Erik Goormaghtigh

Toxic prefibrillar α-synuclein amyloid oligomers adopt a distinctive antiparallel β-sheet structure.

Parkinsons disease is an age-related movement disorder characterized by the presence in the mid-brain of amyloid deposits of the 140-amino-acid protein AS (α-synuclein). AS fibrillation follows a nucleation polymerization pathway involving diverse transient prefibrillar species varying in size and morphology. Similar to other neurodegenerative diseases, cytotoxicity is currently attributed to these prefibrillar species rather than to the insoluble aggregates. Nevertheless, the underlying molecular mechanisms responsible for cytotoxicity remain elusive and structural studies may contribute to the understanding of both the amyloid aggregation mechanism and oligomer-induced toxicity. It is already recognized that soluble oligomeric AS species adopt β-sheet structures that differ from those characterizing the fibrillar structure. In the present study we used ATR (attenuated total reflection)-FTIR (Fourier-transform infrared) spectroscopy, a technique especially sensitive to β-sheet structure, to get a deeper insight into the β-sheet organization within oligomers and fibrils. Careful spectral analysis revealed that AS oligomers adopt an antiparallel β-sheet structure, whereas fibrils adopt a parallel arrangement. The results are discussed in terms of regions of the protein involved in the early β-sheet interactions and the implications of such conformational arrangement for the pathogenicity associated with AS oligomers.

Relevance of Protein Thin Films Prepared for Attenuated Total Reflection Fourier Transform Infrared Spectroscopy: Significance of the pH

Amide hydrogen–deuterium exchange rates were recorded on thin lysozyme films by attenuated total reflection Fourier transform infrared spectroscopy as a function of the pH of the solution from which the films were prepared. We describe the appropriate spectral corrections for atmospheric water absorption and for lateral side-chain contribution in the amide I/amide II region required to correctly evaluate the exchange rate from the amide II integrated intensity decrease. We show that the pH dependence of the exchange rate in our experimental conditions (0.4 g water/g protein) parallels the solution behavior. This observation confirms that the technique can be used to monitor pH-sensitive effects and allows exchange curves to be made which include most of the protein amide protons.

Acid phospholipid vesicles produce conformational changes on the antitumour protein α-sarcin

The antitumour protein alpha-sarcin interacts at neutral pH with acid phospholipid vesicles promoting their aggregation and fusion. This interaction produces conformational changes on the protein molecule. Circular dichroism and infrared spectroscopy have been used to analyze the secondary structure of the protein molecule. The obtained results show an increased alpha-helix content upon interaction with the lipid vesicles. Detergents and halogenated alcohols have also been considered as an approach to the study of the conformational changes produced upon alpha-sarcin-phospholipid vesicles interaction. SDS treatment as well as trifluoroethanol also increase the helical content of alpha-sarcin. Intrinsic fluorescence of the protein has also been measured for the analysis of the conformational changes produced. The above helicogenic treatments produce a decrease on the structural quenching in alpha-sarcin which is consistent with the existence of hydrophobic protein-lipid interactions. The observed conformational changes are interpreted in terms of a shielding from polar groups caused by the lipids, which promotes intrachain hydrogen bonding and decreased static quenching. The reported conformational changes are discussed in terms of electrostatic and hydrophobic interactions involved in the fusion of lipid vesicles promoted by alpha-sarcin, and potentially in the passage of the protein across membrane cells.

Intraspecific variability of cadmium tolerance and accumulation, and cadmium-induced cell wall modifications in the metal hyperaccumulator Arabidopsis halleri

Highlight A huge variability in Cd tolerance and accumulation exist within A. halleri, and the relationship between tolerance, accumulation, and edaphic type is not straightforward. Cd-induced cell wall modifications suggest various shoot detoxification mechanisms.

Organization and dynamics of Fas transmembrane domain in raft membranes and modulation by ceramide.

To comprehend the molecular processes that lead to the Fas death receptor clustering in lipid rafts, a 21-mer peptide corresponding to its single transmembrane domain (TMD) was reconstituted into mammalian raft model membranes composed of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol. The peptide membrane lateral organization and dynamics, and its influence on membrane properties, were studied by steady-state and time-resolved fluorescence techniques and by attenuated total reflection Fourier transformed infrared spectroscopy. Our results show that Fas TMD is preferentially localized in liquid-disordered membrane regions and undergoes a strong reorganization as the membrane composition is changed toward the liquid-ordered phase. This results from the strong hydrophobic mismatch between the length of the peptide hydrophobic stretch and the hydrophobic thickness of liquid-ordered membranes. The stability of nonclustered Fas TMD in liquid-disordered domains suggests that its sequence may have a protective function against nonligand-induced Fas clustering in lipid rafts. It has been reported that ceramide induces Fas oligomerization in lipid rafts. Here, it is shown that neither Fas TMD membrane organization nor its conformation is affected by ceramide. These results are discussed within the framework of Fas membrane signaling events.

