Microbial Genetic Screens for Monitoring Protein Misfolding Associated with Neurodegeneration: Tools for Identifying Disease-Relevant Genes and for Screening Synthetic and Natural Compound Libraries for the Discovery of Potential Therapeutics.

Neurodegenerative Diseases (ND) are a major threat to the aging population and the lack of a single preventive or disease-modifying agent only serves to increase their impact. In the past few years, protein misfolding and the subsequent formation of neurotoxic oligomeric/aggregated protein species have emerged as a unifying theme underlying the pathology of these complex diseases. Recently developed microbial genetic screens and selection systems for monitoring ND-associated protein misfolding have allowed the establishment of highthroughput assays for the identification of cellular factors and processes that are important mediators of NDassociated proteotoxicities.

Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production.

Membrane proteins perform critical cellular functions in all living organisms and constitute major targets for drug discovery. Escherichia coli has been the most popular overexpression host for membrane protein biochemical/structural studies. Bacterial production of recombinant membrane proteins, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low final biomass and minute volumetric yields.

Why bother with a COST Action? The benefits of networking in science.

A COST Action is a consortium of -mainly- European scientists (but open to international cooperation) working on a common research area, with the same subject; COST provides funding to the Actions for networking and dissemination activities, thus the participating scientists must have secured research funding from other national or European sources. COST funding is in the scale of approximately 100 kEuros per year and in this vein, it is often criticized both in that it does not fund research and the core science and in that its funding is 'limited'. However, COST with its instruments is an integral pillar of the European Research Area, and it is through its mission that a variety of aspects of the research environment, fundamental to the success of the research, are catered for; these include scientific networking, collaboration/exchange/training and dissemination activities.

Towards antigen-specific apheresis of pathogenic autoantibodies as a further step in the treatment of myasthenia gravis by plasmapheresis.

Myasthenia gravis (MG), a prototypic antibody-mediated autoimmune disease, presents an excellent target for scientific research aimed at a better understanding of the disease itself and the source that triggers an autoimmune reaction in an organism. MG is a neuromuscular disease caused mainly by an autoimmune response against the nicotinic acetylcholine receptor (AChR) which interferes with neuromuscular transmission. This review focuses on our studies on the extracellular domains of human muscle AChR subunits in an effort to develop an approach for the specific therapeutic apheresis of autoantibodies from patients' sera using the immobilized subunits as immunoadsorbents.

Antigen-specific apheresis of pathogenic autoantibodies from myasthenia gravis sera.

Myasthenia gravis (MG) is usually caused by autoantibodies against muscle nicotinic acetylcholine receptor (AChR), which is composed of five subunits (alpha(2)betagammadelta or alpha(2)betaepsilondelta). Current treatments, including plasmapheresis, are nonspecific, causing several side effects. We aim to develop an antigen-specific alternative to plasmapheresis, since the latter removes indispensable plasma components in addition to anti-AChR antibodies.

Mutant forms of the extracellular domain of the human acetylcholine receptor gamma-subunit with improved solubility and enhanced antigenicity. The importance of the Cys-loop.

The muscle nicotinic acetylcholine receptor (AChR) is the prototype of the ligand-gated ion channels (or Cys-loop receptors), formed by 5 homologous subunits (alpha2betagammadelta or alpha2betagammaepsilon), and is the major autoantigen in the autoimmune disease, myasthenia gravis. Previously, we expressed the wild-type extracellular domain (ECD) of the gamma-subunit (gammaECD) of the AChR in yeast Pichia pastoris at 0.3-0.

Extracellular domains of the beta, gamma and epsilon subunits of the human acetylcholine receptor as immunoadsorbents for myasthenic autoantibodies: a combination of immunoadsorbents results in increased efficiency.

Myasthenia gravis (MG) is usually caused by autoantibodies against the human muscle acetylcholine receptor (AChR). Plasmapheresis offers a therapeutic option, but, as well as removing the pathogenic anti-AChR autoantibodies, it non-specifically removes indispensable immunoglobulins. An attractive alternative to plasmapheresis would be the extracorporeal specific removal of the autoantibodies using AChR-based immunoadsorbents.

Circular dichroism studies of extracellular domains of human nicotinic acetylcholine receptors provide an insight into their structure.

The extracellular domains (ECDs) of human nicotinic acetylcholine receptors (nAChRs) are of major pharmacological interest as drug targets in the autoimmune disease myasthenia gravis and in various neurological disorders. We have previously expressed and purified the human muscle alpha1-, beta1-, gamma- and epsilon-nAChR-ECDs, as well as the wild type and a mutant of neuronal alpha7-ECD, in yeast Pichia pastoris. The far-UV circular dichroism (CD) studies of these ECDs, presented here, revealed a major prevalence of beta-sheet ( approximately 40%) and a small proportion of alpha-helical ( approximately 5%) structure for all ECDs, in good agreement with the secondary structure composition of the Torpedo muscle-type nAChR-ECDs and in less, but considerable, agreement with that of the homologous invertebrate acetylcholine-binding proteins (AChBPs).

Isolation and functional characterization of anti-acetylcholine receptor subunit-specific autoantibodies from myasthenic patients: receptor loss in cell culture.

The muscle nicotinic acetylcholine receptor (nAChR) is the major autoantigen in the autoimmune disease myasthenia gravis (MG), in which autoantibodies bind to, and cause loss of, nAChRs. Antibody-mediated nAChR loss is caused by the action of complement and by the acceleration of nAChR internalization caused by antibody-induced cross-linking of nAChR molecules (antigenic modulation). To obtain an insight into the role of the various anti-nAChR antibody specificities in MG, we have studied nAChR antigenic modulation caused by isolated anti-subunit autoantibodies.

Expression and characterization of soluble forms of the extracellular domains of the beta, gamma and epsilon subunits of the human muscle acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel found in muscles and neurons. Muscle AChR, formed by five homologous subunits (alpha2 beta gamma delta or alpha2 beta gamma epsilon), is the major antigen in the autoimmune disease, myasthenia gravis (MG), in which pathogenic autoantibodies bind to, and inactivate, the AChR. The extracellular domain (ECD) of the human muscle alpha subunit has been heterologously expressed and extensively studied.

DNA recognition by the KorA proteins of IncP-1 plasmids RK2 and R751.

The KorA repressor proteins of IncP-1 plasmids belong to a growing family of plasmid-encoded repressors that regulate partitioning genes, and in the IncP-1 plasmids coordinate these with expression of replication and transfer genes as well. Both KorA(RK2) (IncP-1 alpha) and KorA(R751) (IncP-1 beta) recognise the 5'-GTTTAGCTAAAC-3' palindrome. Reporter gene assays showed that KorA proteins from these two main subgroups of IncP-1 plasmids show specificity for their own promoter/operators and this preference was confirmed with in vitro binding studies using gel mobility shift assays on one representative promoter.