Altered fatty acid composition of dopaminergic neurons expressing alpha-synuclein and human brains with alpha-synucleinopathies

Alpha-synuclein (alphaS) is an abundant neuronal protein that accumulates in insoluble inclusions in Parkinson's disease (PD) and the related disorder, dementia with Lewy bodies (DLB). A central question about the role of alphaS in the pathogenesis of PD and DLB concerns how this normally soluble protein assembles into insoluble aggregates associated with neuronal dysfunction. We recently detected highly soluble oligomers of alphaS in normal brain supernatants and observed their augmentation in PD and DLB brains. Further, we found that polyunsaturated fatty acids (PUFAs) enhanced alphaS oligomerization in intact mesencephalic neuronal cells. We now report the presence of elevated PUFA levels in PD and DLB brain soluble fractions. Higher PUFA levels were also detected in the supernatants and high-speed membrane fractions of neuronal cells over-expressing wild-type or PD-causing mutant alphaS. This increased PUFA content in the membrane fraction was accompanied by increased membrane fluidity in the alphaS overexpressing neurons. In accord, membrane fluidity and the levels of certain PUFAs were decreased in the brains of mice genetically deleted of alphaS. Together with our earlier observations, these results suggest that alphaS-PUFA interactions help regulate neuronal PUFA levels as well as the oligomerization state of alphaS, both normally and in human synucleinopathies.     

The extracellular N-terminal domain and transmembrane domains 1 and 2 mediate oligomerization of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form homodimers/oligomers and/or heterodimers/oligomers. The mechanisms used to form specific GPCR oligomers are poorly understood because the domains that mediate such interactions and the step(s) in the secretory pathway where oligomerization occurs have not been well characterized. Here we have used subcellular fractionation and fluorescence resonance energy transfer (FRET) experiments to show that oligomerization of a GPCR (alpha-factor receptor; STE2 gene product) of the yeast Saccharomyces cerevisiae occurs in the endoplasmic reticulum. To identify domains of this receptor that mediate oligomerization, we used FRET and endocytosis assays of oligomerization in vivo to analyze receptor deletion mutants. A mutant lacking the N-terminal extracellular domain and transmembrane (TM) domain 1 was expressed at the cell surface but did not self-associate. In contrast, a receptor fragment containing only the N-terminal extracellular domain and TM1 could self-associate and heterodimerize with wild type receptors. Analysis of other mutants suggested that oligomerization is facilitated by the N-terminal extracellular domain and TM2. Therefore, the N-terminal extracellular domain, TM1, and TM2 appear to stabilize alpha-factor receptor oligomers. These domains may form an interface in contact or domain-swapped oligomers. Similar domains may mediate dimerization of certain mammalian GPCRs.     

Self-assembly is important for TIP47 function in mannose 6-phosphate receptor transport

TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of mannose 6-phosphate receptors and is required for their transport from endosomes to the trans- Golgi network in vitro and in living cells. TIP47 occurs in cytosol as an oligomer; it chromatographs with an apparent mass of ∼ 300 kDa and displays an S -value of ∼ 13. Recombinant TIP47 forms homo-oligomers that are likely to represent hexamers, as determined by chemical cross-linking. Removal of TIP47 residues 1–151 yields a protein that behaves as a monomer upon gel filtration, yet is fully capable of binding mannose 6-phosphate receptor cytoplasmic domains. The presence of an oligomerization domain in the N-terminus of TIP47 was confirmed by expression of N-terminal residues 1–133 or 1–257 in mammalian cells. Co-expression of full-length TIP47 with either of these fragments led to the formation of higher-order aggregates of wild-type TIP47. Furthermore, the N-terminal domains expressed alone also occurred as oligomers. These studies reveal an N-terminal oligomerization domain in TIP47, and show that oligomerization is not required for TIP47 recognition of mannose 6-phosphate receptors. However, oligomerization is required for TIP47 stimulation of mannose 6-phosphate receptor transport from endosomes to the trans- Golgi in vivo .     