Structures of intermediate transport states of ZneA, a Zn(II)/proton antiporter

Significance Transmembrane efflux pumps belonging to the resistance–nodulation–cell division (RND) superfamily are found in all kingdoms of life, and transport substrates out of cells, powered by an electrochemical proton gradient. Here we report two X-ray crystal structures of a Zn(II) efflux pump, ZneA, that capture different intermediate states along the transport cycle. The structures show how passage of substrates through ZneA is regulated by a series of conformational changes in the efflux pump. By comparing the structures of ZneA with other RND efflux pumps, we present a coherent mechanistic model for RND-mediated substrate efflux, which ensures efficient transport of substrates out of the cell. Efflux pumps belonging to the ubiquitous resistance–nodulation–cell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two X-ray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters.

Antimitotics induced cardiolipin cluster formation possible role in mitochondrial enzyme inactivation

We demonstrate here that drugs which inactivate cytochrome c oxidase are able to segregate cardiolipin essential for the enzyme activity, in a separate phase inaccessible for the enzyme. A molecular explanation of the drug-induced aggregation process is proposed.

Metal binding properties and structure of a type III metallothionein from the metal hyperaccumulator plant Noccaea caerulescens

Metallothioneins (MT) are low molecular weight proteins with cysteine-rich sequences that bind heavy metals with remarkably high affinities. Plant MTs differ from animal ones by a peculiar amino acid sequence organization consisting of two short Cys-rich terminal domains (containing from 4 to 8 Cys each) linked by a Cys free region of about 30 residues. In contrast with the current knowledge on the 3D structure of animal MTs, there is a striking lack of structural data on plant MTs. We have expressed and purified a type III MT from Noccaea caerulescens (previously Thlaspi caerulescens). This protein is able to bind a variety of cations including Cd(2+), Cu(2+), Zn(2+) and Pb(2+), with different stoichiometries as shown by mass spectrometry. The protein displays a complete absence of periodic secondary structures as measured by far-UV circular dichroism, infrared spectroscopy and hydrogen/deuterium exchange kinetics. When attached onto a BIA-ATR biosensor, no significant structural change was observed upon removing the metal ions.

Infrared imaging in histopathology: Is a unified approach possible?

BACKGROUND: infrared imaging has emerged as a new promising tool in histopathology to provide label free analysis of tissue sections. Interestingly, infrared imaging has the potential to measure many markers at the same time, on one section, without staining. It has been demonstrated to deliver accurate results in numerous cancer pathologies. Yet, today, it is not used in routine diagnostics. The gap between the demonstrated potential and the applications is striking. The reasons why FTIR imaging is not used in the clinics are multiple but one of them is a major obstacle: the diversity of sample preparation, image recording parameters and pre-analytical methods used by the different research groups. This diversity prevents comparison of data and thereby the large scale validation necessary to enter the medical world. OBJECTIVE: we will briefly review here the main aspects of data acquisition and processing used in infrared imaging of tissue sections for which a common approach should be considered. RESULTS: considering requirement for spectral histopathology, the development of the technology and the literature on this topic, guidelines ruling sample preparation and pre-analytical methods do emerge. CONCLUSIONS: consensus values are proposed for most parameters whose current diversity prevents the exchange of data among institutions and thereby the validation of the method on a large scale.

Analysis of protein microarrays by FTIR imaging

BACKGROUND: Proteins are sensitive to environmental conditions. Whether they are produced for therapeutic purposes or for fundamental research, the integrity of their structure and post-traductional modifications are key issues. Measuring glycosylation or phosphorylation level as well as their secondary structure most often rely on complex and indirect experiments. Infrared spectroscopy presents a series of advantages related to its multivariate character. There is a lack of high-throughput methods able to analyse these parameters. OBJECTIVE: In this paper we attempted to combine protein microarrays and infrared imaging for high throughput analysis of proteins. METHODS: A protein microarrayer was used to produce protein microarrays on BaF2 slides transparent in the mid-infrared. Spot density was about 25 spots/mm 2 . A 128 × 128 focal plane array infrared detector was used to record images of the protein microarrays. RESULTS: We show that 100 µm diameter spot are easily analyzed. Spots obtained with low protein concentrations, resulting in an average of a single protein monolayer (ca 3 fg/µm 2 for a 66 kDa protein) provided good quality spectra. CONCLUSIONS: Infrared imaging is a label free, high throughput method, able to analyse protein microarrays and to take advantage from the wide information available in the infrared spectra.