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, β-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.     

Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis

Deposition of β cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mm glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that β cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of β cells.     

Small Liposomes Accelerate the Fibrillation of Amyloid β (1–40)

The deposition of amyloid β (Aβ) peptides is a pathological hallmark of Alzheimer disease. Aβ peptides were previously considered to interact specifically with ganglioside-containing membranes. Several studies have suggested that Aβ peptides also bind to phosphatidylcholine membranes, which lead to deformation of membranes and fibrillation of Aβ. Moreover, the role of membrane curvature, one type of deformation produced by binding of proteins to a membrane, in the binding and fibrillation of Aβ remains unclear. To clearly understand the relationship between the binding, consequent membrane deformation, and fibrillation of Aβ, we examined the amyloid fibrillation of Aβ-(1–40) in the presence of liposomes of various sizes. Membrane curvature increased with a decrease in the size of the liposomes. We used liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine to eliminate electrostatic effects. The results obtained showed that liposomes of smaller sizes (≤50 nm) significantly accelerated the nucleation step, thereby shortening the lag time of fibrillation. On the other hand, liposomes of larger sizes decreased the amount of fibrils but did not notably affect the lag time. The morphologies of fibrils, which were monitored by total internal reflection fluorescence microscopy, atomic force microscopy, and transmission electron microscopy, revealed that the length of Aβ-(1–40) fibrils became shorter and the amount of amorphous aggregates became larger as liposomes increased in size. These results suggest that the curvature of membranes coupled with an increase in water-accessible hydrophobic regions is important for binding and concentrating Aβ monomers, leading to amyloid nucleation. Furthermore, amyloid fibrillation on membranes may compete with non-productive binding to produce amorphous aggregates.     

Neprilysin Impedes Islet Amyloid Formation by Inhibition of Fibril Formation Rather Than Peptide Degradation

Deposition of islet amyloid polypeptide (IAPP) as islet amyloid in type 2 diabetes contributes to loss of β-cell function and mass, yet the mechanism for its occurrence is unclear. Neprilysin is a metallopeptidase known to degrade amyloid in Alzheimer disease. We previously demonstrated neprilysin to be present in pancreatic islets and now sought to determine whether it plays a role in degrading islet amyloid. We used an in vitro model where cultured human IAPP (hIAPP) transgenic mouse islets develop amyloid and thereby have increased β-cell apoptosis. Islet neprilysin activity was inhibited or up-regulated using a specific inhibitor or adenovirus encoding neprilysin, respectively. Following neprilysin inhibition, islet amyloid deposition and β-cell apoptosis increased by 54 and 75%, respectively, whereas when neprilysin was up-regulated islet amyloid deposition and β-cell apoptosis both decreased by 79%. To determine if neprilysin modulated amyloid deposition by cleaving hIAPP, analysis of hIAPP incubated with neprilysin was performed by mass spectrometry, which failed to demonstrate neprilysin-induced cleavage. Rather, neprilysin may act by reducing hIAPP fibrillogenesis, which we showed to be the case by fluorescence-based thioflavin T binding studies and electron microscopy. In summary, neprilysin decreases islet amyloid deposition by inhibiting hIAPP fibril formation, rather than degrading hIAPP. These findings suggest that targeting the role of neprilysin in IAPP fibril assembly, in addition to IAPP cleavage by other peptidases, may provide a novel approach to reduce and/or prevent islet amyloid deposition in type 2 diabetes.     

Matrix Metalloproteinase-9 Reduces Islet Amyloid Formation by Degrading Islet Amyloid Polypeptide

Deposition of islet amyloid polypeptide (IAPP) as amyloid is a pathological hallmark of the islet in type 2 diabetes, which is toxic to β-cells. We previously showed that the enzyme neprilysin reduces islet amyloid deposition and thereby reduces β-cell apoptosis, by inhibiting fibril formation. Two other enzymes, matrix metalloproteinase (MMP)-2 and MMP-9, are extracellular gelatinases capable of degrading another amyloidogenic peptide, Aβ, the constituent of amyloid deposits in Alzheimer disease. We therefore investigated whether MMP-2 and MMP-9 play a role in reducing islet amyloid deposition. MMP-2 and MMP-9 mRNA were present in mouse islets but only MMP-9 activity was detectable. In an islet culture model where human IAPP (hIAPP) transgenic mouse islets develop amyloid but nontransgenic islets do not, a broad spectrum MMP inhibitor (GM6001) and an MMP-2/9 inhibitor increased amyloid formation and the resultant β-cell apoptosis. In contrast, a specific MMP-2 inhibitor had no effect on either amyloid deposition or β-cell apoptosis. Mass spectrometry demonstrated that MMP-9 degraded amyloidogenic hIAPP but not nonamyloidogenic mouse IAPP. Thus, MMP-9 constitutes an endogenous islet protease that limits islet amyloid deposition and its toxic effects via degradation of hIAPP. Because islet MMP-9 mRNA levels are decreased in type 2 diabetic subjects, islet MMP-9 activity may also be decreased in human type 2 diabetes, thereby contributing to increased islet amyloid deposition and β-cell loss. Approaches to increase islet MMP-9 activity could reduce or prevent amyloid deposition and its toxic effects in type 2 diabetes.     

