Fluorescent protein tagging promotes phase separation and alters the aggregation pathway of huntingtin exon-1.

Fluorescent protein tags are convenient tools for tracking the aggregation states of amyloidogenic or phase separating proteins, but the effect of the tags is often not well understood. Here, we investigated the impact of a C-terminal red fluorescent protein (RFP) tag on the phase separation of huntingtin exon-1 (Httex1), an N-terminal portion of the huntingtin protein that aggregates in Huntington's disease. We found that the RFP-tagged Httex1 rapidly formed micron-sized, phase separated states in the presence of a crowding agent. The formed structures had a rounded appearance and were highly dynamic according to electron paramagnetic resonance and fluorescence recovery after photobleaching, suggesting that the phase separated state was largely liquid in nature. Remarkably, the untagged protein did not undergo any detectable liquid condensate formation under the same conditions. In addition to strongly promoting liquid-liquid phase separation, the RFP tag also facilitated fibril formation, as the tag-dependent liquid condensates rapidly underwent a liquid-to-solid transition. The rate of fibril formation under these conditions was significantly faster than that of the untagged protein. When expressed in cells, the RFP-tagged Httex1 formed larger aggregates with different antibody staining patterns compared to untagged Httex1. Collectively, these data reveal that the addition of a fluorescent protein tag significantly impacts liquid and solid phase separations of Httex1 in vitro and leads to altered aggregation in cells. Considering that the tagged Httex1 is commonly used to study the mechanisms of Httex1 misfolding and toxicity, our findings highlight the importance to validate the conclusions with untagged protein.

Molecular basis of Q-length selectivity for the MW1 antibody-huntingtin interaction.

Huntington's disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein. Huntingtin exon 1 (Httex1), as well as other naturally occurring N-terminal huntingtin fragments with expanded polyQ are prone to aggregation, forming potentially cytotoxic oligomers and fibrils. Antibodies and other N-terminal huntingtin binders are widely explored as biomarkers and possible aggregation-inhibiting therapeutics. A monoclonal antibody, MW1, is known to preferentially bind to huntingtin fragments with expanded polyQ lengths, but the molecular basis of the polyQ length specificity remains poorly understood. Using solution NMR, electron paramagnetic resonance, and other biophysical methods, we investigated the structural features of the Httex1-MW1 interaction. Rather than recognizing residual α-helical structure, which is promoted by expanded Q-lengths, MW1 caused the formation of a new, non-native, conformation in which the entire polyQ is largely extended. This non-native polyQ structure allowed the formation of large mixed Httex1-MW1 multimers (600-2900 kD), when Httex1 with pathogenic Q-length (Q46) was used. We propose that these multivalent, entropically favored interactions, are available only to proteins with longer Q-lengths and represent a major factor governing the Q-length preference of MW1. The present study reveals that it is possible to target proteins with longer Q-lengths without having to stabilize a natively favored conformation. Such mechanisms could be exploited in the design of other Q-length specific binders.

An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles.

Positive-strand RNA viruses universally remodel host intracellular membranes to form membrane-bound viral replication complexes, where viral offspring RNAs are synthesized. In the majority of cases, viral replication proteins are targeted to and play critical roles in the modulation of the designated organelle membranes. Many viral replication proteins do not have transmembrane domains, but contain single or multiple amphipathic alpha-helices. It has been conventionally recognized that these helices serve as an anchor for viral replication protein to be associated with membranes. We report here that a peptide representing the amphipathic α-helix at the N-terminus of the poliovirus 2C protein not only binds to liposomes, but also remodels spherical liposomes into tubules. The membrane remodeling ability of this amphipathic alpha-helix is similar to that recognized in other amphipathic alpha-helices from cellular proteins involved in membrane remodeling, such as BAR domain proteins. Mutations affecting the hydrophobic face of the amphipathic alpha-helix severely compromised membrane remodeling of vesicles with physiologically relevant phospholipid composition. These mutations also affected the ability of poliovirus to form plaques indicative of reduced viral replication, further underscoring the importance of membrane remodeling by the amphipathic alpha-helix in possible relation to the formation of viral replication complexes.

