Teresa J. T. Pinheiro

Albumin as a zinc carrier: properties of its high-affinity zinc-binding site

Although details of the molecular mechanisms for the uptake of the essential nutrient zinc into the bloodstream and its subsequent delivery to zinc-requiring organs and cells are poorly understood, it is clear that in vertebrates the majority of plasma zinc (9-14 microM; approx. 75-85%) is bound to serum albumin, constituting part of the so-called exchangeable pool. The binding of metal ions to serum albumins has been the subject of decades of studies, employing a multitude of techniques, but only recently has the identity and putative structure of the major zinc site on albumin been reported. Intriguingly, this site is located at the interface between two domains, and involves two residues from each of domains I and II. Comparisons of X-ray crystal structures of free and fatty-acid bound human serum albumin suggest that zinc binding to this site and fatty acid binding to one of the five major sites may be interdependent. Interactive binding of zinc and long-chain fatty acids to albumin may therefore have physiological implications.

Structural analysis of prion proteins by means of drift cell and traveling wave ion mobility mass spectrometry

The prion protein (PrP) is implicitly involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). The conversion of normal cellular PrP (PrPC), a protein that is predominantly α-helical, to a β-sheet-rich isoform (PrPSc), which has a propensity to aggregate, is the key molecular event in prion diseases. During its short life span, PrP can experience two different pH environments; a mildly acidic environment, whilst cycling within the cell, and a neutral pH when it is glycosyl phosphatidylinositol (GPI)-anchored to the cell membrane. Ion mobility (IM) combined with mass spectrometry has been employed to differentiate between two conformational isoforms of recombinant Syrian hamster prion protein (SHaPrP). The recombinant proteins studied were α-helical SHaPrP(90-231) and β-sheet-rich SHaPrP(90-231) at pH 5.5 and pH 7.0. The recombinant proteins have the same nominal mass-to-charge ratio (m/z) but differ in their secondary and tertiary structures. A comparison of traveling-wave (T-Wave) ion mobility and drift cell ion mobility (DCIM) mass spectrometry estimated and absolute cross-sections showed an excellent agreement between the two techniques. The use of T-Wave ion mobility as a shape-selective separation technique enabled differentiation between the estimated cross-sections and arrival time distributions (ATDs) of α-helical SHaPrP(90-231) and β-sheet-rich SHaPrP(90-231) at pH 5.5. No differences in cross-section or ATD profiles were observed between the protein isoforms at pH 7.0. The findings have potential implications for a new ante-mortem screening assay, in bodily fluids, for prion misfolding diseases such as TSEs.

Synthesis and structural characterization of a mimetic membrane-anchored prion protein.

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C‐terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP–GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane‐anchored PrP. We show that the structure of PrP–GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor‐free forms of PrP represent the structure of cellular, membrane‐anchored PrP. The availability of a lipid‐anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP.

Conformational stability of Syrian hamster prion protein PrP(90-231).

Many transmissible spongiform encephalopathies (TSEs) are believed to be caused by a misfolded form of the normal cellular prion protein (PrP(C)) known as PrP(Sc). While PrP(Sc) is known to be exceptionally stable and resistant to protease degradation, PrP(C) has not shown these same unusual characteristics. However, using ion mobility spectrometry mass spectrometry (IMS-MS), we found evidence for at least one very stable conformation of a truncated form of recombinant PrP(C) consisting of residues 90-231, which resists unfolding in the absence of solvent at high injection energies and at temperatures in excess of 600 K. We also report the first absolute collision cross sections measured for recombinant Syrian hamster prion protein PrP(90-231).

Insight into early events in the aggregation of the prion protein on lipid membranes.

The key molecular event underlying prion diseases is the conversion of the monomeric and alpha-helical cellular form of the prion protein (PrP(C)) to the disease-associated state, which is aggregated and rich in beta-sheet (PrP(Sc)). The molecular details associated with the conversion of PrP(C) into PrP(Sc) are not fully understood. The prion protein is attached to the cell membrane via a GPI lipid anchor and evidence suggests that the lipid environment plays an important role in prion conversion and propagation. We have previously shown that the interaction of the prion protein with anionic lipid membranes induces beta-sheet structure and promotes prion aggregation, whereas zwitterionic membranes stabilize the alpha-helical form of the protein. Here, we report on the interaction of recombinant sheep prion protein with planar lipid membranes in real-time, using dual polarization interferometry (DPI). Using this technique, the simultaneous evaluation of multiple physical properties of PrP layers on membranes was achieved. The deposition of prion on membranes of POPC and POPC/POPS mixtures was studied. The properties of the resulting protein layers were found to depend on the lipid composition of the membranes. Denser and thicker protein deposits formed on lipid membranes containing POPS compared to those formed on POPC. DPI thus provides a further insight on the organization of PrP at the surface of lipid membranes.

Globular and pre-fibrillar prion aggregates are toxic to neuronal cells and perturb their electrophysiology.