The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson's disease

Accumulation of misfolded proteins as insoluble aggregates occurs in several neurodegenerative diseases. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), alpha-synuclein (alpha S) accumulates in insoluble inclusions. To identify soluble alpha S oligomers that precede insoluble aggregates, we probed the cytosols of mesencephalic neuronal (MES) cells, normal and alpha S-transgenic mouse brains, and normal, PD, and DLB human brains. All contained highly soluble oligomers of alpha S whose detection was enhanced by delipidation. Exposure of living MES neurons to polyunsaturated fatty acids (PUFAs) increased alpha S oligomer levels, whereas saturated FAs decreased them. PUFAs directly promoted oligomerization of recombinant alphaS. Transgenic mice accumulated soluble oligomers with age. PD and DLB brains had elevated amounts of the soluble, lipid-dependent oligomers. We conclude that alpha S interacts with PUFAs in vivo to promote the formation of highly soluble oligomers that precede the insoluble alpha S aggregates associated with neurodegeneration.     

Conformational properties of alpha-synuclein in its free and lipid-associated states

alpha-Synuclein (alphaS) is a presynaptic terminal protein that is believed to play an important role in the pathogenesis of Parkinson's disease (PD). We have used NMR spectroscopy to characterize the conformational properties of alphaS in solution as a free monomer and when bound to lipid vesicles and lipid-mimetic detergent micelles. Free wild-type alphaS is largely unfolded in solution, but exhibits a region with a preference for helical conformations that may be important in the aggregation of alphaS into fibrils. The N-terminal region of alphaS binds to synthetic lipid vesicles and detergent micelles in vitro and adopts a highly helical conformation, consistent with predictions based on sequence analysis. The C-terminal part of the protein does not associate with either vesicles or micelles, remaining free and unfolded. These results suggest that one function of alphaS may be to tether as of yet unidentified partners to lipid surfaces via interactions with its C-terminal tail.     

Characterization of the stomatin domain involved in homo-oligomerization and lipid raft association

The cytoplasmically oriented monotopic integral membrane protein stomatin forms high-order oligomers and associates with lipid rafts. To characterize the domains that are involved in oligomerization and detergent-resistant membrane (DRM) association, we expressed truncation and point mutants of stomatin and analyzed their size and buoyancy by ultracentrifugation methods. A small C-terminal region of stomatin that is largely hydrophobic, Ser-Thr-Ile-Val-Phe-Pro-Leu-Pro-Ile (residues 264-272), proved to be crucial for oligomerization, whereas the N-terminal domain (residues 1-20) and the last 12 C-terminal amino acids (residues 276-287) were not essential. The introduction of alanine substitutions in the region 264-272 resulted in the appearance of monomers. Remarkably, only three of these residues, Ile-Val-Phe (residues 266-268), were found to be indispensable for the DRM association. Interestingly, the exchange of Pro-269 and to some extent the residues 270-272, which are essential for oligomerization, did not affect the DRM association of stomatin. This suggests that the formation of oligomers is not necessary for the association of stomatin with DRMs. Internal deletions near the membrane anchoring domain resulted in the formation of intermediate size oligomers suggesting a conformational interdependence of large parts of the C-terminal region. Fluorescence recovery after photobleaching analysis of the tagged, monomeric, non-DRM mutant ST-(1-262)-green fluorescent protein and wild type stomatin StomGFP showed a significantly higher lateral mobility of the truncation mutant in the plasma membrane suggesting a membrane interaction of the respective C-terminal region also in vivo.     

Mutational analysis of the leucine zipper-like motif of the human immunodeficiency virus type 1 envelope transmembrane glycoprotein.

The N-terminal region of the envelope (env) transmembrane protein of human immunodeficiency virus type 1 (HIV-1) has a leucine zipper-like motif. This highly conserved zipper motif, which consists of a heptad repeat of leucine or isoleucine residues, has been suggested to play a role in HIV-1 env glycoprotein oligomerization. This hypothesis was tested by replacing the highly conserved leucine or isoleucine residues in the zipper motif with a strong alpha-helix breaker, proline. We report here that such substitutions did not abolish the ability of env protein to form oligomers, indicating that this highly conserved zipper motif does not have a crucial role in env protein oligomerization. However, the mutant viruses all showed impaired infectivity, suggesting that this conserved zipper motif can have an important role in the virus life cycle.     

