Petra Parizek

Binding of disease-associated prion protein to plasminogen

Transmissible spongiform encephalopathies are associated with accumulation of PrPSc, a conformer of a cellular protein called PrP C. PrPSc is thought to replicate by imparting its conformation onto PrPC (ref. 1), yet conformational discrimination between PrPC and PrPSc has remained elusive. Because deposition of PrPSc alone is not enough to cause neuropathology, PrPSc probably damages the brain by interacting with other cellular constituents. Here we find activities in human and mouse blood which bind PrPSc and prion infectivity, but not PrPC. We identify plasminogen, a pro-protease implicated in neuronal excitotoxicity, as a PrPSc-binding protein. Binding is abolished if the conformation of PrPSc is disrupted by 6M urea or guanidine. The isolated lysine binding site 1 of plasminogen (kringles I–III) retains this binding activity, and binding can be competed for with lysine. Therefore, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes.

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

We have selected designed ankyrin repeat proteins (DARPins) from a synthetic library by using ribosome display that selectively bind to the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) in either its nonphosphorylated (inactive) or doubly phosphorylated (active) form. They do not bind to other kinases tested. Crystal structures of complexes with two DARPins, each specific for one of the kinase forms, were obtained. The two DARPins bind to essentially the same region of the kinase, but recognize the conformational change within the activation loop and an adjacent area, which is the key structural difference that occurs upon activation. Whereas the rigid phosphorylated activation loop remains in the same form when bound by the DARPin, the more mobile unphosphorylated loop is pushed to a new position. The DARPins can be used to selectively precipitate the cognate form of the kinases from cell lysates. They can also specifically recognize the modification status of the kinase inside the cell. By fusing the kinase with Renilla luciferase and the DARPin to GFP, an energy transfer from luciferase to GFP can be observed in COS-7 cells upon intracellular complex formation. Phosphorylated ERK2 is seen to increase by incubation of the COS-7 cells with FBS and to decrease upon adding the ERK pathway inhibitor PD98509. Furthermore, the anti-ERK2 DARPin is seen to inhibit ERK phosphorylation as it blocks the target inside the cell. This strategy of creating activation-state–specific sensors and kinase-specific inhibitors may add to the repertoire to investigate intracellular signaling in real time.

Designed Ankyrin Repeat Proteins (DARPins) as Novel Isoform-Specific Intracellular Inhibitors of c-Jun N-Terminal Kinases

The c-Jun N-terminal kinases (JNKs) are involved in many biological processes such as proliferation, differentiation, apoptosis, and inflammation and occur in highly similar isoforms in eukaryotic cells. Isoform-specific functions and diseases have been reported for individual JNK isoforms mainly from gene-knockout studies in mice. There is, however, a high demand for intracellular inhibitors with high selectivity to improve the understanding of isoform-specific mechanisms and for use as therapeutic tools. The commonly used JNK inhibitors are based on small molecules or peptides that often target the conserved ATP binding site or docking sites and thus show only moderate selectivity. To target novel binding epitopes, we used designed ankyrin repeat proteins (DARPins) to generate alternative intracellular JNK inhibitors that discriminate two very similar isoforms, JNK1 and JNK2. DARPins are small binding proteins that are well expressed, stable, and cysteine-free, which makes them ideal candidates for applications in the reducing intracellular environment. We performed ribosome display selections against JNK1α1 and JNK2α1 using highly diverse combinatorial libraries of DARPins. The selected binders specifically recognize either JNK1 or JNK2 or both isoforms in vitro and in mammalian cells. All analyzed DARPins show affinities in the low nanomolar range and isoform-specific inhibition of JNK activation in vitro at physiological ATP concentrations. Importantly, DARPins that selectively inhibit JNK activation in human cells were also identified. These results emphasize the great potential of DARPins as a novel class of highly specific intracellular inhibitors of distinct enzyme isoforms for use in biological studies and as possible therapeutic leads.

Neuroinvasion of Prions: Insights from Mouse Models

The prion was defined by Stanley B. Prusiner as the infectious agent that causes transmissible spongiform encephalopathies. A pathological protein accumulating in the brain of scrapie‐infected hamsters was isolated in 1982 and termed prion protein (PrPSc). Its cognate gene Prnp was identified more than a decade ago by Charles Weissmann, and shown to encode the host protein PrPC. Since the latter discovery, transgenic mice have contributed many important insights into the field of prion biology, including the understanding of the molecular basis of the species barrier for prions. By disrupting the Prnp gene, it was shown that an organism that lacks PrPC is resistant to infection by prions. Introduction of mutant PrP genes into PrP‐deficient mice was used to investigate the structure‐activity relationship of the PrP gene with regard to scrapie susceptibility. Ectopic expression of PrP in PrP knockout mice proved a useful tool for the identification of host cells competent for prion replication. Finally, the availability of PrP knockout mice and transgenic mice overexpressing PrP allows selective reconstitution experiments aimed at expressing PrP in neurografts or in specific populations of haemato‐ and lymphopoietic cells. The latter studies have allowed us to clarify some of the mechanisms of prion spread and disease pathogenesis.

Spongiform encephalopathies: insights from transgenic models.

Publisher Summary This chapter summarizes some of the transgenic mouse models that contributed to the current understanding of the pathogenesis of transmissible spongiform encephalopathies. Prion diseases, or transmissible spongiform encephalopathies, are neurological disorders caused by transmissible pathogens termed prions. Human prion diseases are characterized by extended incubation periods ranging from several months to decades that are followed by a progressive clinical phase presenting with severe dementia and ataxia. Clinical disease is always lethal: death can occur within as short a period as a few weeks but occasionally in a period of up to a few years. Peripheral prion pathogenesis, and ultimately neuroinvasion are dependent on the components of the host-immune system. Collectively, these processes require either B cells or their products. At least one B cell-dependent event is the acquisition of a functional follicular dendritic cell network within the germinal centers of peripheral lymphoid tissue. These cells are the major sites of extraneuronal prion protein (PrP c ) expression and probably the principal sites of PrP Sc accumulation.

Amyloid-β Peptide-specific DARPins as a Novel Class of Potential Therapeutics for Alzheimer Disease

Background: The amyloid-β peptide (Aβ) is crucially involved in the onset and progression of Alzheimer disease (AD). Results: A designed ankyrin repeat protein (DARPin) was selected to bind and neutralize Aβ. Conclusion: DARPins can prevent amyloid formation and associated neurotoxic effects of Aβ. Significance: DARPins provide a therapeutic potential in the treatment of AD. Passive immunization with anti-amyloid-β peptide (Aβ) antibodies is effective in animal models of Alzheimer disease. With the advent of efficient in vitro selection technologies, the novel class of designed ankyrin repeat proteins (DARPins) presents an attractive alternative to the immunoglobulin scaffold. DARPins are small and highly stable proteins with a compact modular architecture ideal for high affinity protein-protein interactions. In this report, we describe the selection, binding profile, and epitope analysis of Aβ-specific DARPins. We further showed their ability to delay Aβ aggregation and prevent Aβ-mediated neurotoxicity in vitro. To demonstrate their therapeutic potential in vivo, mono- and trivalent Aβ-specific DARPins (D23 and 3×D23) were infused intracerebroventricularly into the brains of 11-month-old Tg2576 mice over 4 weeks. Both D23 and 3×D23 treatments were shown to result in improved cognitive performance and reduced soluble Aβ levels. These findings demonstrate the therapeutic potential of Aβ-specific DARPins for the treatment of Alzheimer disease.