Overall Sulfation of Heparan Sulfate from Pancreatic Islet β-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide

Islet amyloid, a pathologic feature of type 2 diabetes, contains the islet β-cell peptide islet amyloid polypeptide (IAPP) as its unique amyloidogenic component. Islet amyloid also contains heparan sulfate proteoglycans (HSPGs) that may contribute to amyloid formation by binding IAPP via their heparan sulfate (HS) chains. We hypothesized that β-cells produce HS that bind IAPP via regions of highly sulfated disaccharides. Unexpectedly, HS from the β-cell line β-TC3 contained fewer regions of highly sulfated disaccharides compared with control normal murine mammary gland (NMuMG) cells. The proportion of HS that bound IAPP was similar in both cell lines (∼65%). The sulfation pattern of IAPP-bound versus non-bound HS from β-TC3 cells was similar. In contrast, IAPP-bound HS from NMuMG cells contained frequent highly sulfated regions, whereas the non-bound material demonstrated fewer sulfated regions. Fibril formation from IAPP was stimulated equally by IAPP-bound β-TC3 HS, non-bound β-TC3 HS, and non-bound NMuMG HS but was stimulated to a greater extent by the highly sulfated IAPP-bound NMuMG HS. Desulfation of HS decreased the ability of both β-TC3 and NMuMG HS to stimulate IAPP maximal fibril formation, but desulfated HS from both cell types still accelerated fibril formation relative to IAPP alone. In summary, neither binding to nor acceleration of fibril formation from the amyloidogenic peptide IAPP is dependent on overall sulfation in HS synthesized by β-TC3 cells. This information will be important in determining approaches to reduce HS-IAPP interactions and ultimately prevent islet amyloid formation and its toxic effects in type 2 diabetes.     

The Antimicrobial Mechanism of Action of Epsilon-Poly-l-Lysine

Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL''s mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL''s effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL''s binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles.     

Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function

Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane β-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ∼1/40 nm⁻¹) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (∼40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ∼50 mN/m and a curvature-imposed protein deformation energy of ∼7 k_BT. Such substantial energies have been shown to conformationally activate, or unfold, β-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general.     

Roles of Amphipathic Helices and the Bin/Amphiphysin/Rvs (BAR) Domain of Endophilin in Membrane Curvature Generation

Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63–86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63–75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic α-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.     

Impact of membrane curvature on amyloid aggregation

The misfolding, amyloid aggregation, and fibril formation of intrinsically disordered proteins/peptides (or amyloid proteins) have been shown to cause a number of disorders. The underlying mechanisms of amyloid fibrillation and structural properties of amyloidogenic precursors, intermediates, and amyloid fibrils have been elucidated in detail; however, in-depth examinations on physiologically relevant contributing factors that induce amyloidogenesis and lead to cell death remain challenging. A large number of studies have attempted to characterize the roles of biomembranes on protein aggregation and membrane-mediated cell death by designing various membrane components, such as gangliosides, cholesterol, and other lipid compositions, and by using various membrane mimetics, including liposomes, bicelles, and different types of lipid-nanodiscs.We herein review the dynamic effects of membrane curvature on amyloid generation and the inhibition of amyloidogenic proteins and peptides, and also discuss how amyloid formation affects membrane curvature and integrity, which are key for understanding relationships with cell death. Small unilamellar vesicles with high curvature and large unilamellar vesicles with low curvature have been demonstrated to exhibit different capabilities to induce the nucleation, amyloid formation, and inhibition of amyloid-β peptides and α-synuclein. Polymorphic amyloidogenesis in small unilamellar vesicles was revealed and may be viewed as one of the generic properties of interprotein interaction-dominated amyloid formation. Several mechanical models and phase diagrams are comprehensively shown to better explain experimental findings. The negative membrane curvature-mediated mechanisms responsible for the toxicity of pancreatic β cells by the amyloid aggregation of human islet amyloid polypeptide (IAPP) and binding of the precursors of the semen-derived enhancer of viral infection (SEVI) are also described. The curvature-dependent binding modes of several types of islet amyloid polypeptides with high-resolution NMR structures are also discussed.     

