Anja Stefanovic - Academia.edu (original) (raw)

Uploads

Papers by Anja Stefanovic

Physical Review Letters

In water, networks of semiflexible fibrils of the protein α-synuclein stiffen significantly with ... more In water, networks of semiflexible fibrils of the protein α-synuclein stiffen significantly with increasing temperature. We make plausible that this reversible stiffening is a result of hydrophobic contacts between the fibrils that become more prominent with increasing temperature. The good agreement of our experimentally observed temperature dependence of the storage modulus of the network with a scaling theory linking network elasticity with reversible cross-linking enables us to quantify the endothermic binding enthalpy and estimate the effective size of hydrophobic patches on the fibril surface. Our findings may not only shed light on the role of amyloid deposits in disease conditions, but can also inspire new approaches for the design of thermoresponsive materials.

Biochemistry, 2015

Single-amino acid mutations in the human α-synuclein (αS) protein are related to early onset Park... more Single-amino acid mutations in the human α-synuclein (αS) protein are related to early onset Parkinson's disease (PD). In addition to the well-known A30P, A53T, and E46K mutants, recently a number of new familial disease-related αS mutations have been discovered. How these mutations affect the putative physiological function of αS and the disease pathology is still unknown. Here we focus on the H50Q and G51D familial mutants and show that like wild-type αS, H50Q and G51D monomers bind to negatively charged membranes, form soluble partially folded oligomers with an aggregation number of ∼30 monomers under specific conditions, and can aggregate into amyloid fibrils. We systematically studied the ability of these isolated oligomers to permeabilize membranes composed of anionic phospholipids (DOPG) and membranes mimicking the mitochondrial phospholipid composition (CL:POPE:POPC) using a calcein release assay. Small-angle X-ray scattering studies of isolated oligomers show that oligomers formed from wild-type αS and the A30P, E46K, H50Q, G51D, and A53T disease-related mutants are composed of a similar number of monomers. However, although the binding affinity of the monomeric protein and the aggregation number of the oligomers formed under our specific protocol are comparable for wild-type αS and H50Q and G51D αS, G51D oligomers cannot disrupt negatively charged and physiologically relevant model membranes. Replacement of the membrane-immersed glycine with a negatively charged aspartic acid at position 51 apparently abrogates membrane destabilization, whereas a mutation in the proximal but solvent-exposed part of the membrane-bound α-helix such as that found in the H50Q mutant has little effect on the bilayer disrupting properties of oligomers.

Small (Weinheim an der Bergstrasse, Germany), Jan 15, 2015

Multivalent membrane binding sites on the α-synuclein oligomer result in clustering of vesicles a... more Multivalent membrane binding sites on the α-synuclein oligomer result in clustering of vesicles and hemifusion of negatively charged model membranes. These multivalent, biological nanoparticles are reminiscent of inorganic nanoparticles in their interactions with membranes. Alpha-synuclein oligomers induce lipid exchange efficiently, with fewer than 10 oligomers/vesicle required to complete hemifusion. No full fusion or vesicle content mixing is observed.

FEBS Letters, 2014

We studied α-synuclein (αS) aggregation in giant vesicles, and observed dramatic membrane disinte... more We studied α-synuclein (αS) aggregation in giant vesicles, and observed dramatic membrane disintegration, as well as lipid incorporation into micrometer-sized suprafibrillar aggregates. In the presence of dye-filled vesicles, dye leakage and fibrillization happen concurrently. However, growing fibrils do not impair the integrity of phospholipid vesicles that have a low affinity for αS. Seeding αS aggregation accelerates dye leakage, indicating that oligomeric species are not required to explain the observed effect. The evolving picture suggests that fibrils that appear in solution bind membranes and recruit membrane-bound monomers, resulting in lipid extraction, membrane destabilization and the formation of lipid-containing suprafibrillar aggregates.

