Jesper Søndergaard Pedersen

Chaperone networks: Tipping the balance in protein folding diseases

Adult-onset neurodegeneration and other protein conformational diseases are associated with the appearance, persistence, and accumulation of misfolded and aggregation-prone proteins. To protect the proteome from long-term damage, the cell expresses a highly integrated protein homeostasis (proteostasis) machinery to ensure that proteins are properly expressed, folded, and cleared, and to recognize damaged proteins. Molecular chaperones have a central role in proteostasis as they have been shown to be essential to prevent the accumulation of alternate folded proteotoxic states as occurs in protein conformation diseases exemplified by neurodegeneration. Studies using invertebrate models expressing proteins associated with Huntingtons disease, Alzheimers disease, ALS, and Parkinsons disease have provided insights into the genetic networks and stress signaling pathways that regulate the proteostasis machinery to prevent cellular dysfunction, tissue pathology, and organismal failure. These events appear to be further amplified by aging and provide evidence that age-related failures in proteostasis may be a common element in many diseases.

Amyloid—a state in many guises: Survival of the fittest fibril fold

Under appropriate conditions, essentially all proteins are able to aggregate to form long, well‐ordered and β‐sheet‐rich arrays known as amyloid‐like fibrils. These fibrils consist of varying numbers of intertwined protofibrils and can for any given protein exhibit a wealth of different forms at the ultrastructural level. Traditionally, this structural variability or polymorphism has been attributed to differences in the assembly of a common protofibril structure. However, recent work on glucagon, insulin, and the Aβ peptide suggests that this polymorphism can occur at the level of secondary structure. Simple variations in either solvent conditions such as temperature, protein concentration, and ionic strength or external mechanical influences such as agitation can lead to formation of fibrils with markedly different characteristics. In some cases, these characteristics can be passed on to new fibrils in a strain‐specific manner, similar to what is known for prions. The preferred structure of fibrils formed can be explained in terms of selective pressure and survival of the fittest; the most populated types of fibrils we observe at the end of an experiment are those that had the fastest overall growth rate under the given conditions. Fibrillar polymorphism is probably a consequence of the lack of structural restraints on a nonfunctional conformational state.

Amyloid structure – one but not the same: the many levels of fibrillar polymorphism

Many proteins and peptides can form amyloid‐like structures both in vivo and in vitro. Although strikingly similar fibrillar structures can be observed across a variety of amino acid sequences, the fibrils formed often exhibit a stunning wealth of polymorphisms at the level of electron or atomic force microscopy. This appears to violate the Anfinsen principle seen for globular proteins, where each protein sequence codes for just one well‐defined fold. To a large extent, polymorphism reflects variable packing of a single protofilament structure in the mature fibrils. However, we and others have recently demonstrated that polymorphism can also reflect real structural differences in the molecular packing of the polypeptide chains leading to several possible protofilament structures and diverse mature fibrillar structures. Glucagon has been a particularly useful model system for studying the fibrillogenesis mechanisms that lead to the formation of structural polymorphism, thanks to its single tryptophan residue and the availability of large quantities at pharmaceutical‐grade quality. Combinations of structural investigations and seed extension experiments have revealed the reproducible formation of at least five different self‐propagating fibril types from subtle variations in growth conditions. These reflect the underlying complexity of the peptide conformational landscape and provide a link to natively disordered proteins, where structure is dictated by context in the form of different binding partners. Here we review some of the latest advances in the study of glucagon fibrillar polymorphism and their implications for mechanisms of fibril formation in general.

Functional improvement of antibody fragments using a novel phage coat protein III fusion system.

Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.

The Nature of Amyloid-like Glucagon Fibrils

Protein aggregation and formation of amyloid fibrils is a phenomenon usually associated with proteotoxicity and degenerative diseases, such as type 2 diabetes, Alzheimers disease, and prion diseases. However, several protein and peptide hormones are known to have a high propensity to form amyloid-like fibrils in vitro raising concerns about safety and stability of pharmaceutical protein solutions. Comprehensive understanding of the aggregation mechanisms is an important prerequisite to the design of strategies to prevent fibril formation. Detailed kinetic, spectroscopic, and morphological studies have revealed that glucagon can form several types of fibrils that differ at the level of molecular packing of the peptide. Each type forms through distinct nucleation-dependent aggregation pathways Influenced by solution conditions and can be self-propagated by seeding. An increasing number of functional amyloid-like structures have been discovered in nature, and it has recently been proposed that an amyloid-like state of glucagon may be utilized by the pancreatic α-cells as in vivo storage form. This article reviews the current state of our knowledge about the nature of the different types of amyloid-like glucagon fibrils, the mechanisms by which they form, and discusses implications for formulation strategies and the safety of glucagon pharmaceuticals.

Integrating XML data in the TARGIT OLAP system

We present work on logical integration of OLAP and XML data sources, carried out in cooperation between TARGIT, a Danish OLAP client vendor, and Aalborg University. A prototype has been developed that allows XML data on the WWW to be used as dimensions and measures in the OLAP system in the same way as ordinary dimensions and measures, providing a powerful and flexible way to handle unexpected or short-term data requirements as well as rapidly changing data. Compared to earlier work, we present several major extensions that resulted from TARGITs requirements. These include the ability to use XML data as measures, as well as a novel multigranular data model and query language that formalizes and extends the TARGIT data model and query language.

Directed Evolution of Barnase Stability Using Proteolytic Selection

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistance of the mutants toward proteolysis. Susceptibility toward proteolysis has been correlated with flexibility and unfolding, and is thus expected to yield mutants with increased thermal stability. Enrichment of barnase mutants with specific combinations of amino acid residues at four of the randomised positions was observed. Three of these enriched amino acid residues are present in neither barnase nor binase. For some of the mutations, the improvement in proteolytic stability does not lead to a pronounced improvement in thermodynamic stability, indicating that the factors governing the proteolytic stability in some cases may be different from those governing the thermodynamic stability, e.g. propensity to local unfolding.The results obtained add important knowledge to a novel use of phage display technology for selection of thermodynamically stable proteins. Only by carefully establishing the parameters that can be adjusted, and recognising the influence this will have on the outcome of selection, will it be possible to realise the powerful technique of proteolytic selection.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimers disease (AD), Parkinsons disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Spectroscopic evidence for the existence of an obligate pre‐fibrillar oligomer during glucagon fibrillation

The 29‐residue peptide hormone glucagon has been used as a model system for the study of amyloid‐like fibrils. Atomic force microscopy (AFM) studies have detected putative oligomeric species during this lag phase, but this has not been confirmed by any spectroscopic technique. Here we use an attached pyrene group to detect association (excimer formation) between individual glucagon molecules. Our data show that excimer formation precedes fibrillation both at different pHs and with sulfate, and support our original proposal that glucagon fibril formation is preceded by oligomer formation. We suggest that pyrene‐labelling may be a useful way to monitor oligomer formation during protein fibrillation.