Erich E. Wanker

EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers.

The accumulation of β-sheet–rich amyloid fibrils or aggregates is a complex, multistep process that is associated with cellular toxicity in a number of human protein misfolding disorders, including Parkinsons and Alzheimers diseases. It involves the formation of various transient and intransient, on- and off-pathway aggregate species, whose structure, size and cellular toxicity are largely unclear. Here we demonstrate redirection of amyloid fibril formation through the action of a small molecule, resulting in off-pathway, highly stable oligomers. The polyphenol (−)-epigallocatechin gallate efficiently inhibits the fibrillogenesis of both α-synuclein and amyloid-β by directly binding to the natively unfolded polypeptides and preventing their conversion into toxic, on-pathway aggregation intermediates. Instead of β-sheet–rich amyloid, the formation of unstructured, nontoxic α-synuclein and amyloid-β oligomers of a new type is promoted, suggesting a generic effect on aggregation pathways in neurodegenerative diseases.

EGCG remodels mature α-synuclein and amyloid-β fibrils and reduces cellular toxicity

Protein misfolding and formation of β-sheet-rich amyloid fibrils or aggregates is related to cellular toxicity and decay in various human disorders including Alzheimer’s and Parkinson’s disease. Recently, we demonstrated that the polyphenol (-)-epi-gallocatechine gallate (EGCG) inhibits α-synuclein and amyloid-β fibrillogenesis. It associates with natively unfolded polypeptides and promotes the self-assembly of unstructured oligomers of a new type. Whether EGCG disassembles preformed amyloid fibrils, however, remained unclear. Here, we show that EGCG has the ability to convert large, mature α-synuclein and amyloid-β fibrils into smaller, amorphous protein aggregates that are nontoxic to mammalian cells. Mechanistic studies revealed that the compound directly binds to β-sheet-rich aggregates and mediates the conformational change without their disassembly into monomers or small diffusible oligomers. These findings suggest that EGCG is a potent remodeling agent of mature amyloid fibrils.

The hunt for huntingtin function: interaction partners tell many different stories

Huntingtons disease (HD) is a neurodegenerative disorder caused by an abnormally elongated polyglutamine (polyQ) tract in the large protein huntingtin (htt). Currently, both the normal function of htt in neurons and the molecular mechanism by which the expanded polyQ sequence in htt causes selective neurodegeneration remain elusive. Research in past years has identified several htt-interacting proteins such as htt-interacting protein 1, Src homology region 3-containing Grb2-like protein 3, protein kinase C and casein kinase substrate in neurons 1, htt-associated protein 1, postsynaptic density-95, FIP-2 (for 14.7K-interacting protein), specificity protein 1 and nuclear receptor co-repressor. These proteins play roles in clathrin-mediated endocytosis, apoptosis, vesicle transport, cell signalling, morphogenesis and transcriptional regulation, suggesting that htt is also involved in these processes.

Small-molecule conversion of toxic oligomers to nontoxic β-sheet–rich amyloid fibrils

Several lines of evidence indicate that prefibrillar assemblies of amyloid-β (Aβ) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimers disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of Aβ fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in Aβ peptides and stabilizes the self-assembly of seeding-competent, β-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic Aβ oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by Aβ oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells.

A Directed Protein Interaction Network for Investigating Intracellular Signal Transduction

Effective prediction of the direction of signal flow in an interaction network enables modeling of signaling dynamics and identification of regulatory proteins. Finding More Pieces to the Signaling Puzzle Even well-studied pathways are likely to be incomplete in terms of our knowledge of all the components and their relationships, and the larger interconnected network that represents the true cellular regulatory landscape remains woefully unknown. Vinayagam et al. used an automated yeast two-hybrid interaction mating assay to identify protein-protein interactions (PPIs) among human proteins and then integrated that PPI data set with previously published data to create an undirected human PPI network connecting 9832 proteins through 39,641 interactions. The authors then applied a Bayesian learning strategy to assign direction to the interactions among the proteins. The resulting directed network enabled them to evaluate growth factor–induced protein phosphorylation dynamics and to identify previously unknown modulators of the extracellular signal–regulated protein kinase pathway, of which 18 were validated with cell-based assays. This strategy should prove useful in completing the puzzle of the cellular regulatory network. Cellular signal transduction is a complex process involving protein-protein interactions (PPIs) that transmit information. For example, signals from the plasma membrane may be transduced to transcription factors to regulate gene expression. To obtain a global view of cellular signaling and to predict potential signal modulators, we searched for protein interaction partners of more than 450 signaling-related proteins by means of automated yeast two-hybrid interaction mating. The resulting PPI network connected 1126 proteins through 2626 PPIs. After expansion of this interaction map with publicly available PPI data, we generated a directed network resembling the signal transduction flow between proteins with a naïve Bayesian classifier. We exploited information on the shortest PPI paths from membrane receptors to transcription factors to predict input and output relationships between interacting proteins. Integration of directed PPI with time-resolved protein phosphorylation data revealed network structures that dynamically conveyed information from the activated epidermal growth factor and extracellular signal–regulated kinase (EGF/ERK) signaling cascade to directly associated proteins and more distant proteins in the network. From the model network, we predicted 18 previously unknown modulators of EGF/ERK signaling, which we validated in mammalian cell-based assays. This generic experimental and computational approach provides a framework for elucidating causal connections between signaling proteins and facilitates the identification of proteins that modulate the flow of information in signaling networks.

