Marcus Bokvist

How is protein aggregation in amyloidogenic diseases modulated by biological membranes

The fate of proteins with amyloidogenic properties depends critically on their immediate biochemical environment. However, the role of biological interfaces such as membrane surfaces, as promoters of pathological aggregation of amyloidogenic proteins, is rarely studied and only established for the amyloid-β protein (Aβ) involved in Alzheimer’s disease, and α-synuclein in Parkinsonism. The occurrence of binding and misfolding of these proteins on membrane surfaces, is poorly understood, not at least due to the two-dimensional character of this event. Clearly, the nature of the folding pathway for Aβ protein adsorbed upon two-dimensional aggregation templates, must be fundamentally different from the three-dimensional situation in solution. Here, we summarize the current research and focus on the function of membrane interfaces as aggregation templates for amyloidogenic proteins (and even prionic ones). One major aspect will be the relationship between membrane properties and protein association and the consequences for amyloidogenic products. The other focus will be on a general understanding of protein folding pathways on two-dimensional templates on a molecular level. Finally, we will demonstrate the potential importance of membrane-mediated aggregation for non-amphiphatic soluble amyloidogenic proteins, by using the SOD1 protein involved in the amyotrophic lateral sclerosis syndrome.

Disordered Proteins: Biological Membranes as Two-Dimensional Aggregation Matrices

Aberrant folded proteins and peptides are hallmarks of amyloidogenic diseases. However, the molecular processes that cause these proteins to adopt non-native structures in vivo and become cytotoxic are still largely unknown, despite intense efforts to establish a general molecular description of their behavior. Clearly, the fate of these proteins is ultimately linked to their immediate biochemical environment in vivo. In this review, we focus on the role of biological membranes, reactive interfaces that not only affect the conformational stability of amyloidogenic proteins, but also their aggregation rates and, probably, their toxicity. We first provide an overview of recent work, starting with findings regarding the amphiphatic amyloid-β protein (Aβ), which give evidence that membranes can directly promote aggregation, and that the effectiveness in this process can be related to the presence of specific neuronal ganglioside lipids. In addition, we discuss the implications of recent research (medin as an detailed example) regarding putative roles of membranes in the misfolding behavior of soluble, non-amphiphatic proteins, which are attracting increasing interest. The potential role of membranes in exerting the toxic action of misfolded proteins will also be highlighted in a molecular context. In this review, we discuss novel NMR-based approaches for exploring membrane–protein interactions, and findings obtained using them, which we use to develop a molecular concept to describe membrane-mediated protein misfolding as a quasi-two-dimensional process rather than a three-dimensional event in a biochemical environment. The aim of the review is to provide researchers with a general understanding of the involvement of membranes in folding/misfolding processes in vivo, which might be quite universal and important for future research concerning amyloidogenic and misfolding proteins, and possible ways to prevent their toxic actions.

Association of amyloid-β peptide with membrane surfaces monitored by solid state NMR

Amyloid-β peptide (Aβ), a key substance in Alzheimer’s disease (AD), is characterized by its abnormal folding into neurotoxic aggregates. Since Aβ comprises an extracellular and transmembrane domain, some of its neurotoxic actions might be exerted via interactions with neuronal membranes. Wideline and magic angle spinning 14N and 31P NMR have been used in combination with differential scanning calorimetry and circular dichroism spectroscopy to investigate the association between Aβ1–40 peptide and membranes with different electrostatic surface potentials. Calorimetric measurements showed that all membrane systems were in the liquid crystalline state at 308 K. Binding of Aβ1–40 at a 30 ∶ 1 lipid/peptide ratio to membranes composed of neutral dimyristoyl-phosphatidylcholine (DMPC) and negatively charged dimyristoylphosphatidylglycerol (DMPG) at a 4 ∶ 1 molar ratio is mainly driven electrostatically, reflected in characteristic changes of the isotropic 31P chemical shift values for both lipids. In addition, the average orientation of the choline headgroup of DMPC, with its electric P−–N+(CH3)3 dipole, changed directly in response to the reduced negative membrane surface potential. The deviation in tilt angle of the PN vector relative to the membrane surface is manifested in the observed 14N NMR quadrupole splitting and can therefore be described semiquantitatively. Adding Aβ1–40 to membranes with nominal neutral surface charge, but composed of a ternary mixture of DMPC with DMPG and the cationic amphiphile didodecyldimethyl–ammonium bromide (DDAB) at a 3 ∶ 1 ∶ 1 molar ratio revealed surprisingly electrostatic interactions visible in the NMR spectra. Since Aβ1–40 does not bind to neutral DMPC bilayers a model is proposed, in which on a molecular level the charged residues of Aβ1–40 peptide can interact independently with lipid headgroups of various charges in these microscopically heterogeneous systems.

Electrostatic peptide-lipid interactions of amyloid-β peptide and pentalysine with membrane surfaces monitored by 31P MAS NMR

High-resolution 31P magic angle spinning (MAS) NMR spectroscopy is presented as a direct and non-perturbing method for measuring changes in surface charge density occurring in mixed phospholipid membranes upon binding of charged surface-active peptides. 31P MAS NMR was used to investigate mixed lipid membranes of neutral phosphatidylcholine and negatively charged phosphatidylglycerol where the molar fraction of the charged lipid was varied from 0 to 1. The chemical shifts of the individual membrane lipids showed a simple variation in response to changes in the fraction of the negatively charged component phosphatidylglycerol. Addition of the positively charged amyloid-β1-40 peptide, a key substance in Alzheimers disease, resulted in changes in the isotropic chemical shifts of the membrane lipid phosphates in a way consistent with reduction in the negative surface charge of the mixed lipid bilayers. Binding of different amounts of the positively charged peptide pentalysine to L-α-dioleoylphosphatidylcholine/L-α-dioleoylphosphatidylglycerol (DOPC/DOPG) vesicles (2:1 molar ratio) also showed a systematic variation of both chemical shift values. These changes were described by a simple two-site model and indicate purely electrostatic binding of pentalysine.