General Amyloid Inhibitors? A Critical Examination of the Inhibition of IAPP Amyloid Formation by Inositol Stereoisomers

Islet amyloid polypeptide (IAPP or amylin) forms amyloid deposits in the islets of Langerhans; a process that is believed to contribute to the progression of type 2 diabetes and to the failure of islet transplants. An emerging theme in amyloid research is the hypothesis that the toxic species produced during amyloid formation by different polypeptides share common features and exert their effects by common mechanisms. If correct, this suggests that inhibitors of amyloid formation by one polypeptide might be effective against other amyloidogenic sequences. IAPP and Aβ, the peptide responsible for amyloid formation in Alzheimer''s disease, are particularly interesting in this regard as they are both natively unfolded in their monomeric states and share some common characteristics. Comparatively little effort has been expended on the design of IAPP amyloid inhibitors, thus it is natural to inquire if Aβ inhibitors are effective against IAPP, especially since no IAPP inhibitors have been clinically approved. A range of compounds inhibit Aβ amyloid formation, including various stereoisomers of inositol. Myo-, scyllo-, and epi-inositol have been shown to induce conformational changes in Aβ and prevent Aβ amyloid fibril formation by stabilizing non-fibrillar β-sheet structures. We investigate the ability of inositol stereoisomers to inhibit amyloid formation by IAPP. The compounds do not induce a conformational change in IAPP and are ineffective inhibitors of IAPP amyloid formation, although some do lead to modest apparent changes in IAPP amyloid fibril morphology. Thus not all classes of Aβ inhibitors are effective against IAPP. This work provides a basis of comparison to work on polyphenol based inhibitors of IAPP amyloid formation and helps provide clues as to the features which render them effective. The study also helps provide information for further efforts in rational inhibitor design.     

Fiber-Dependent and -Independent Toxicity of Islet Amyloid Polypeptide

The 37-residue peptide hormone islet amyloid polypeptide (IAPP) plays a central role in diabetes pathology. Although its amyloid fiber aggregation kinetics and cytotoxicity to β-cells are well documented, few reports have directly assessed the role of fibers in cell-based toxicity experiments. Here, we report that amyloid formation of IAPP can be strongly inhibited by the extracellular environment of live cells. For example, fiber formation is more strongly suppressed in cell culture medium than in aqueous buffer. The serum component of the medium is responsible for this inhibition. Although amyloid formation was previously shown to be catalyzed by both synthetic and chloroform-extracted phospholipid surfaces, it is instead inhibited by membrane surfaces prepared directly from the plasma membranes of an immortal β-cell line. This disparity is reconciled by direct assessment of fibers in cell-culture-based toxicity experiments. We discovered that fibers are nontoxic if they are washed free of adsorbed nonfibrillar components. Moreover, toxicity is not only rescued when monomers are added back to fibers but is greater than what is observed from the precursor alone. Our results are interpreted in light of the capacity of the fiber surface to template amyloid nucleation.     

Amyloids of Alpha-Synuclein Affect the Structure and Dynamics of Supported Lipid Bilayers

Interactions of monomeric alpha-synuclein (αS) with lipid membranes have been suggested to play an important role in initiating aggregation of αS. We have systematically analyzed the distribution and self-assembly of monomeric αS on supported lipid bilayers. We observe that at protein/lipid ratios higher than 1:10, αS forms micrometer-sized clusters, leading to observable membrane defects and decrease in lateral diffusion of both lipids and proteins. An αS deletion mutant lacking amino-acid residues 71–82 binds to membranes, but does not observably affect membrane integrity. Although this deletion mutant cannot form amyloid, significant amyloid formation is observed in the wild-type αS clusters. These results suggest that the process of amyloid formation, rather than binding of αS on membranes, is crucial in compromising membrane integrity.     