Structural Model of the Proline-Rich Domain of Huntingtin Exon-1 Fibrils.

Huntington's disease is a heritable neurodegenerative disease that is caused by a CAG expansion in the first exon of the huntingtin gene. This expansion results in an elongated polyglutamine domain that increases the propensity of huntingtin exon-1 to form cross-ß fibrils. Although the polyglutamine domain is important for fibril formation, the dynamic, C-terminal proline-rich domain (PRD) of huntingtin exon-1 makes up a large fraction of the fibril surface. Because potential fibril toxicity has to be mediated by interactions of the fibril surface with its cellular environment, we wanted to model the conformational space adopted by the PRD. We ran 800-ns long molecular dynamics simulations of the PRD using an explicit water model optimized for intrinsically disordered proteins. These simulations accurately predicted our previous solid-state NMR data and newly acquired electron paramagnetic resonance double electron-electron resonance distances, lending confidence in their accuracy. The simulations show that the PRD generally forms an imperfect polyproline (polyP) II helical conformation. The two polyP regions within the PRD stay in a polyP II helix for most of the simulation, whereas occasional kinks in the proline-rich linker region cause an overall bend in the PRD structure. The dihedral angles of the glycine at the end of the second polyP region are very variable, effectively decoupling the highly dynamic 12 C-terminal residues from the rest of the PRD.

E46K-like α-synuclein mutants increase lipid interactions and disrupt membrane selectivity.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, and both genetic and histopathological evidence have implicated the ubiquitous presynaptic protein α-synuclein (αSyn) in its pathogenesis. Recent work has investigated how disrupting αSyn's interaction with membranes triggers trafficking defects, cellular stress, and apoptosis. Special interest has been devoted to a series of mutants exacerbating the effects of the E46K mutation (associated with autosomal dominant PD) through homologous Glu-to-Lys substitutions in αSyn's N-terminal region (i.e. E35K and E61K). Such E46K-like mutants have been shown to cause dopaminergic neuron loss and severe but L-DOPA-responsive motor defects in mouse overexpression models, presenting enormous translational potential for PD and other "synucleinopathies." In this work, using a variety of biophysical techniques, we characterize the molecular pathology of E46K-like αSyn mutants by studying their structure and membrane-binding and remodeling abilities. We find that, although a slight increase in the mutants' avidity for synaptic vesicle-like membranes can be detected, most of their deleterious effects are connected to their complete disruption of αSyn's curvature selectivity. Indiscriminate binding can shift αSyn's subcellular localization away from its physiological interactants at the synaptic bouton toward trafficking vesicles and organelles, as observed in E46K-like cellular and murine models, as well as in human pathology. In conclusion, our findings suggest that a loss of curvature selectivity, rather than increased membrane affinity, could be the critical dyshomeostasis in synucleinopathies.

Construction of Parkinson's disease marker-based weighted protein-protein interaction network for prioritization of co-expressed genes.

BACKGROUND: Parkinson's disease (PD) is a complex neurodegenerative movement disorder that primarily results due to the loss of dopaminergic neurons in the substantia nigra region. Studying gene expression in the substantia nigra region would potentially unravel disease-relevant protein-protein interactions. METHODS: In this study we have used network science approach to prioritize candidate genes by focussing on differentially expressed genes (DEGs) that interact with established PD associated-genes (PDAG). Prioritizing genes that interact with already established PDAG would reduce the probability of spurious protein-protein associations. The dataset GSE54282 with Parkinson's disease affected substantia nigra samples was extracted from Gene Expression Omnibus (GEO) database. Protein-Protein Interaction Network (PPIN) was constructed by retrieving all possible interactions between DEGs from high-throughput experiments and literature data using Bisogenet. This complex PPIN was decomposed to construct a subnetwork of Parkinson's Disease-Protein Interaction Map (PD-PIM) by including PDAG and following well-established concepts of network biology such as degree and betweenness centrality. We then implemented a "two-way analysis" where we selected genes belonging to PDPIM subnetwork with their primary interacting partners and highly coexpressed genes on the basis of Pearson score. RESULTS: A complex PPIN comprised of 5340 nodes (genes) and 39,199 edges (interactions) was obtained. A list of 205 genes (123 PDAGs, 69 hub genes and 13 bottleneck genes) with their respective first level interacting partners were extracted from PPIN interactome to build a PD-specific subnetwork, PD-PIM. This subnetwork PD-PIM comprised of 5078 nodes and 38,357 edges. We then employed a "two-way" gene prioritization method that delineated 267 genes of which 16 genes were found to intersect in the two networks of the "two-way analysis". Of the 16 genes, we narrowed down to 7 novel candidate genes (VCAM1, BACH1, CALM3, EGR1, IKBKE, MYC and YWHAG) displaying significant changes in their network interactions between control and disease samples. Interestingly, these genes were associated with neuroinflammation signaling pathway, MAPK signaling apoptosis pathway, movement disorders and development of neurons that are linked with development of PD. CONCLUSION: We propose that VCAM1, BACH1, CALM3, EGR1, IKBKE, MYC and YWHAG genes might play important roles in PD pathogenesis, as well as facilitate the development of effective targeted therapies. Our integrative and network based approach for finding therapeutic targets in genomic data could accelerate the identification of novel drug targets for Parkinson's disease.

