Jitka Petrlova

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.

The fibrillogenic L178H variant of apolipoprotein A-I forms helical fibrils

A number of amyloidogenic variants of apoA-I have been discovered but most have not been analyzed. Previously, we showed that the G26R mutation of apoA-I leads to increased β-strand structure, increased N-terminal protease susceptibility, and increased fibril formation after several days of incubation. In vivo, this and other variants mutated in the N-terminal domain (residues 26 to ∼90) lead to renal and hepatic accumulation. In contrast, several mutations identified within residues 170 to 178 lead to cardiac, laryngeal, and cutaneous protein deposition. Here, we describe the structural changes in the fibrillogenic variant L178H. Like G26R, the initial structure of the protein exhibits altered tertiary conformation relative to wild-type protein along with decreased stability and an altered lipid binding profile. However, in contrast to G26R, L178H undergoes an increase in helical structure upon incubation at 37°C with a half time (t1/2) of about 12 days. Upon prolonged incubation, the L178H mutant forms fibrils of a diameter of 10 nm that ranges in length from 30 to 120 nm. These results show that apoA-I, known for its dynamic properties, has the ability to form multiple fibrillar conformations, which may play a role in the tissue-specific deposition of the individual variants.

A differential association of Apolipoprotein E isoforms with the amyloid‐β oligomer in solution

The molecular pathogenesis of disorders arising from protein misfolding and aggregation is difficult to elucidate, involving a complex ensemble of intermediates, whose toxicity depends upon their state of progression along distinct processing pathways. To address the complex misfolding and aggregation that initiates the toxic cascade resulting in Alzheimers disease (AD), we have developed a 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid spin‐labeled amyloid‐β (Aβ) peptide to observe its isoform‐dependent interaction with the apoE protein. Although most individuals carry the E3 isoform of apoE, ∼15% of humans carry the E4 isoform, which is recognized as the most significant genetic determinant for Alzheimers. ApoE is consistently associated with the amyloid plaque marker for AD. A vital question centers on the influence of the two predominant isoforms, E3 and E4, on Aβ peptide processing and hence Aβ toxicity. We used electron paramagnetic resonance (EPR) spectroscopy of incorporated spin labels to investigate the interaction of apoE with the toxic oligomeric species of Aβ in solution. EPR spectra of the spin‐labeled side chain report on side chain and backbone dynamics as well as the spatial proximity of spins in an assembly. Our results indicate oligomer binding involves the C‐terminal domain of apoE, with apoE3 reporting a much greater response through this conformational marker. Coupled with SPR binding measurements, apoE3 displays a higher affinity and capacity for the toxic Aβ oligomer. These findings support the hypothesis that apoE polymorphism and Alzheimers risk can largely be attributed to the reduced ability of apoE4 to function as a clearance vehicle for the toxic form of Aβ. Proteins 2011.

Single injections of apoA-I acutely improve in vivo glucose tolerance in insulin-resistant mice

Aims/hypothesisApolipoprotein A-I (apoA-I), the main protein constituent of HDL, has a central role in the reverse cholesterol-transport pathway, which together with the anti-inflammatory properties of apoA-I/HDL provide cardioprotection. Recent findings of direct stimulation of glucose uptake in muscle by apoA-I/HDL suggest that altered apoA-I and HDL functionality may be a contributing factor to the development of diabetes. We have studied the in vivo effects of short treatments with human apoA-I in a high-fat diet fed mouse model. In addition to native apoA-I, we investigated the effects of the cardioprotective Milano variant (Arg173Cys).MethodsMale C57Bl6 mice on a high-fat diet for 2xa0weeks that received a single injection of human apoA-I proteins (wild-type and Milano) were analysed for blood glucose and insulin levels during a 3xa0h incubation followed by glucose tolerance tests. Incorporation of injected human apoA-I protein into HDLs was analysed by native gel electrophoresis.ResultsApoA-I treatment significantly improved insulin secretion and blood glucose clearance in the glucose tolerance test, with an efficiency exceeding that of lean control animals, and led to decreased basal glucose during the 3xa0h incubation. Notably, the two apoA-I variants triggered insulin secretion and glucose clearance to the same extent.Conclusions/interpretationApoA-I treatment leads to insulin- and non-insulin-dependent effects on glucose homeostasis. The experimental model of short-term (2xa0weeks) feeding of a high-fat diet to C57Bl6 mice provides a suitable and time-efficient system to unravel the resulting tissue-specific mechanisms of acute apoA-I treatment that lead to improved glucose homeostasis.