Table of Contents Chapter 1: Introduction 1.1 Alpha synuclein and Parkinson's disease 1.2 Propert... more Table of Contents Chapter 1: Introduction 1.1 Alpha synuclein and Parkinson's disease 1.2 Properties of alpha-synuclein 1.3 Oligomeric alpha-synuclein 1.4 Formation and characterization of oligomeric alpha-synuclein 1.4.1 High concentration-induced oligomers 1.4.2 Metal ion-induced oligomers 1.4.2 Metal ion-induced oligomers 1.4.4 HNE/ONE-induced oligomers 1.5 Alpha-synuclein membrane interactions 1.6 Scope of this thesis 1.7 References Chapter 2: Oligomer binding to bilayers with increasingly complex lipid compositions 2.1 Abstract 2.2 Introduction 2.3 Materials and Methods 2.3.1 Expression and purification of α-synuclein 2.3.2 Labeling of alpha-synuclein 2.3.3 Preparation of labeled αS oligomers 2.3.4 Qualitative oligomer binding assay 2.4 Results 2.4.1 The effect of cholesterol and sphingomyelin on αS oligomer binding in the presence of negatively charged lipids 2.4.2 The effect of lipid headgroup on oligomer binding 2.4.3 The effect of Cardiolipin on αS oligomer binding 2.4.4 The effect of brain lipids on oligomers binding 2.5 Discussion 2.6 References Chapter 3: Alpha-synuclein oligomers distinctively permeabilize complex model membranes 3.1. Abstract 3.2 Introduction 3.3 Materials and methods 3.3.1 Expression and purification of αS 3.3.2 Labeling of αS 3.3.3 Preparation of unlabeled and labeled αS oligomers 3.3.4 LUVs preparation and calcein release assay 3.3.5 Semi-quantitative αS monomer and oligomer binding assay 3.3.6 SUVs preparation and binding of αS oligomers to SUVs 3.4 Results 3.4.1 Binding of αS monomers to bilayers that mimic lipid composition of natural membranes 3.4.2 Do αS oligomers bind to bilayers mimicking the lipid composition of natural membranes and does this binding result in conformational changes? 3.4.3 Kinetics of membrane permeabilization (Dye release assay) 3.5 Discussion 3.6 Acknowledgments 3.7 References Chapter 4: Characterization of oligomers formed from disease-related alpha-synuclein amino acid mutations 4.1 Abstract 4.2 Introduction 4.3 Material and methods 4.3.1 Preparation of oligomeric alpha-synuclein 4.3.2 SUVs preparation and binding of αS monomers to SUVs 4.3.4 LUV preparation and calcein release assay 4.3.5 Small-Angle X-ray Scattering 4.3.6 Kinetics of aggregation 4.3.7 Atomic force microscopy (AFM) 4.4 Results 4.4.1 Binding of αS monomers to SUVs 4.4.2 Aggregation studies 4.4.3 Calcein release assay 4.4.4 Aggregation number 4.5 Discussion 4.6 Acknowledgments 4.7 References Chapter 5: Are alpha-synuclein oligomers toxic species? Chapter 6: Alpha-synuclein amyloid multimers act as multivalent nanoparticles to cause hemifusion in negatively charged bilayers 6.1 Abstract 6.2 Introduction 6.3 Material and Methods 6.3.1 Expression and purification of αS 6.3.2 Labeling of αS-A140C 6.3.3 Preparation of unlabeled and labeled αS oligomers 6.3.4 LUVs preparation 6.3.5 GUVs preparation for clustering experiment and imaging of clustered vesicles 6.3.6 Content mixing 6.3.7 Lipid mixing 96 6.3.8 Fluorescence Correlation Spectroscopy 6.3.9 Estimation of αS oligomers-membrane binding equilibrium 97 6.4 Results 98 6.4.1 αS oligomers induce vesicle clustering 98 6.4.2 Oligomer-induced vesicle fusion 99 6.4.3 Oligomer-induced hemifusion 6.5 Discussion 6.6 Acknowledgments 6.7 References Chapter 7: Conclusion and future recommendations Summary Samenvatting List of abbreviations Acknowledgments/Dankwoord/Zahvalnica List of publications Curriculum vitae Aggregation is the key process where the αS protein loses its putative function and gains toxicity [21-24]. The aggregation of αS starts from intrinsically disordered monomers, which self-assemble and form dimers and then possibly with the help of additional factors convert to (on-and off-pathway) oligomers [25-27], which may assemble into fibrils (Figure 1.2) [28]. The aggregated proteins in the fibrils have a characteristic β-sheet conformation and are packed perpendicular to the fiber axis [29]. Although earlier studies assumed fibrils to be toxic, currently it is suggested that oligomers are the main toxic species involved in the cell death of 1.4.3 Dopamine-induced oligomers In PD, oxidation of dopamine is one of the possible causes of cell death. Oxidation of dopamine gives an excessive production of semiquinones, H 2 O 2 and • HO radicals. In vivo studies have shown that αS is the negative regulator of dopamine neurotransmission [63, 64]. Mosharev et al. [65] confirmed that increased levels of dopamine (and its metabolic products) and Ca 2+ ions in dopaminergic neurons that are overexpressing αS are selectively neurotoxic for these neurons. Furthermore, 1.5 Alpha-synuclein membrane interactions To gain a clearer picture on the interaction between membranes and αS monomers or oligomers, many studies have made use of in vitro model membrane systems. Model membranes, such as lipid vesicles, mimic lipid membranes of cells and organelles. Early studies on the colocalization of αS with synaptic vesicles led to the suggestion that αS can interact with lipids [21]. Others [83] showed that αS also binds to model membranes and suggested that αS-membrane interactions have an important physiological and pathological role. As mentioned before, the N-terminal part of αS plays a crucial role in membrane binding [22, 23]. In solution the monomeric intrinsically disordered protein αS has no secondary structure [84], but upon binding to phospholipid membranes and detergent micelles the protein adopts an α-helical conformation [22, 37, 85-88]. Circular dichroism (CD) experiments show that the α-helical content upon binding to membranes increases from 3 to 80% [85]. According to the literature 41% of the αS protein becomes α-helical when bound to sodium dodecyl sulfate (SDS) micelles [89], while upon the binding to 1,2-dioleoylphosphatidylglycerol (DOPG): 1,2-dipalmitoylphosphatidylglycerol (DPPG) (DOPG:DPPG) vesicles, 61% of the protein adopts α-helical structure [90]. Additionally, binding of monomers to