Gene co-expression network analysis for identifying genetic markers in Parkinson's disease - a three-way comparative approach.

Parkinson's disease (PD) is a neurodegenerative disorder involving progressive deterioration of dopaminergic neurons. Although few genetic markers for familial PD are known, the etiology of sporadic PD remains poorly understood. Microarray data was analysed for induced pluripotent stem cells (iPSCs) derived from PD patients and mature neuronal cells (mDA) differentiated from these iPSCs. Combining expression and semantic similarity, a highly-correlated PD interactome was constructed that included interactions of established Parkinson's disease marker genes. A novel three-way comparative approach was employed, delineating topologically and functionally important genes. These genes showed involvement in pathways like Parkin-ubiquitin proteosomal system (UPS), immune associated biological processes and apoptosis. Of interest are three genes, eEF1A1, CASK, and PSMD6 that are linked to PARK2 activity in the cell and thereby form attractive candidate genes for understanding PD. Network biology approach delineated in this study can be applied to other neurodegenerative disorders for identification of important genetic regulators.

Membranes as modulators of amyloid protein misfolding and target of toxicity.

Abnormal protein aggregation is a hallmark of various human diseases. α-Synuclein, a protein implicated in Parkinson's disease, is found in aggregated form within Lewy bodies that are characteristically observed in the brains of PD patients. Similarly, deposits of aggregated human islet amyloid polypeptide (IAPP) are found in the pancreatic islets in individuals with type 2 diabetes mellitus. Significant number of studies have focused on how monomeric, disaggregated proteins transition into various amyloid structures leading to identification of a vast number of aggregation promoting molecules and processes over the years. Inasmuch as these factors likely enhance the formation of toxic, misfolded species, they might act as risk factors in disease. Cellular membranes, and particularly certain lipids, are considered to be among the major players for aggregation of α-synuclein and IAPP, and membranes might also be the target of toxicity. Past studies have utilized an array of biophysical tools, both in vitro and in vivo, to expound the membrane-mediated aggregation. Here, we focus on membrane interaction of α-synuclein and IAPP, and how various kinds of membranes catalyze or modulate the aggregation of these proteins and how, in turn, these proteins disrupt membrane integrity, both in vitro and in vivo. The membrane interaction and subsequent aggregation has been briefly contrasted to aggregation of α-synuclein and IAPP in solution. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.

Pericyte degeneration causes white matter dysfunction in the mouse central nervous system.

Diffuse white-matter disease associated with small-vessel disease and dementia is prevalent in the elderly. The biological mechanisms, however, remain elusive. Using pericyte-deficient mice, magnetic resonance imaging, viral-based tract-tracing, and behavior and tissue analysis, we found that pericyte degeneration disrupted white-matter microcirculation, resulting in an accumulation of toxic blood-derived fibrin(ogen) deposits and blood-flow reductions, which triggered a loss of myelin, axons and oligodendrocytes. This disrupted brain circuits, leading to white-matter functional deficits before neuronal loss occurs. Fibrinogen and fibrin fibrils initiated autophagy-dependent cell death in oligodendrocyte and pericyte cultures, whereas pharmacological and genetic manipulations of systemic fibrinogen levels in pericyte-deficient, but not control mice, influenced the degree of white-matter fibrin(ogen) deposition, pericyte degeneration, vascular pathology and white-matter changes. Thus, our data indicate that pericytes control white-matter structure and function, which has implications for the pathogenesis and treatment of human white-matter disease associated with small-vessel disease.