Parkinson’s Protein α-Synuclein Binds Efficiently and with a Novel Conformation to Two Natural Membrane Mimics

Binding of human α-Synuclein, a protein associated with Parkinson’s disease, to natural membranes is thought to be crucial in relation to its pathological and physiological function. Here the binding of αS to small unilamellar vesicles mimicking the inner mitochondrial and the neuronal plasma membrane is studied in situ by continuous wave and pulsed electron paramagnetic resonance. Local binding information of αS spin labeled by MTSL at positions 56 and 69 respectively shows that also helix 2 (residues 50–100) binds firmly to both membranes. By double electron-electron resonance (DEER) on the mutant spin labeled at positions 27 and 56 (αS 27/56) a new conformation on the membrane is found with a distance of 3.6 nm/ 3.7 nm between residues 27 and 56. In view of the low negative charge density of these membranes, the strong interaction is surprising, emphasizing that function and pathology of αS could involve synaptic vesicles and mitochondria.     

Spatial Control of Epsin-induced Clathrin Assembly by Membrane Curvature

Epsins belong to the family of highly conserved clathrin-associated sorting proteins that are indispensable for clathrin-mediated endocytosis, but their precise functions remain unclear. We have developed an assay system of budded supported membrane tubes displaying planar and highly curved membrane surfaces to analyze intrinsic membrane curvature preference shown by clathrin-associated sorting proteins. Using real-time fluorescence microscopy, we find that epsin preferentially partitions to and assembles clathrin on highly curved membrane surfaces. Sorting of epsin to regions of high curvature strictly depends on binding to phosphatidylinositol 4,5-bisphosphate. Fluorescently labeled clathrins rapidly assemble as foci, which in turn cluster epsin, while maintaining tube integrity. Clathrin foci grow in intensity with a typical time constant of ∼75 s, similar to the time scales for coated pit formation seen in cells. Epsin therefore effectively senses membrane curvature to spatially control clathrin assembly. Our results highlight the potential role of membrane curvature in orchestrating the myriad molecular interactions necessary for the success of clathrin-mediated membrane budding.     

N-terminal Acetylation Stabilizes N-terminal Helicity in Lipid- and Micelle-bound α-Synuclein and Increases Its Affinity for Physiological Membranes

The Parkinson disease protein α-synuclein is N-terminally acetylated, but most in vitro studies have been performed using unacetylated α-synuclein. Binding to lipid membranes is considered key to the still poorly understood function of α-synuclein. We report the effects of N-terminal acetylation on α-synuclein binding to lipid vesicles of different composition and curvature and to micelles composed of the detergents β-octyl-glucoside (BOG) and SDS. In the presence of SDS, N-terminal acetylation results in a slightly increased helicity for the N-terminal ∼10 residues of the protein, likely due to the stabilization of N-terminal fraying through the formation of a helix cap motif. In the presence of BOG, a detergent used in previous isolations of helical oligomeric forms of α-synuclein, the N-terminally acetylated protein adopts a novel conformation in which the N-terminal ∼30 residues bind the detergent micelle in a partly helical conformation, whereas the remainder of the protein remains unbound and disordered. Binding of α-synuclein to lipid vesicles with high negative charge content is essentially unaffected by N-terminal acetylation irrespective of curvature, but binding to vesicles of lower negative charge content is increased, with stronger binding observed for vesicles with higher curvature. Thus, the naturally occurring N-terminally acetylated form of α-synuclein exhibits stabilized helicity at its N terminus and increased affinity for lipid vesicles similar to synaptic vesicles, a binding target of the protein in vivo. Furthermore, the novel BOG-bound state of N-terminally acetylated α-synuclein may serve as a model of partly helical membrane-bound intermediates with a role in α-synuclein function and dysfunction.     

Amyloid Precursor Protein (APP)/APP-like Protein 2 (APLP2) Expression Is Required to Initiate Endosome-Nucleus-Autophagosome Trafficking of Glypican-1-derived Heparan Sulfate