FEBS Journal, 2014

α-Synuclein oligomers are increasingly considered to be responsible for the death of dopaminergic... more α-Synuclein oligomers are increasingly considered to be responsible for the death of dopaminergic neurons in Parkinson's disease. The toxicity mechanism of α-synuclein oligomers likely involves membrane permeabilization. Even though it is well established that α-synuclein oligomers bind and permeabilize vesicles composed of negatively-charged lipids, little attention has been given to the interaction of oligomers with bilayers of physiologically relevant lipid compositions. We investigated the interaction of α-synuclein with bilayers composed of lipid mixtures that mimic the composition of plasma and mitochondrial membranes. In the present study, we show that monomeric and oligomeric α-synuclein bind to these membranes. The resulting membrane leakage differs from that observed for simple artificial model bilayers. Although the addition of oligomers to negatively-charged lipid vesicles displays fast content release in a bulk permeabilization assay, adding oligomers to vesicles with compositions mimicking mitochondrial membranes shows a much slower loss of content. Oligomers are unable to induce leakage in the artificial plasma membranes, even after long-term incubation. CD experiments indicate that binding to lipid bilayers initially induces conformational changes in both oligomeric and monomeric α-synuclein, which show little change upon long-term incubation of oligomers with membranes. The results of the present study demonstrate that the mitochondrial model membranes are more vulnerable to permeabilization by oligomers than model plasma membranes reconstituted from brain-derived lipids; this preference may imply that increasingly complex membrane components, such as those in the plasma membrane mimic used in the present study, are less vulnerable to damage by oligomers.