Regions of SV40 large T antigen necessary for oligomerization and complex formation with the cellular oncoprotein p53

The simian virus 40 (SV40) T antigen is composed of 708 amino acids and forms monomers and various oligomers and, in small amounts, heterologous complexes with the cellular oncoprotein p53 (T-p53). Using SV40 mutants coding for T antigen fragments which are either deleted in the N-terminal half or truncated by various lengths at the C-terminal end, we found that a region between amino acids 114 and 152 and a C-terminal region up to amino acid 669 are essential for the formation of high Mr oligomers of T antigen. Furthermore, only the C-terminal end up to amino acid 669 is essential for T-p53 complex formation but not the N-terminus up to amino acid 152.     

Investigations of the molecular mechanism of metal-induced Abeta (1-40) amyloidogenesis

Transition-metal ions (Cu2+ and Zn2+) play critical roles in the Abeta plaque formation. However, precise roles of the metal ions in the Abeta amyloidogenesis have been controversial. In this study, the molecular mechanism of the metal-induced Abeta oligomerization was investigated with extensive metal ion titration NMR experiments. Upon additions of the metal ions, the N-terminal region (1-16) of the Abeta (1-40) peptide was selectively perturbed. In particular, polar residues 4-8 and 13-15 were more strongly affected by the metal ions, suggesting that those regions may be the major binding sites of the metal ions. The NMR signal changes of the N-terminal region were dependent on the peptide concentrations (higher peptide concentrations resulted in stronger signal changes), suggesting that the metal ions facilitate the intermolecular contact between the Abeta peptides. The Abeta (1-40) peptides (>30 microM) were eventually oligomerized even at low temperature (3 degrees C), where the Abeta peptides are stable as monomeric forms without the metal ions. The real-time oligomerization process was monitored by 1H/15N HSQC NMR experiments, which provided the first residue-specific structural transition information. Hydrophobic residues 12-21 initially underwent conformational changes due to the intermolecular interactions. After the initial structural rearrangements, the C-terminal residues (32-40) readjusted their conformations presumably for effective oligomerization. Similar structural changes of the metal-free Abeta (1-40) peptides were also observed in the presence of the preformed oligomers, suggesting that the conformational transitions may be the general molecular mechanism of the Abeta (1-40) amyloidogenesis.     

Dissecting amelogenin protein nanospheres: characterization of metastable oligomers

Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of amelogenin nanosphere formation, we manipulated the interactions between amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable amelogenin oligomers. Recombinant porcine amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp(161), Trp(45), and Trp(25)) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (R(H)) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg · ml(-1). We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp(161)) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp(25) and Trp(45) is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite.     

Mass spectrometry analysis of HIV-1 Vif reveals an increase in ordered structure upon oligomerization in regions necessary for viral infectivity

HIV-1 Vif, an accessory protein in the viral genome, performs an important role in viral pathogenesis by facilitating the degradation of APOBEC3G, an endogenous cellular inhibitor of HIV-1 replication. In this study, intrinsically disordered regions are predicted in HIV-1 Vif using sequence-based algorithms. Intrinsic disorder may explain why traditional structure determination of HIV-1 Vif has been elusive, making structure-based drug design impossible. To characterize HIV-1 Vif's structural topology and to map the domains involved in oligomerization we used chemical cross-linking, proteolysis, and mass spectrometry. Cross-linking showed evidence of monomer, dimer, and trimer species via denaturing gel analysis and an additional tetramer via western blot analysis. We identified 47 unique linear peptides and 24 (13 intramolecular; 11 intermolecular) noncontiguous, cross-linked peptides, among the noncross-linked monomer, cross-linked monomer, cross-linked dimer, and cross-linked trimer samples. Almost complete peptide coverage of the N-terminus is observed in all samples analyzed, however reduced peptide coverage in the C-terminal region is observed in the dimer and trimer samples. These differences in peptide coverage or "protections" between dimer and trimer indicate specific differences in packing between the two oligomeric forms. Intramolecular cross-links within the monomer suggest that the N-terminus is likely folded into a compact domain, while the C-terminus remains intrinsically disordered. Upon oligomerization, as evidenced by the intermolecular cross-links, the C-terminus of one Vif protein becomes ordered by wrapping back on the N-terminal domain of another. In addition, the majority of the intramolecular cross-links map to regions that have been previously reported to be necessary for viral infectivity. Thus, this data suggests HIV-1 Vif is in a dynamic equilibrium between the various oligomers potentially allowing it to interact with other binding partners.     