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR.

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single protein can generate different type of membrane curvatures using specific conformations for specific membrane structures and how EPR is a versatile tool well-suited to analyze subtle alterations in structures under such modifying conditions which otherwise would have been difficult using other biophysical tools.

α-Synuclein Amyloid Fibrils with Two Entwined, Asymmetrically Associated Protofibrils.

Parkinson disease and other progressive neurodegenerative conditions are characterized by the intracerebral presence of Lewy bodies, containing amyloid fibrils of α-synuclein. We used cryo-electron microscopy and scanning transmission electron microscopy (STEM) to study in vitro-assembled fibrils. These fibrils are highly polymorphic. Focusing on twisting fibrils with an inter-crossover spacing of 77 nm, our reconstructions showed them to consist of paired protofibrils. STEM mass per length data gave one subunit per 0.47 nm axial rise per protofibril, consistent with a superpleated ß-structure. The STEM images show two thread-like densities running along each of these fibrils, which we interpret as ladders of metal ions. These threads confirmed the two-protofibril architecture of the 77-nm twisting fibrils and allowed us to identify this morphotype in STEM micrographs. Some other, but not all, fibril morphotypes also exhibit dense threads, implying that they also present a putative metal binding site. We propose a molecular model for the protofibril and suggest that polymorphic variant fibrils have different numbers of protofibrils that are associated differently.

Early sodium dodecyl sulfate induced collapse of α-synuclein correlates with its amyloid formation.

The aggregation of α-synuclein (A-syn) has been implicated strongly in Parkinson's disease (PD). In vitro studies established A-syn to be a member of the intrinsically disordered protein (IDP) family. This protein undergoes structural interconversion between an extended and a compact state, and this equilibrium influences the mechanism of its aggregation. A combination of fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) has been used to study the membrane induced conformational reorganization and aggregation of A-syn. Different structural and conformational events, including the early collapse, the formation of the secondary structure, and aggregation have been identified and characterized using FCS and other biophysical methods. In addition, the concentrations of glycerol and urea have been varied to study the effect of solution conditions on the above conformational events. Further, we have extended this study on a number of A-syn mutants, namely, A30P, A53T, and E46K. These mutants are chosen because of their known implications in the disease pathology. The variation of solution conditions and mutational analyses suggest a strong correlation between the extent of early collapse and the onset of aggregation in PD.

Nonaggregated α-synuclein influences SNARE-dependent vesicle docking via membrane binding.

α-Synuclein (α-Syn), a major component of Lewy body that is considered as the hallmark of Parkinson's disease (PD), has been implicated in neuroexocytosis. Overexpression of α-Syn decreases the neurotransmitter release. However, the mechanism by which α-Syn buildup inhibits the neurotransmitter release is still unclear. Here, we investigated the effect of nonaggregated α-Syn on SNARE-dependent liposome fusion using fluorescence methods. In ensemble in vitro assays, α-Syn reduces lipid mixing mediated by SNAREs. Furthermore, with the more advanced single-vesicle assay that can distinguish vesicle docking from fusion, we found that α-Syn specifically inhibits vesicle docking, without interfering with the fusion. The inhibition in vesicle docking requires α-Syn binding to acidic lipid containing membranes. Thus, these results imply the existence of at least two mechanisms of inhibition of SNARE-dependent membrane fusion: at high concentrations, nonaggregated α-Syn inhibits docking by binding acidic lipids but not v-SNARE; on the other hand, at much lower concentrations, large α-Syn oligomers inhibit via a mechanism that requires v-SNARE interaction [ Choi et al. Proc. Natl. Acad. Sci. U. S. A. 2013 , 110 ( 10 ), 4087 - 4092 ].