Characterization of De Novo Synthesized GPCRs Supported in Nanolipoprotein Discs

The protein family known as G-protein coupled receptors (GPCRs) comprises an important class of membrane-associated proteins, which remains a difficult family of proteins to characterize because their function requires a native-like lipid membrane environment. This paper focuses on applying a single step method leading to the formation of nanolipoprotein particles (NLPs) capable of solubilizing functional GPCRs for biophysical characterization. NLPs were used to demonstrate increased solubility for multiple GPCRs such as the Neurokinin 1 Receptor (NK1R), the Adrenergic Receptor â2 (ADRB2) and the Dopamine Receptor D1 (DRD1). All three GPCRs showed affinity for their specific ligands using a simple dot blot assay. The NK1R was characterized in greater detail to demonstrate correct folding of the ligand pocket with nanomolar specificity. Electron paramagnetic resonance (EPR) spectroscopy validated the correct folding of the NK1R binding pocket for Substance P (SP). Fluorescence correlation spectroscopy (FCS) was used to identify SP-bound NK1R-containing NLPs and measure their dissociation rate in an aqueous environment. The dissociation constant was found to be 83 nM and was consistent with dot blot assays. This study represents a unique combinational approach involving the single step de novo production of a functional GPCR combined with biophysical techniques to demonstrate receptor association with the NLPs and binding affinity to specific ligands. Such a combined approach provides a novel path forward to screen and characterize GPCRs for drug discovery as well as structural studies outside of the complex cellular environment.

Discoidal HDL and apoA-I-derived peptides improve glucose uptake in skeletal muscle

Lipid-free apoA-I and mature spherical HDL have been shown to induce glucose uptake in skeletal muscle. To exploit apoA-I and HDL states for diabetes therapy, further understanding of interaction between muscle and apoA-I is required. This study has examined whether nascent discoidal HDL, in which apoA-I attains a different conformation from mature HDL and lipid-free states, could induce muscle glucose uptake and whether a specific domain of apoA-I can mediate this effect. Using L6 myotubes stimulated with synthetic reconstituted discoidal HDL (rHDL), we show a glucose uptake effect comparable to insulin. Increased plasma membrane GLUT4 levels in ex vivo rHDL-stimulated myofibers from HA-GLUT4-GFP transgenic mice support this observation. rHDL increased phosphorylation of AMP kinase (AMPK) and acetyl-coA carboxylase (ACC) but not Akt. A survey of domain-specific peptides of apoA-I showed that the lipid-free C-terminal 190–243 fragment increases plasma membrane GLUT4, promotes glucose uptake, and activates AMPK signaling but not Akt. This may be explained by changes in α-helical content of 190–243 fragment versus full-length lipid-free apoA-I as assessed by circular dichroism spectroscopy. Discoidal HDL and the 190–243 peptide of apoA-I are potent agonists of glucose uptake in skeletal muscle, and the C-terminal α-helical content of apoA-I may be an important determinant of this effect.

Binding of Apolipoprotein E Inhibits the Oligomer Growth of Amyloid-β Peptide in Solution as Determined by Fluorescence Cross-correlation Spectroscopy