Physical Review Letters

In water, networks of semiflexible fibrils of the protein α-synuclein stiffen significantly with ... more In water, networks of semiflexible fibrils of the protein α-synuclein stiffen significantly with increasing temperature. We make plausible that this reversible stiffening is a result of hydrophobic contacts between the fibrils that become more prominent with increasing temperature. The good agreement of our experimentally observed temperature dependence of the storage modulus of the network with a scaling theory linking network elasticity with reversible cross-linking enables us to quantify the endothermic binding enthalpy and estimate the effective size of hydrophobic patches on the fibril surface. Our findings may not only shed light on the role of amyloid deposits in disease conditions, but can also inspire new approaches for the design of thermoresponsive materials.

Biochemistry, 2015

Single-amino acid mutations in the human α-synuclein (αS) protein are related to early onset Park... more Single-amino acid mutations in the human α-synuclein (αS) protein are related to early onset Parkinson's disease (PD). In addition to the well-known A30P, A53T, and E46K mutants, recently a number of new familial disease-related αS mutations have been discovered. How these mutations affect the putative physiological function of αS and the disease pathology is still unknown. Here we focus on the H50Q and G51D familial mutants and show that like wild-type αS, H50Q and G51D monomers bind to negatively charged membranes, form soluble partially folded oligomers with an aggregation number of ∼30 monomers under specific conditions, and can aggregate into amyloid fibrils. We systematically studied the ability of these isolated oligomers to permeabilize membranes composed of anionic phospholipids (DOPG) and membranes mimicking the mitochondrial phospholipid composition (CL:POPE:POPC) using a calcein release assay. Small-angle X-ray scattering studies of isolated oligomers show that oligomers formed from wild-type αS and the A30P, E46K, H50Q, G51D, and A53T disease-related mutants are composed of a similar number of monomers. However, although the binding affinity of the monomeric protein and the aggregation number of the oligomers formed under our specific protocol are comparable for wild-type αS and H50Q and G51D αS, G51D oligomers cannot disrupt negatively charged and physiologically relevant model membranes. Replacement of the membrane-immersed glycine with a negatively charged aspartic acid at position 51 apparently abrogates membrane destabilization, whereas a mutation in the proximal but solvent-exposed part of the membrane-bound α-helix such as that found in the H50Q mutant has little effect on the bilayer disrupting properties of oligomers.

Small (Weinheim an der Bergstrasse, Germany), Jan 15, 2015

Multivalent membrane binding sites on the α-synuclein oligomer result in clustering of vesicles a... more Multivalent membrane binding sites on the α-synuclein oligomer result in clustering of vesicles and hemifusion of negatively charged model membranes. These multivalent, biological nanoparticles are reminiscent of inorganic nanoparticles in their interactions with membranes. Alpha-synuclein oligomers induce lipid exchange efficiently, with fewer than 10 oligomers/vesicle required to complete hemifusion. No full fusion or vesicle content mixing is observed.

FEBS Letters, 2014

We studied α-synuclein (αS) aggregation in giant vesicles, and observed dramatic membrane disinte... more We studied α-synuclein (αS) aggregation in giant vesicles, and observed dramatic membrane disintegration, as well as lipid incorporation into micrometer-sized suprafibrillar aggregates. In the presence of dye-filled vesicles, dye leakage and fibrillization happen concurrently. However, growing fibrils do not impair the integrity of phospholipid vesicles that have a low affinity for αS. Seeding αS aggregation accelerates dye leakage, indicating that oligomeric species are not required to explain the observed effect. The evolving picture suggests that fibrils that appear in solution bind membranes and recruit membrane-bound monomers, resulting in lipid extraction, membrane destabilization and the formation of lipid-containing suprafibrillar aggregates.

