ABSTRACT
Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Motor Neurons/metabolism , Protein Structure, Tertiary , RNA-Binding Proteins/genetics , Spinal Cord/cytology , TATA-Binding Protein Associated Factors/genetics , Animals , Cells, Cultured , Computational Biology , Drosophila melanogaster/genetics , Genetic Association Studies/methods , Humans , Immunohistochemistry , Mutation, Missense/genetics , Saccharomyces cerevisiae/genetics , TATA-Binding Protein Associated Factors/metabolismABSTRACT
Small-molecule drug discovery can be hindered by the formation of aggregates that act as non-selective inhibitors of drug targets. Such aggregates appear as false positives in high-throughput screening campaigns and can bedevil structure-activity relationships during compound optimization. Protocols are described for resonant waveguide grating (RWG) and dynamic light scattering (DLS) as microplate-based high-throughput approaches to identify compound aggregation. Resonant waveguide grating and dynamic light scattering give equivalent results for the compound test set, as assessed with Bland-Altman analysis. © 2019 The Authors. Basic Protocol 1: Resonant waveguide grating (RWG) in 384-well or 1536-well plate format to detect compound aggregation Basic Protocol 2: Dynamic light scattering (DLS) in 384-well plate format to detect compound aggregation.
Subject(s)
Artifacts , High-Throughput Screening Assays/methods , Pharmaceutical Preparations/chemistry , Small Molecule Libraries/analysis , Small Molecule Libraries/chemistry , Drug Discovery/methods , Dynamic Light ScatteringABSTRACT
A prevalent observation in high-throughput screening and drug discovery programs is the inhibition of protein function by small-molecule compound aggregation. Here, we present the X-ray structural description of aggregation-based inhibition of a protein-protein interaction involving tumor necrosis factor α (TNFα). An ordered conglomerate of an aggregating small-molecule inhibitor (JNJ525) induces a quaternary structure switch of TNFα that inhibits the protein-protein interaction between TNFα and TNFα receptors. SPD-304 may employ a similar mechanism of inhibition.