Tomasz Borowik

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.

Fluorescence Techniques for Determination of the Membrane Potentials in High Throughput Screening

The characterization of small molecules requires identification and evaluation of several predictive parameters, when selecting compounds for pharmacological applications and/or determining their toxicity. A number of them are correlated with the compound interaction with biological membranes and/or capacity to cross them. The knowledge of the extent of adsorption, partition coefficient and permeability along with the compound ability to alter membrane properties are critical for such studies. Lipid bilayers are frequently used as the adequate experimental models of a biological membrane despite their simple structure and a limited number of components. A significant number of the biologically relevant lipid bilayer properties are related to its electrostatics. Three electrostatic potentials were defined for the lipid bilayer; the intrinsic or induced surface electrostatic potential, the dipole potential and the membrane potential. Each of them was measured with dedicated methodologies. The complex measurement protocols and technically demanding instrumentation made the development of efficient HTS approaches for complete characterization of membrane electrostatics practically impossible. However, the rapid development of fluorescence techniques accompanied by rapid growth in diversity and number of dedicated fluorescent probes enabled characterization of lipid bilayer electrostatics in a moderately simple manner. Technically advanced, compact and automated workstations, capable of measuring practically all fluorescence parameters, are now available. Therefore, the proper selection of fluorescent probes with measuring procedures can be designed to evaluate drug candidates in context of their ability to alter membrane electrostatics. In the paper we present a critical review of available fluorescence methods, useful for the membrane electrostatics evaluation and discuss the feasibility of their adaptation to HTS procedures. The significance of the presented methodology is even greater considering the rapid growth of advanced drug formulations, where electrostatics is an important parameter for production processes and pharmacokinetics of the product. Finally, the potential of the membrane electrostatics to emerge as a viable pharmacological target is indicated and fluorescence techniques capable to evaluate this potential are presented.

The effect of the lipid-binding site of the ankyrin-binding domain of erythroid β-spectrin on the properties of natural membranes and skeletal structures

It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of β-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482–94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na+K+ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the β-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.

Neuroprotective effects of tempol acyl esters against retinal ganglion cell death in a rat partial optic nerve crush model.

Purpose:  The aim of this study is to search for more effective derivatives of the superoxide dismutase mimetic tempol (4‐hydroxy‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). Although tempol is neuroprotective in a rat partial optic nerve crush (PONC) model, relatively high doses are required to exert this effect.

The cellular internalization of liposome encapsulated protoporphyrin IX by HeLa cells.

The proper lipid composition of liposomes designed to carry drugs determines their surface properties ensuring their accumulation within selected tissue. The electrostatic potential and surface topology of liposomes affect the internalization by single cells. The high-resolution imaging of cancer cells and the distribution of protoporphyrin-loaded liposomes within the cytoplasm and its dependence on the liposome surface properties are presented. In the paper, HeLa cells were used to investigate the uptake of porphyrin-loaded liposomes and liposomes alone by means of confocal and differential interference contrast microscopies. The effect of liposomes surface electrostatic potential and surface topology on their intracellular distribution was evaluated. The time evolution of the intracellular distribution of liposomes labelled with Rhodamine-PE was examined on HeLa cells. These studies allow for the identification of the liposome lipid composition so the efficient delivery of the active substance to cancer cells will be achieved. The obtained results showed that neutral PC-liposomes are the most efficiently internalized by HeLa cells. Moreover, results showed that properties of liposomes affect not only the internalization efficiency of the photosensitizer but also its distribution within the cells, as revealed by colocalization measurements.

Application of liposome based sensors in high-throughput screening systems.

High throughout screening is an approach based on the concept which assumes that when sufficiently large library of compounds are tested, the chance of discovering a new active compound is increased. In order to meet this expectation, proper testing criteria need to be devised. Those criteria should be related to the fate of a compound in the organism to have any predictive power. Not long ago, the main criteria were based exclusively on parameters defined by the maximum activity (QSAR). In this system the activity criteria have not been included therefore the compound ability to reach the target is not accounted for. Considering that, the construction of yet another set of parameters has been initiated (QSPR). The parameters are in fact semi-empirical numbers which need to be tested on real, physical models. Whereas the activity tests are straightforward, the pharmacokinetic ones are difficult and controversial. One such parameter describes the critical property of an active compound, namely its ability to cross biological membranes. This review describes new concepts in the determination of the permeability coefficient with the help of methods which are based on liposome biosensors. Two methods using fluorescence probes incorporated in the lipid bilayer of liposome are described in detail and compared to other currently available techniques.

Chapter three - A Multi Time-Scale Approach of the Lipid Bilayer Dynamics

Abstract The lipid bilayer is a supramolecular aggregate with complex dynamics. Independent and collective motions of lipid molecules span a wide range of timescales. In order to understand the lipid bilayer functioning, the correlation between various motions needs to be understood. Molecules with low molecular weight, when placed in the lipid bilayer, may alter membrane properties depending on their location. The effect of two membrane active molecules, ethanol and lidocaine, on the lipid bilayer was investigated in three different timescales by means of dedicated fluorescence techniques. The interfacial water dynamics was measured using “solvent relaxation” technique at picosecond timescale. The lateral lipid molecule mobility along the lipid bilayer surface was determined with fluorescence correlation spectroscopy at millisecond timescale, and collective lipid processes, such as spontaneous lipid pore formation, were monitored with fluorescence stopped-flow technique at the timescale of seconds and longer. When the lipid bilayer was exposed to the ethanol molecules, changes at all probed timescales have been detected, whereas in the presence of lidocaine, only the alterations in interfacial water mobility and spontaneous lipid pore formation were observed. The introduction of lidocaine left the lateral lipid mobility unaffected. These results indicate that there might happen loose correlations between processes occurring in different timescales within the same lipid bilayer.

A method to evaluate a propidium iodide association with oligonucleotides and oligonucleotide-cationic lipid interactions.

Complex molecular ensembles are frequently considered an element of new pharmacological formulations. This is especially evident in the therapies based on genetic information. In order to obtain an effective drug, it is necessary to associate a nucleic acid molecule with the components to ensure the desired aggregate structure and properties. To evaluate the progress of the supromolecular aggregate formation a range of methodologies and techniques are needed to test the quality and uniformity of the formulations. In this paper we propose a procedure which measures the association of a small molecule with nucleic acid using propidium iodide and oligonucleotides as an example. To measure propidium iodide binding constant the oligonucleotide was covalently labeled with fluorescein and then the changes in fluorescence resonance energy transfer (FRET) were determined and handled according to the acceptor-donor titration methodology. The calculated binding constants were in a good agreement with the values published previously. The developed method was then used to evaluate the extent of an oligonucleotide association with the lipid aggregates. It was found that two populations of oligonucleotides are present in all lipid samples studied. The fraction of oligonucleotides associated with liposomes rises with the increase of a cationic lipid content, reaching the constant value when the fraction of cationic lipid exceeded 20 mol%. Energy transfer data combined with these obtained in quenching experiments show that the orientation of the oligonucleotide associated with a lipid bilayer depends on the amount of surface charge.