Table of Contents Chapter 1: Introduction 1.1 Alpha synuclein and Parkinson's disease 1.2 Propert... more Table of Contents Chapter 1: Introduction 1.1 Alpha synuclein and Parkinson's disease 1.2 Properties of alpha-synuclein 1.3 Oligomeric alpha-synuclein 1.4 Formation and characterization of oligomeric alpha-synuclein 1.4.1 High concentration-induced oligomers 1.4.2 Metal ion-induced oligomers 1.4.2 Metal ion-induced oligomers 1.4.4 HNE/ONE-induced oligomers 1.5 Alpha-synuclein membrane interactions 1.6 Scope of this thesis 1.7 References Chapter 2: Oligomer binding to bilayers with increasingly complex lipid compositions 2.1 Abstract 2.2 Introduction 2.3 Materials and Methods 2.3.1 Expression and purification of α-synuclein 2.3.2 Labeling of alpha-synuclein 2.3.3 Preparation of labeled αS oligomers 2.3.4 Qualitative oligomer binding assay 2.4 Results 2.4.1 The effect of cholesterol and sphingomyelin on αS oligomer binding in the presence of negatively charged lipids 2.4.2 The effect of lipid headgroup on oligomer binding 2.4.3 The effect of Cardiolipin on αS oligomer binding 2.4.4 The effect of brain lipids on oligomers binding 2.5 Discussion 2.6 References Chapter 3: Alpha-synuclein oligomers distinctively permeabilize complex model membranes 3.1. Abstract 3.2 Introduction 3.3 Materials and methods 3.3.1 Expression and purification of αS 3.3.2 Labeling of αS 3.3.3 Preparation of unlabeled and labeled αS oligomers 3.3.4 LUVs preparation and calcein release assay 3.3.5 Semi-quantitative αS monomer and oligomer binding assay 3.3.6 SUVs preparation and binding of αS oligomers to SUVs 3.4 Results 3.4.1 Binding of αS monomers to bilayers that mimic lipid composition of natural membranes 3.4.2 Do αS oligomers bind to bilayers mimicking the lipid composition of natural membranes and does this binding result in conformational changes? 3.4.3 Kinetics of membrane permeabilization (Dye release assay) 3.5 Discussion 3.6 Acknowledgments 3.7 References Chapter 4: Characterization of oligomers formed from disease-related alpha-synuclein amino acid mutations 4.1 Abstract 4.2 Introduction 4.3 Material and methods 4.3.1 Preparation of oligomeric alpha-synuclein 4.3.2 SUVs preparation and binding of αS monomers to SUVs 4.3.4 LUV preparation and calcein release assay 4.3.5 Small-Angle X-ray Scattering 4.3.6 Kinetics of aggregation 4.3.7 Atomic force microscopy (AFM) 4.4 Results 4.4.1 Binding of αS monomers to SUVs 4.4.2 Aggregation studies 4.4.3 Calcein release assay 4.4.4 Aggregation number 4.5 Discussion 4.6 Acknowledgments 4.7 References Chapter 5: Are alpha-synuclein oligomers toxic species? Chapter 6: Alpha-synuclein amyloid multimers act as multivalent nanoparticles to cause hemifusion in negatively charged bilayers 6.1 Abstract 6.2 Introduction 6.3 Material and Methods 6.3.1 Expression and purification of αS 6.3.2 Labeling of αS-A140C 6.3.3 Preparation of unlabeled and labeled αS oligomers 6.3.4 LUVs preparation 6.3.5 GUVs preparation for clustering experiment and imaging of clustered vesicles 6.3.6 Content mixing 6.3.7 Lipid mixing 96 6.3.8 Fluorescence Correlation Spectroscopy 6.3.9 Estimation of αS oligomers-membrane binding equilibrium 97 6.4 Results 98 6.4.1 αS oligomers induce vesicle clustering 98 6.4.2 Oligomer-induced vesicle fusion 99 6.4.3 Oligomer-induced hemifusion 6.5 Discussion 6.6 Acknowledgments 6.7 References Chapter 7: Conclusion and future recommendations Summary Samenvatting List of abbreviations Acknowledgments/Dankwoord/Zahvalnica List of publications Curriculum vitae Aggregation is the key process where the αS protein loses its putative function and gains toxicity [21-24]. The aggregation of αS starts from intrinsically disordered monomers, which self-assemble and form dimers and then possibly with the help of additional factors convert to (on-and off-pathway) oligomers [25-27], which may assemble into fibrils (Figure 1.2) [28]. The aggregated proteins in the fibrils have a characteristic β-sheet conformation and are packed perpendicular to the fiber axis [29]. Although earlier studies assumed fibrils to be toxic, currently it is suggested that oligomers are the main toxic species involved in the cell death of 1.4.3 Dopamine-induced oligomers In PD, oxidation of dopamine is one of the possible causes of cell death. Oxidation of dopamine gives an excessive production of semiquinones, H 2 O 2 and • HO radicals. In vivo studies have shown that αS is the negative regulator of dopamine neurotransmission [63, 64]. Mosharev et al. [65] confirmed that increased levels of dopamine (and its metabolic products) and Ca 2+ ions in dopaminergic neurons that are overexpressing αS are selectively neurotoxic for these neurons. Furthermore, 1.5 Alpha-synuclein membrane interactions To gain a clearer picture on the interaction between membranes and αS monomers or oligomers, many studies have made use of in vitro model membrane systems. Model membranes, such as lipid vesicles, mimic lipid membranes of cells and organelles. Early studies on the colocalization of αS with synaptic vesicles led to the suggestion that αS can interact with lipids [21]. Others [83] showed that αS also binds to model membranes and suggested that αS-membrane interactions have an important physiological and pathological role. As mentioned before, the N-terminal part of αS plays a crucial role in membrane binding [22, 23]. In solution the monomeric intrinsically disordered protein αS has no secondary structure [84], but upon binding to phospholipid membranes and detergent micelles the protein adopts an α-helical conformation [22, 37, 85-88]. Circular dichroism (CD) experiments show that the α-helical content upon binding to membranes increases from 3 to 80% [85]. According to the literature 41% of the αS protein becomes α-helical when bound to sodium dodecyl sulfate (SDS) micelles [89], while upon the binding to 1,2-dioleoylphosphatidylglycerol (DOPG): 1,2-dipalmitoylphosphatidylglycerol (DPPG) (DOPG:DPPG) vesicles, 61% of the protein adopts α-helical structure [90]. Additionally, binding of monomers to