Background: ApoE is the most significant risk factor for Alzheimer disease, with known effects on Aβ deposition in the brain. Results: ApoE binds to aggregating Aβ peptides and maintains a faster diffusion rate for the Aβ peptide over time. Conclusion: Binding of apoE to Aβ slows the oligomerization of Aβ. Significance: FCCS measurements quantify isoform-dependent differences in apoE binding to Aβ in solution. One of the primary neuropathological hallmarks of Alzheimer disease is the presence of extracellular amyloid plaques resulting from the aggregation of amyloid-β (Aβ) peptides. The intrinsic disorder of the Aβ peptide drives self-association and progressive reordering of the conformation in solution, and this dynamic distribution of Aβ complicates biophysical studies. This property poses a challenge for understanding the interaction of Aβ with apolipoprotein E (apoE). ApoE plays a pivotal role in the aggregation and clearance of Aβ peptides in the brain, and the ϵ4 allele of APOE is the most significant known genetic modulator of Alzheimer risk. Understanding the interaction between apoE and Aβ will provide insight into the mechanism by which different apoE isoforms determine Alzheimer disease risk. Here we applied alternating laser excitation fluorescence cross-correlation spectroscopy to observe the single molecule interaction of Aβ with apoE in the hydrated state. The diffusion time of freely diffusing Aβ in the absence of apoE shows significant self-aggregation, whereas in the presence of apoE, binding of the protein results in a more stable complex. These results show that apoE slows down the oligomerization of Aβ in solution and provide direct insight into the process by which apoE influences the deposition and clearance of Aβ peptides in the brain. Furthermore, by developing an approach to remove signals arising from very large Aβ aggregates, we show that real-time single particle observations provide access to information regarding the fraction of apoE bound and the stoichiometry of apoE and Aβ in the complex.

Ganglioside embedded in reconstituted lipoprotein binds cholera toxin with elevated affinity

The ability to exogenously present cell-surface receptors in high-affinity conformations in a synthetic system offers an opportunity to provide host cells with protection from pathogenic toxins. This strategy requires improvement of the synthetic receptor binding affinity against its native counterpart, particularly with polyvalent toxins where clustering of membrane receptors can hinder binding. Here we demonstrate that reconstituted lipoprotein, nanometer-sized discoidal lipid bilayers bounded by apolipoprotein and functionalized by incorporation of pathogen receptors, provides a means to enhance toxin-receptor binding through molecular-level control over the receptor microenvironment (specifically, its rigidity, composition, and heterogeneity). Using a Foerster Resonance Energy Transfer (FRET)-based assay, we found that reconstituted lipoprotein incorporating low concentrations of ganglioside monosialotetrahexosylganglioside (GM1) binds polymeric cholera toxin with significantly higher affinity than liposomes or supported lipid bilayers, most likely a result of the enhanced control over receptor clustering provided by the lipoprotein platform. Using wide-area epifluorescence, we found that this enhanced binding capacity can be effectively utilized to divert cholera toxin away from populations of healthy mammalian cells. In summary, we found that reconstitutions of high-density lipoprotein can be engineered to include specific pathogen receptors; that their pathogen binding affinity is altered, presumably due to attenuation of receptor aggregation; and that these assemblies are effective at protecting cells from biological toxins.

The secondary structure of apolipoprotein A-I on 9.6-nm reconstituted high-density lipoprotein determined by EPR spectroscopy

Apolipoprotein A‐I (ApoA‐I) is the major protein component of high‐density lipoprotein (HDL), and is critical for maintenance of cholesterol homeostasis. During reverse cholesterol transport, HDL transitions between an array of subclasses, differing in size and composition. This process requires ApoA‐I to adapt to changes in the shape of the HDL particle, transiting from an apolipoprotein to a myriad of HDL subclass‐specific conformations. Changes in ApoA‐I structure cause alterations in HDL‐specific enzyme and receptor‐binding properties, and thereby direct the HDL particle through the reverse cholesterol transport pathway. In this study, we used site‐directed spin label spectroscopy to examine the conformational details of the ApoA‐I central domain on HDL. The motional dynamics and accessibility to hydrophobic/hydrophilic relaxation agents of ApoA‐I residues 99–163 on 9.6‐nm reconstituted HDL was analyzed by EPR. In previous analyses, we examined residues 6–98 and 164–238 (of ApoA‐Is 243 residues), and combining these findings with the current results, we have generated a full‐length map of the backbone structure of reconstituted HDL‐associated ApoA‐I. Remarkably, given that the majority of ApoA‐Is length is composed of amphipathic helices, we have identified nonhelical residues, specifically the presence of a β‐strand (residues 149–157). The significance of these nonhelical residues is discussed, along with the other features, in the context of ApoA‐I function in contrast to recent models derived by other methods.

The influence of spin-labeled fluorene compounds on the assembly and toxicity of the aβ peptide.

Background The deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimers disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species. Methodology/Principal Findings To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species. Conclusions Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they posses both antioxidant properties and the ability to disrupt AβO species.