by a combination of NMR and MD approaches revealed that the oligomers adopt an elongated, almost straight, structure, stabilized by inter-residue H-bonds, one of which is a non-conventional C−H⋅⋅⋅O hydrogen bond between H5 of the residue (i+1) and O3 of the residue (i). The structures position the C-2 substituents almost perpendicular to the oligosaccharide main chain axis, pointing to the bulk solvent and available for interactions with antibodies or other binding partners.     

Structural and mechanistic studies on γ-butyrobetaine hydroxylase

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Highlights? Crystal structure of γ-butyrobetaine hydroxylase in complex with substrate and inhibitor ? N-terminal Zn-binding domain involved in dimerization ? The clinically used inhibitor, THP, is also a substrate for BBOX ? BBOX catalyzed rearrangement reaction