The early events of alpha-synuclein oligomerization revealed by photo-induced cross-linking

Assembly of alpha-synuclein (alpha-Syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of Parkinson's disease. Studying the early events of alpha-Syn aggregation, such as oligomerization and nucleation, is indispensable to understanding the complicated process. Here, photo-induced cross-linking of unmodified proteins (PICUP) technique is applied to elucidate the early-stage oligomerization of alpha-Syn. Results show that alpha-Syn in solution exhibits a mixture of various species, including at least monomers, dimers and trimers. Aggregation of alpha-Syn probably originates from the dimeric and trimeric seeds. Furthermore, the N-terminal amphipathic region is proposed to be required for the oligomerization (dimerization and trimerization) process. This observation may extend our knowledge on the early events of alpha-Syn aggregation and the neurotoxic aggregation species.     

Modulation of beta-amyloid aggregation by engineering the sequence connecting beta-strand forming domains

Aggregation of beta-amyloid (Aβ) into oligomers and fibrils is associated with the pathology of Alzheimer's disease. The major structural characteristics of Aβ fibrils include the presence of β sheet-loop-β sheet conformations. Several lines of study suggested a potentially important role of the Aβ loop forming sequence (referred to as the Aβ linker region) in Aβ aggregation. Effects of mutations in several charged residues within the Aβ linker region on aggregation have been extensively studied. However, little is known about oligomerization effects of sequence variation in other residues within the Aβ linker region. Moreover, modulation effects of the Aβ linker mutants on Aβ aggregation have yet to be characterized. Here, we created and characterized Aβ linker variants containing sequences preferentially found in specific β turn conformations. Our results indicate that a propensity to form oligomers may be changed by local sequence variation in the Aβ linker region without mutating the charged residues. Strikingly, one Aβ linker variant rapidly formed protofibrillar oligomers, which did not convert to fibrillar aggregates in contrast to Aβ aggregating to fibrils under similar incubation conditions. Moreover, our results suggest that molecular forces critical in oligomerization and fibrillization may differ at least for those involved in the linker region. When co-incubated with Aβ, some Aβ linker variants were found to induce accumulation of Aβ oligomers. Our results suggest that engineering of the Aβ linker region as described in this paper may represent a novel approach to control Aβ oligomerization and create Aβ oligomerization modulators.     

Dampening of Hyperexcitability in CA1 Pyramidal Neurons by Polyunsaturated Fatty Acids Acting on Voltage-Gated Ion Channels

A ketogenic diet is an alternative treatment of epilepsy in infants. The diet, rich in fat and low in carbohydrates, elevates the level of polyunsaturated fatty acids (PUFAs) in plasma. These substances have therefore been suggested to contribute to the anticonvulsive effect of the diet. PUFAs modulate the properties of a range of ion channels, including K and Na channels, and it has been hypothesized that these changes may be part of a mechanistic explanation of the ketogenic diet. Using computational modelling, we here study how experimentally observed PUFA-induced changes of ion channel activity affect neuronal excitability in CA1, in particular responses to synaptic input of high synchronicity. The PUFA effects were studied in two pathological models of cellular hyperexcitability associated with epileptogenesis. We found that experimentally derived PUFA modulation of the A-type K (K_A) channel, but not the delayed-rectifier K channel, restored healthy excitability by selectively reducing the response to inputs of high synchronicity. We also found that PUFA modulation of the transient Na channel was effective in this respect if the channel''s steady-state inactivation was selectively affected. Furthermore, PUFA-induced hyperpolarization of the resting membrane potential was an effective approach to prevent hyperexcitability. When the combined effect of PUFA on the K_A channel, the Na channel, and the resting membrane potential, was simulated, a lower concentration of PUFA was needed to restore healthy excitability. We therefore propose that one explanation of the beneficial effect of PUFAs lies in its simultaneous action on a range of ion-channel targets. Furthermore, this work suggests that a pharmacological cocktail acting on the voltage dependence of the Na-channel inactivation, the voltage dependences of K_A channels, and the resting potential can be an effective treatment of epilepsy.     

