Jesús Zurdo

Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.

A range of human degenerative conditions, including Alzheimers disease, light-chain amyloidosis and the spongiform encephalopathies, is associated with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those associated with clinical amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidyl-inositol-3′-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-associated proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in the origins of this family of protein deposition diseases.

Cryo‐electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing

Amyloid fibrils are assemblies of misfolded proteins and are associated with pathological conditions such as Alzheimers disease and the spongiform encephalopathies. In the amyloid diseases, a diverse group of normally soluble proteins self‐assemble to form insoluble fibrils. X‐ray fibre diffraction studies have shown that the protofilament cores of fibrils formed from the various proteins all contain a cross‐β‐scaffold, with β‐strands perpendicular and β‐sheets parallel to the fibre axis. We have determined the threedimensional structure of an amyloid fibril, formed by the SH3 domain of phosphatidylinositol‐3′‐kinase, using cryo‐electron microscopy and image processing at 25 Å resolution. The structure is a double helix of two protofilament pairs wound around a hollow core, with a helical crossover repeat of ∼600 Å and an axial subunit repeat of ∼27 Å. The native SH3 domain is too compact to fit into the fibril density, and must unfold to adopt a longer, thinner shape in the amyloid form. The 20×40‐Å protofilaments can only accommodate one pair of flat β‐sheets stacked against each other, with very little inter‐strand twist. We propose a model for the polypeptide packing as a basis for understanding the structure of amyloid fibrils in general.

De novo designed peptide-based amyloid fibrils

Identification of therapeutic strategies to prevent or cure diseases associated with amyloid fibril deposition in tissue (Alzheimers disease, spongiform encephalopathies, etc.) requires a rational understanding of the driving forces involved in the formation of these organized assemblies rich in β-sheet structure. To this end, we used a computer-designed algorithm to search for hexapeptide sequences with a high propensity to form homopolymeric β-sheets. Sequences predicted to be highly favorable on this basis were found experimentally to self-associate efficiently into β-sheets, whereas point mutations predicted to be unfavorable for this structure inhibited polymerization. However, the property to form polymeric β-sheets is not a sufficient requirement for fibril formation because, under the conditions used here, preformed β-sheets from these peptides with charged residues form well defined fibrils only if the total net charge of the molecule is ±1. This finding illustrates the delicate balance of interactions involved in the formation of fibrils relative to more disordered aggregates. The present results, in conjunction with x-ray fiber diffraction, electron microscopy, and Fourier transform infrared measurements, have allowed us to propose a detailed structural model of the fibrils.

Molecular recycling within amyloid fibrils

Amyloid fibrils are thread-like protein aggregates with a core region formed from repetitive arrays of β-sheets oriented parallel to the fibril axis. Such structures were first recognized in clinical disorders, but more recently have also been linked to a variety of non-pathogenic phenomena ranging from the transfer of genetic information to synaptic changes associated with memory. The observation that many proteins can convert into similar structures in vitro has suggested that this ability is a generic feature of polypeptide chains. Here we have probed the nature of the amyloid structure by monitoring hydrogen/deuterium exchange in fibrils formed from an SH3 domain using a combination of nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. The results reveal that under the conditions used in this study, exchange is dominated by a mechanism of dissociation and re-association that results in the recycling of molecules within the fibril population. This insight into the dynamic nature of amyloid fibrils, and the ability to determine the parameters that define this behaviour, have important implications for the design of therapeutic strategies directed against amyloid disease.

Protein folding and misfolding: a paradigm of self-assembly and regulation in complex biological systems.

Understanding biological complexity is one of the grand scientific challenges for the future. A living organism is a highly evolved system made up of a large number of interwoven molecular networks. These networks primarily involve proteins, the macromolecules that enable and control virtually every chemical process that takes place in the cell. Proteins are also key elements in the essential characteristic of living systems, their ability to function and replicate themselves through controlled molecular interactions. Recent progress in understanding the most fundamental aspect of polypeptide self–organization, the process by which proteins fold to attain their active conformations, provides a global platform to gain knowledge about the function of biological systems and the regulatory mechanisms that underpin their ability to adapt to changing conditions. In order to exploit such progress effectively, we are developing a variety of approaches, including procedures that use experimental data to restrain the properties of complex systems in computer simulations, to describe their behaviour under a wide variety of conditions. We believe that such approaches can lead to significant advances in understanding biological complexity, in general, and protein folding and misfolding in particular. These advances would contribute to: a more effective exploitation of the information from genome sequences; more rational therapeutic approaches to diseases, particularly those associated with ageing; the responsible control of our own evolution; and the development of new technologies based on mimicking the principles of biological self–assembly, for instance in nanotechnology. More fundamentally, we believe that this research will result in a more coherent understanding of the origin, evolution and functional properties of living systems.

Competing intrachain interactions regulate the formation of beta-sheet fibrils in bovine PrP peptides.

At the heart of the pathogenesis of transmissible spongiform encephalopathies (TSEs), such as BSE, scrapie, and Creutzfeldt–Jakob disease, lies a poorly understood structural rearrangement of PrP, an abundant glycoprotein of the nervous and lymphoid systems. The normal form (PrPC), rich in α‐helix, converts into an aberrant β‐sheet‐dominated form (PrPSc), which seems to be at the center of the pathotoxic symptoms observed in TSEs. To understand this process better at a molecular level, we have studied the interactions between different peptides derived from bovine PrP and their structural significance. We show that two unstructured peptides derived from the central region of bovine PrP, residues 115–133 and 140–152, respectively, interact stoichiometrically under physiological conditions to generate β‐sheet‐dominated fibrils. However, when both peptides are incubated in the presence of a third peptide derived from an adjoining α‐helical region (residues 153–169), the formation of β‐sheet‐rich fibrils is abolished. These data indicate that native PrPC helix 1 might inhibit the strong intrinsic β‐sheet‐forming propensity of sequences immediately N‐terminal to the globular core of PrPC, by keeping in place intrachain interactions that would prevent these amyloidogenic regions from triggering aggregation. Moreover, these results indicate new ways in which PrPSc formation could be prevented.

Amyloidogenicity and Aggregate Cytotoxicity of Human Glucagon-Like Peptide-1 (hGLP-1)

The potential of human glucagon-like peptide-1 (hGLP-1) as a therapeutic agent is limited by its high aggregation propensity. We show that hGLP-1 forms amyloid-like structures that are preceded by cytotoxic aggregates, suggesting that aggregation of biopharmaceuticals could present a cytotoxic risk to patients besides the reported increased risk in immunogenicity.