Anhydromannose (anMan)-containing heparan sulfate (HS) derived from the proteoglycan glypican-1 is generated in endosomes by an endogenously or ascorbate-induced S-nitrosothiol-catalyzed reaction. Processing of the amyloid precursor protein (APP) and APP-like protein 2 (APLP2) by β- and γ-secretases into amyloid β (Aβ) and Aβ-like peptides also takes place in these compartments. Moreover, anMan-containing HS suppresses the formation of toxic Aβ assemblies in vitro. We showed by using deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody as well as ³⁵S labeling experiments that expression of APP/APLP2 is required for ascorbate-induced transport of HS from endosomes to the nucleus. Nuclear translocation was observed in wild-type mouse embryonic fibroblasts (WT MEFs), Tg2576 MEFs, and N2a neuroblastoma cells but not in APP^−/− and APLP2^−/− MEFs. Transfection of APP^−/− cells with a vector encoding APP restored nuclear import of anMan-containing HS. In WT MEFs and N2a neuroblastoma cells exposed to β- or γ-secretase inhibitors, nuclear translocation was greatly impeded, suggesting involvement of APP/APLP2 degradation products. In Tg2576 MEFs, the β-inhibitor blocked transport, but the γ-inhibitor did not. During chase in ascorbate-free medium, anMan-containing HS disappeared from the nuclei of WT MEFs. Confocal immunofluorescence microscopy showed that they appeared in acidic, LC3-positive vesicles in keeping with an autophagosomal location. There was increased accumulation of anMan-containing HS in nuclei and cytosolic vesicles upon treatment with chloroquine, indicating that HS was degraded in lysosomes. Manipulations of APP expression and processing may have deleterious effects upon HS function in the nucleus.     

Serum albumin impedes the amyloid aggregation and hemolysis of human islet amyloid polypeptide and alpha synuclein

Protein aggregation is a ubiquitous phenomenon underpinning the origins of a range of human diseases. The amyloid aggregation of human islet amyloid polypeptide (IAPP) and alpha synuclein (αS), specifically, is a hallmark of type 2 diabetes (T2D) and Parkinson's disease impacting millions of people worldwide. Although IAPP and αS are strongly associated with pancreatic β-cell islets and presynaptic terminals, they have also been found in blood circulation and the gut. While extensive biophysical and biochemical studies have been focused on IAPP and αS interacting with cell membranes or model lipid vesicles, the roles of plasma proteins on the amyloidosis and membrane association of these two major types of amyloid proteins have rarely been examined. Using a thioflavin T kinetic assay, transmission electron microscopy and a hemolysis assay here we show that human serum albumin, the most abundant protein in the plasma, impeded the fibrillization and mitigated membrane damage of both IAPP and αS. This study offers a new insight on the native inhibition of amyloidosis.     

The interplay between lipids and dopamine on α-synuclein oligomerization and membrane binding

The deposition of α-syn (α-synuclein) as amyloid fibrils and the selective loss of DA (dopamine) containing neurons in the substantia nigra are two key features of PD (Parkinson''s disease). α-syn is a natively unfolded protein and adopts an α-helical conformation upon binding to lipid membrane. Oligomeric species of α-syn have been proposed to be the pathogenic species associated with PD because they can bind lipid membranes and disrupt membrane integrity. DA is readily oxidized to generate reactive intermediates and ROS (reactive oxygen species) and in the presence of DA, α-syn form of SDS-resistant soluble oligomers. It is postulated that the formation of the α-syn:DA oligomers involves the cross-linking of DA-melanin with α-syn, via covalent linkage, hydrogen and hydrophobic interactions. We investigate the effect of lipids on DA-induced α-syn oligomerization and studied the ability of α-syn:DA oligomers to interact with lipids vesicles. Our results show that the interaction of α-syn with lipids inhibits the formation of DA-induced α-syn oligomers. Moreover, the α-syn:DA oligomer cannot interact with lipid vesicles or cause membrane permeability. Thus, the formation of α-syn:DA oligomers may alter the actions of α-syn which require membrane association, leading to disruption of its normal cellular function.     

Effect of Proline Mutations on the Monomer Conformations of Amylin

The formation of human islet amyloid polypeptide (hIAPP) is implicated in the loss of pancreatic β-cells in type II diabetes. Rat amylin, which differs from human amylin at six residues, does not lead to formation of amyloid fibrils. Pramlintide is a synthetic analog of human amylin that shares three proline substitutions with rat amylin. Pramlintide has a much smaller propensity to form amyloid aggregates and has been widely prescribed in amylin replacement treatment. It is known that the three prolines attenuate β-sheet formation. However, the detailed effects of these proline substitutions on full-length hIAPP remain poorly understood. In this work, we use molecular simulations and bias-exchange metadynamics to investigate the effect of proline substitutions on the conformation of the hIAPP monomer. Our results demonstrate that hIAPP can adopt various β-sheet conformations, some of which have been reported in experiments. The proline substitutions perturb the formation of long β-sheets and reduce their stability. More importantly, we find that all three proline substitutions of pramlintide are required to inhibit β conformations and stabilize the α-helical conformation. Fewer substitutions do not have a significant inhibiting effect.