HIPPIE: Integrating protein interaction networks with experiment based quality scores.

Protein function is often modulated by protein-protein interactions (PPIs) and therefore defining the partners of a protein helps to understand its activity. PPIs can be detected through different experimental approaches and are collected in several expert curated databases. These databases are used by researchers interested in examining detailed information on particular proteins. In many analyses the reliability of the characterization of the interactions becomes important and it might be necessary to select sets of PPIs of different confidence levels. To this goal, we generated HIPPIE (Human Integrated Protein-Protein Interaction rEference), a human PPI dataset with a normalized scoring scheme that integrates multiple experimental PPI datasets. HIPPIEs scoring scheme has been optimized by human experts and a computer algorithm to reflect the amount and quality of evidence for a given PPI and we show that these scores correlate to the quality of the experimental characterization. The HIPPIE web tool (available at http://cbdm.mdc-berlin.de/tools/hippie) allows researchers to do network analyses focused on likely true PPI sets by generating subnetworks around proteins of interest at a specified confidence level.

Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates.

Publisher Summary The accumulation of polyglutamine-containing protein aggregates in neuronal intranuclear inclusions (NIIs) has been demonstrated for several progressive neurodegenerative diseases such as Huntingtons disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), and spinocerebellar ataxia (SCA) types 1, 3, and 7. Furthermore, it has been shown in vitro that the proteolytic cleavage of fusion proteins of glutathione S-transferase (GST) and the polyglutamine-containing huntingtin peptide coded for by the first exon of the HD gene s leads to the formation of insoluble high molecular weight protein aggregates with a fibrillar or ribbonlike morphology reminiscent of β -amyloid fibrils in Alzheimers disease and scrapie prion rods. This chapter demonstrates that the cellulose acetate filter retardation assay can be a useful tool for the identification, structural characterization, and quantification of SDS-insoluble polyglutamine-containing protein aggregates formed in vitro and in vivo. In addition to the histochemical identification of amyloids, it useful in detecting insoluble protein aggregates in all types of human and animal amyloidoses, including the polyglutamine diseases, and also in screening compound libraries for potential aggregation inhibitors. Currently, attempts to develop a microtiter plate-based high-throughput filter retardation assay to identify chemical compounds that slow down the rate of formation of polyglutamine-containing fibrils in vitro are in progress. The amyloid-binding agents arising from this screen then will be tested in a HD cell culture model system and in the HD animal model for their therapeutic potential.

An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis

Arginine/lysine‐rich motifs typically function as targeting signals for the translocation of proteins to the nucleus. Here, we demonstrate that such a motif consisting of four basic amino acids in the polyglutamine protein ataxin‐3 (Atx‐3) serves as a recognition site for the interaction with the molecular chaperone VCP. Through this interaction, VCP modulates the fibrillogenesis of pathogenic forms of Atx‐3 in a concentration‐dependent manner, with low concentrations of VCP stimulating fibrillogenesis and excess concentrations suppressing it. No such effect was observed with a mutant Atx‐3 variant, which does not contain a functional VCP interaction motif. Strikingly, a stretch of four basic amino acids in the ubiquitin chain assembly factor E4B was also discovered to be critical for VCP binding, indicating that arginine/lysine‐rich motifs might be generally utilized by VCP for the targeting of proteins. In vivo studies with Drosophila models confirmed that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic Atx‐3. Together, these results define the VCP–Atx‐3 association as a potential target for therapeutic intervention and suggest that it might influence the progression of spinocerebellar ataxia type 3.

Polyglutamine fibrillogenesis: The pathway unfolds

Nine neurodegenerative diseases are caused by expanding CAG repeats coding for polyglutamine (polyGln) (1–4). These include Huntingtons disease, dentatorubral and pallidoluysian atrophy, several forms of spino-cerebellar ataxia, and spinal and bulbar muscular atrophy. Within the central nervous system, each disease has a distinctive pattern of degeneration, with considerable overlap among the diseases (5, 6). The genes containing CAG repeats show no homology to each other outside of the glutamine repeats, and most are genes of unknown function. Thus, speculation concerning pathogenesis has focused on the polyGln expansion itself.

UniHI: an entry gate to the human protein interactome.

Systematic mapping of protein–protein interactions has become a central task of functional genomics. To map the human interactome, several strategies have recently been pursued. The generated interaction datasets are valuable resources for scientists in biology and medicine. However, comparison reveals limited overlap between different interaction networks. This divergence obstructs usability, as researchers have to interrogate numerous heterogeneous datasets to identify potential interaction partners for proteins of interest. To facilitate direct access through a single entry gate, we have started to integrate currently available human protein interaction data in an easily accessible online database. It is called UniHI (Unified Human Interactome) and is available at . At present, it is based on 10 major interaction maps derived by computational and experimental methods. It includes more than 150 000 distinct interactions between more than 17 000 unique human proteins. UniHI provides researchers with a flexible integrated tool for finding and using comprehensive information about the human interactome.