Of Bits and Bugs — On the Use of Bioinformatics and a Bacterial Crystal Structure to Solve a Eukaryotic Repeat-Protein Structure

Pur-α is a nucleic acid-binding protein involved in cell cycle control, transcription, and neuronal function. Initially no prediction of the three-dimensional structure of Pur-α was possible. However, recently we solved the X-ray structure of Pur-α from the fruitfly Drosophila melanogaster and showed that it contains a so-called PUR domain. Here we explain how we exploited bioinformatics tools in combination with X-ray structure determination of a bacterial homolog to obtain diffracting crystals and the high-resolution structure of Drosophila Pur-α. First, we used sensitive methods for remote-homology detection to find three repetitive regions in Pur-α. We realized that our lack of understanding how these repeats interact to form a globular domain was a major problem for crystallization and structure determination. With our information on the repeat motifs we then identified a distant bacterial homolog that contains only one repeat. We determined the bacterial crystal structure and found that two of the repeats interact to form a globular domain. Based on this bacterial structure, we calculated a computational model of the eukaryotic protein. The model allowed us to design a crystallizable fragment and to determine the structure of Drosophila Pur-α. Key for success was the fact that single repeats of the bacterial protein self-assembled into a globular domain, instructing us on the number and boundaries of repeats to be included for crystallization trials with the eukaryotic protein. This study demonstrates that the simpler structural domain arrangement of a distant prokaryotic protein can guide the design of eukaryotic crystallization constructs. Since many eukaryotic proteins contain multiple repeats or repeating domains, this approach might be instructive for structural studies of a range of proteins

Testing of the toxicity of volatile compounds on human lung cells using the Air/Liquid Interface (ALI) culturing and exposure technique: A prevalidation study

Actually, precision cut lung slices are under extensive investigation to serve as a model for acute inhalation toxicology. Slices can be employed as an ex-vivo/in-vitro model as alternative method in the context of REACH and the 3Rs. Moreover, this in vitro model represents in vivo tissue including all naturally occurring cell types of the respiratory tract situated in their physiological environment. For the reason of these beneficial characteristics, PCLS may be an in vitro model of first choice in the future, especially with respect to studies on inflammatory changes in the lung. However, since safety control and toxicology demand studies on genotoxicity, we also started to establish a Comet assay with live lung tissue in vitro from mouse lungs to expand the experimental possibilities from investigations with PCLS on toxicity and inflammatory responses to genotoxicity. The work focused on optimization of a simple and fast cell separation method by enzymatic digestion of the lung tissue, application of the alkaline Comet assay and exposure of lung slices to culture media (negative control) and test compounds in a first series of experiments. Ethyl methanesulfonate (EMS) and formalin (FA) were tested as model substances known to induce DNA damage. These alterations can be observed in the Comet assay as, respectively, an increased rate of DNA breaks (EMS) or a reduced rate of DNA breaks by induction of cross-links (FA). Factors of concern for the reproducibility and meaningfulness of results obtained with the method established here to investigate genotoxicity induced in this in-vivo-like live complex cell system by application of the Comet assay to PCLS are discussed. These include the slice-to-slice- and individual-to-individual-reproducibility as well as the background signal and the dose-dependency of the effects induced by the model substances. In summary, the experiments showed that dose-dependent effects induced by EMS and FA could be detected in mouse PCLS, and by application of the Comet assay, effects from strand-breaking and cross-linking substances could be reproducibly discriminated

Whole exome sequencing in family trios reveals de novo mutations in PURA as a cause of severe neurodevelopmental delay and learning disability

BACKGROUND: De novo mutations are emerging as an important cause of neurocognitive impairment, and whole exome sequencing of case-parent trios is a powerful way of detecting them. Here, we report the findings in four such trios. METHODS: The Deciphering Developmental Disorders study is using whole exome sequencing in family trios to investigate children with severe, sporadic, undiagnosed developmental delay. Three of our patients were ascertained from the first 1133 children to have been investigated through this large-scale study. Case 4 was a phenotypically isolated case recruited into an undiagnosed rare disorders sequencing study. RESULTS: Protein-altering de novo mutations in PURA were identified in four subjects. They include two different frameshifts, one inframe deletion and one missense mutation. PURA encodes Pur-alpha, a highly conserved multifunctional protein that has an important role in normal postnatal brain development in animal models. The associated human phenotype of de novo heterozygous mutations in this gene is variable, but moderate to severe neurodevelopmental delay and learning disability are common to all. Neonatal hypotonia, early feeding difficulties and seizures, or 'seizure-like' movements, were also common. Additionally, it is suspected that anterior pituitary dysregulation may be within the spectrum of this disorder. Psychomotor developmental outcomes appear variable between patients, and we propose a possible genotype-phenotype correlation, with disruption of Pur repeat III resulting in a more severe phenotype. CONCLUSIONS: These findings provide definitive evidence for the role of PURA in causing a variable syndrome of neurodevelopmental delay, learning disability, neonatal hypotonia, feeding difficulties, abnormal movements and epilepsy in humans, and help clarify the role of PURA in the previously described 5q31.3 microdeletion phenotype.This article is available via Open Access. Please click on the 'Additional Link' above to access the full-text