FEBS Journal, 2014

α-Synuclein oligomers are increasingly considered to be responsible for the death of dopaminergic... more α-Synuclein oligomers are increasingly considered to be responsible for the death of dopaminergic neurons in Parkinson's disease. The toxicity mechanism of α-synuclein oligomers likely involves membrane permeabilization. Even though it is well established that α-synuclein oligomers bind and permeabilize vesicles composed of negatively-charged lipids, little attention has been given to the interaction of oligomers with bilayers of physiologically relevant lipid compositions. We investigated the interaction of α-synuclein with bilayers composed of lipid mixtures that mimic the composition of plasma and mitochondrial membranes. In the present study, we show that monomeric and oligomeric α-synuclein bind to these membranes. The resulting membrane leakage differs from that observed for simple artificial model bilayers. Although the addition of oligomers to negatively-charged lipid vesicles displays fast content release in a bulk permeabilization assay, adding oligomers to vesicles with compositions mimicking mitochondrial membranes shows a much slower loss of content. Oligomers are unable to induce leakage in the artificial plasma membranes, even after long-term incubation. CD experiments indicate that binding to lipid bilayers initially induces conformational changes in both oligomeric and monomeric α-synuclein, which show little change upon long-term incubation of oligomers with membranes. The results of the present study demonstrate that the mitochondrial model membranes are more vulnerable to permeabilization by oligomers than model plasma membranes reconstituted from brain-derived lipids; this preference may imply that increasingly complex membrane components, such as those in the plasma membrane mimic used in the present study, are less vulnerable to damage by oligomers.