Prion diseases are characterised at autopsy by neuronal loss and accumulation of amorphous protein aggregates and/or amyloid fibrils in the brains of humans and animals. These protein deposits result from the conversion of the cellular, mainly alpha-helical prion protein (PrP(C)) to the beta-sheet-rich isoform (PrP(Sc)). Although the pathogenic mechanism of prion diseases is not fully understood, it appears that protein aggregation is itself neurotoxic and not the product of cell death. The precise nature of the neurotoxic species and mechanism of cell death are yet to be determined, although recent studies with other amyloidogenic proteins suggest that ordered pre-fibrillar or oligomeric forms may be responsible for cellular dysfunction. In this study we have refolded recombinant prion protein (rPrP) to two distinct forms rich in beta-sheet structure with an intact disulphide bond. Here we report on the structural properties of globular aggregates and pre-fibrils of rPrP and show that both states are toxic to neuronal cells in culture. We show that exogenous rPrP aggregates are internalised by neuronal cells and found in the cytoplasm. We also measured the changes in electrophysiological properties of cultured neuronal cells on exposure to exogenous prion aggregates and discuss the implications of these findings.

A molecular mechanism for modulating plasma Zn speciation by fatty acids.

Albumin transports both fatty acids and zinc in plasma. Competitive binding studied by isothermal titration calorimetry revealed that physiologically relevant levels of fatty acids modulate the Zn-binding capacity of albumin, with far-reaching implications for biological zinc speciation. The molecular mechanism for this effect is likely due to a large conformational change elicited by fatty acid binding to a high-affinity interdomain site that disrupts at least one Zn site. Albumin may be a molecular device to “translate” certain aspects of the organismal energy state into global zinc signals.

The elusive intermediate on the folding pathway of the prion protein.

A key molecular event in prion diseases is the conversion of the cellular conformation of the prion protein (PrPC) to an altered disease‐associated form, generally denoted as scrapie isoform (PrPSc). The molecular details of this conformational transition are not fully understood, but it has been suggested that an intermediate on the folding pathway of PrPC may be recruited to form PrPSc. In order to investigate the folding pathway of PrP we designed and expressed two mutants, each possessing a single strategically located tryptophan residue. The secondary structure and folding properties of the mutants were examined. Using conventional analyses of folding transition data determined by fluorescence and CD, and novel phase‐diagram analyses, we present compelling evidence for the presence of an intermediate species on the folding pathway of PrP. The potential role of this intermediate in prion conversion is discussed.

Nimesulide interaction with membrane model systems : are membrane physical effects involved in nimesulide mitochondrial toxicity?

Nimesulide (NIM), a widely used nonsteroidal anti-inflammatory drug (NSAID), is known to interfere with mitochondrial physiology and to cause idiosyncratic hepatotoxicity. In this study, we characterized the effects of NIM on the physical properties of membrane models containing the main phospholipid classes of the inner mitochondrial membrane: phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL). NIM binding/incorporation was observed with the mitochondrial membrane mimicking model composed of dioleoyl PC (DOPC), dioleoyl PE (DOPE) and tetraoleoyl CL (TOCL) at a 1:1:1M ratio, as well as an increase of membrane permeability, monitored by calcein release, and an increase of lipid disorder, evaluated by fluorescence anisotropy of DPH-PA. Consistently, DSC thermograms of dipalmitoyl PC (DPPC) and a mixture of dipalmitoyl PE (DPPE) and TOCL (7:3 M ratio) showed a NIM-induced decrease of the cooperativity of the phase transition and a shift of the DPPC endotherm to lower temperatures. On the other hand, (31)P NMR studies with the ternary lipid model indicated a stabilizing effect of NIM on the lipid bilayer structure. Quenching of the fluorescent probes DPH and DPH-PA revealed a peripheral insertion of NIM in the hydrophobic portion of the bilayer. Our data indicate that NIM may influence mitochondria physiological processes by interfering with membrane structure and dynamics. The relevance of these findings will be discussed in terms of the reported NIM effects on mitochondria transmembrane potential, protonophoresis, and induction of the permeability transition pore.

Phospholipid Composition of Membranes Directs Prions Down Alternative Aggregation Pathways

Prion diseases are neurodegenerative disorders of the central nervous system that are associated with the misfolding of the prion protein (PrP). PrP is glycosylphosphatidylinositol-anchored, and therefore the hydrophobic membrane environment may influence the process of prion conversion. This study investigates how the morphology and mechanism of growth of prion aggregates on membranes are influenced by lipid composition. Atomic force microscopy is used to image the aggregation of prions on supported lipid bilayers composed of mixtures of the zwitterionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS). Circular dichroism shows that PrP interactions with POPS membranes result in an increase in beta-sheet structure, whereas interactions with POPC do not influence PrP structure. Prion aggregation is observed on both zwitterionic and anionic membranes, and the morphology of the aggregates formed is dependent on the anionic phospholipid content of the membrane. The aggregates that form on POPC membranes have uniform dimensions and do not disrupt the lipid bilayer. The presence of POPS results in larger aggregates with a distinctive sponge-like morphology that are disruptive to membranes. These data provide detailed information on the aggregation mechanism of PrP on membranes, which can be described by classic models of growth.