Oligomerization propensity and flexibility of yeast frataxin studied by X-ray crystallography and small-angle X-ray scattering

Frataxin is a mitochondrial protein with a central role in iron homeostasis. Defects in frataxin function lead to Friedreich's ataxia, a progressive neurodegenerative disease with childhood onset. The function of frataxin has been shown to be closely associated with its ability to form oligomeric species; however, the factors controlling oligomerization and the types of oligomers present in solution are a matter of debate. Using small-angle X-ray scattering, we found that Co²⁺, glycerol, and a single amino acid substitution at the N-terminus, Y73A, facilitate oligomerization of yeast frataxin, resulting in a dynamic equilibrium between monomers, dimers, trimers, hexamers, and higher-order oligomers. Using X-ray crystallography, we found that Co²⁺ binds inside the channel at the 3-fold axis of the trimer, which suggests that the metal has an oligomer-stabilizing role. The results reveal the types of oligomers present in solution and support our earlier suggestions that the trimer is the main building block of yeast frataxin oligomers. They also indicate that different mechanisms may control oligomer stability and oligomerization in vivo.     

The Molecular Basis of Iron-induced Oligomerization of Frataxin and the Role of the Ferroxidation Reaction in Oligomerization

The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis, and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle x-ray scattering and x-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and Escherichia coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher order oligomers induced at higher iron-to-protein ratios. The x-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A/D82A variant of Yfh1, iron-dependent oligomerization may still take place, although >50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization, and the structure of oligomers formed during assembly.     

Upregulation of SIRT1 deacetylase in phenylephrine-treated cardiomyoblasts

Deposition of amyloid fibrils consisting of amyloid β (Aβ) protein as senile plaques in the brain is a pathological hallmark of Alzheimer’s disease. However, a growing body of evidence shows that soluble Aβ oligomers correlate better with dementia than fibrils, suggesting that Aβ oligomers may be the primary toxic species. The structure and oligomerization mechanism of these Aβ oligomers are crucial for developing effective therapeutics. Here we investigated the oligomerization of Aβ42 in the context of a fusion protein containing GroES and ubiquitin fused to the N-terminus of Aβ sequence. The presence of fusion protein partners, in combination with a denaturing buffer containing 8 M urea at pH 10, is unfavorable for Aβ42 aggregation, thus allowing only the most stable structures to be observed. Transmission electron microscopy showed that Aβ42 fusion protein formed globular oligomers, which bound weakly to thioflavin T and Congo red. SDS–PAGE shows that Aβ42 fusion protein formed SDS-resistant hexamers and tetramers. In contrast, Aβ40 fusion protein remained as monomers on SDS gel, suggesting that the oligomerization of Aβ42 fusion protein is not due to the fusion protein partners. Cysteine scanning mutagenesis at 22 residue positions further revealed that single cysteine substitutions of the C-terminal hydrophobic residues (I31, I32, L34, V39, V40, and I41) led to disruption of hexamer and tetramer formation, suggesting that hydrophobic interactions between these residues are most critical for Aβ42 oligomerization.     

Identification of the coiled-coil domains of Enterococcus faecalis DivIVA that mediate oligomerization and their importance for biological function

Bacillus subtilis (Bs) DivIVA comprises coiled-coil structures and self-associates forming a 10-12 mer complex in vitro. Using bioinformatic approaches, we determined that Enterococcus faecalis (Ef) DivIVA comprises four coiled-coil domains, one at the N-terminus, the second and the third in the central region of the protein and the fourth at the C-terminus. We determined that DivIVA(Ef) self-interacts and forms a 10-12 multimeric complex. Point mutations or deletions of the central regions predicted bioinformatically to disrupt the coiled-coil structures either eliminated or weakened DivIVA(Ef) self-interaction and reduced oligomerization. Mutations disrupting the N- and C-terminal coiled-coils of DivIVA(Ef) did not affect DivIVA(Ef) oligomerization. The introduction of DivIVA(Ef) mutations to both the N-terminal and the central coiled-coil domains were lethal unless rescued by expressing wild-type DivIVA(Ef) in trans. E. faecalis cells expressing these mutations displayed aberrant cell morphology, indicating disruption of the normal cell division phenotype. The results in E. faecalis also indicate that both the N-terminal and the central coiled-coil structures of DivIVA(Ef) are indispensable for proper biological function. Overexpression of wild-type DivIVA(Ef) in both rod-shaped and round Escherichia coli cells resulted in morphological changes, while the overexpression of DivIVA(Ef) mutations failed to induce such alterations.