Hydration dynamics as an intrinsic ruler for refining protein structure at lipid membrane interfaces.

Knowing the topology and location of protein segments at water-membrane interfaces is critical for rationalizing their functions, but their characterization is challenging under physiological conditions. Here, we debut a unique spectroscopic approach by using the hydration dynamics gradient found across the phospholipid bilayer as an intrinsic ruler for determining the topology, immersion depth, and orientation of protein segments in lipid membranes, particularly at water-membrane interfaces. This is achieved through the site-specific quantification of translational diffusion of hydration water using an emerging tool, (1)H Overhauser dynamic nuclear polarization (ODNP)-enhanced NMR relaxometry. ODNP confirms that the membrane-bound region of α-synuclein (αS), an amyloid protein known to insert an amphipathic α-helix into negatively charged phospholipid membranes, forms an extended α-helix parallel to the membrane surface. We extend the current knowledge by showing that residues 90-96 of bound αS, which is a transition segment that links the α-helix and the C terminus, adopt a larger loop than an idealized α-helix. The unstructured C terminus gradually threads through the surface hydration layers of lipid membranes, with the beginning portion residing within 5-15 Å above the phosphate level, and only the very end of C terminus surveying bulk water. Remarkably, the intrinsic hydration dynamics gradient along the bilayer normal extends to 20-30 Å above the phosphate level, as demonstrated with a peripheral membrane protein, annexin B12. ODNP offers the opportunity to reveal previously unresolvable structure and location of protein segments well above the lipid phosphate, whose structure and dynamics critically contribute to the understanding of functional versatility of membrane proteins.

α-Synuclein oligomers with broken helical conformation form lipoprotein nanoparticles.

α-Synuclein (αS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as "discs." Previously, it has been unclear whether αS also possesses this property. Using cryo-electron microscopy and light scattering, we found that αS can remodel phosphatidylglycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7-10-nm range) resemble those formed by apolipoproteins. The molar ratio of αS to lipid in nanoparticles is ∼1:20, and αS is oligomeric (including trimers and tetramers). Similar nanoparticles form when αS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by αS. Circular dichroism and four-pulse double electron electron resonance experiments revealed that in nanoparticles αS assumes a broken helical conformation distinct from the extended helical conformation adopted when αS is bound to intact vesicles or membrane tubules. We also observed αS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in a similar manner. αS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein.

Rational design of potent domain antibody inhibitors of amyloid fibril assembly.

Antibodies hold significant potential for inhibiting toxic protein aggregation associated with conformational disorders such as Alzheimer's and Huntington's diseases. However, near-stoichiometric antibody concentrations are typically required to completely inhibit protein aggregation. We posited that the molecular interactions mediating amyloid fibril formation could be harnessed to generate antibodies with potent antiaggregation. Here we report that grafting small amyloidogenic peptides (6-10 residues) into the complementarity-determining regions of a single-domain (V(H)) antibody yields potent domain antibody inhibitors of amyloid formation. Grafted AMyloid-Motif AntiBODIES (gammabodies) presenting hydrophobic peptides from Aß (Alzheimer's disease), α-Synuclein (Parkinson's disease), and islet amyloid polypeptide (type 2 diabetes) inhibit fibril assembly of each corresponding polypeptide at low substoichiometric concentrations (1:10 gammabody:monomer molar ratio). In contrast, sequence- and conformation-specific antibodies that were obtained via immunization are unable to prevent fibrillization at the same substoichiometric concentrations. Gammabodies prevent amyloid formation by converting monomers and/or fibrillar intermediates into small complexes that are unstructured and benign. We expect that our antibody design approach--which eliminates the need for immunization or screening to identify sequence-specific domain antibody inhibitors--can be readily extended to generate potent aggregation inhibitors of other amyloidogenic polypeptides linked to human disease.

Remodeling of lipid vesicles into cylindrical micelles by α-synuclein in an extended α-helical conformation.

α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ~50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150-500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70-100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation.

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.

Membrane curvature induction and tubulation are common features of synucleins and apolipoproteins.

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, ß-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.