Cystathionine beta-synthase mutants exhibit changes in protein unfolding: conformational analysis of misfolded variants in crude cell extracts

Protein misfolding has been proposed to be a common pathogenic mechanism in many inborn errors of metabolism including cystathionine β-synthase (CBS) deficiency. In this work, we describe the structural properties of nine CBS mutants that represent a common molecular pathology in the CBS gene. Using thermolysin in two proteolytic techniques, we examined conformation of these mutants directly in crude cell extracts after expression in E. coli. Proteolysis with thermolysin under native conditions appeared to be a useful technique even for very unstable mutant proteins, whereas pulse proteolysis in a urea gradient had limited values for the study of the majority of CBS mutants due to their instability. Mutants in the active core had either slightly increased unfolding (p.A114V, p.E302K and p.G307S) or extensive unfolding with decreased stability (p.H65R, p.T191M, p.I278T and p.R369C). The extent of the unfolding inversely correlated with the previously determined degree of tetrameric assembly and with the catalytic activity. In contrast, mutants bearing aminoacid substitutions in the C-terminal regulatory domain (p.R439Q and p.D444N) had increased global stability with decreased flexibility. This study shows that proteolytic techniques can reveal conformational abnormalities even for CBS mutants that have activity and/or a degree of assembly similar to the wild-type enzyme. We present here a methodological strategy that may be used in cell lysates to evaluate properties of proteins that tend to misfold and aggregate and that may be important for conformational studies of disease-causing mutations in the field of inborn errors of metabolism

Types and effects of protein variations

Variations in proteins have very large number of diverse effects affecting sequence, structure, stability, interactions, activity, abundance and other properties. Although protein-coding exons cover just over 1 % of the human genome they harbor an disproportionately large portion of disease-causing variants. Variation ontology (VariO) has been developed for annotation and description of variation effects, mechanisms and consequences. A holistic view for variations in proteins is made available along with examples of real cases. Protein variants can be of genetic origin or emerge at protein level. Systematic names are provided for all variation types, a more detailed description can be made by explaining changes to protein function, structure and properties. Examples are provided for the effects and mechanisms, usually in relation to human diseases. In addition, the examples are selected so that protein 3D structural changes, when relevant, are included and visualized. Here, systematics is described for protein variants based on VariO. It will benefit the unequivocal description of variations and their effects and further reuse and integration of data from different sources

Gene delivery to respiratory epithelial cells by magnetofection.

BACKGROUND: For the topical application of DNA vector complexes to the airways, specific extracellular barriers play a major role. In particular, short contact time of complexes with the cell surface caused by the mucociliary clearance hinders cellular uptake of complexes. The aim of this study was to evaluate the ability of magnetofection, a technique based on the principle of magnetic drug targeting, to overcome these barriers in comparison with conventional nonviral gene transfer methods such as lipofection and polyfection. METHODS: Experiments were carried out on permanent (16HBE14o-) and primary airway epithelial cells (porcine and human), and native porcine airway epithelium ex vivo. Transfection efficiency and dose-response relationship of magnetofection were examined by luciferase reporter gene expression. Sedimentation patterns and uptake of gene transfer complexes were characterized by fluorescence and electron microscopy, respectively. RESULTS: We show that (i) application of a magnetic field allows the magnetofectins to sediment and to enrich at the cell surface within a few minutes, (ii) magnetofection bears an improved dose-response relationship, (iii) magnetofection enhances transfection efficiency in both, permanent and primary airway epithelial cells, and (iv) magnetofection leads to significant transgene expression at very short incubation times in an ex vivo airway epithelium organ model. CONCLUSIONS: Magnetofection provides a potential novel method, which may overcome fundamental limitations of nonviral gene transfer to the airways. Due to the accelerated enrichment at the cell surface it may be of major interest for in vivo applications, where long-term incubation times at the target tissue are hardly achievable

Einsatz agiler Methoden zur Gestaltung strukturierter Weiterbildungsprogramme

Postgraduate medical education (PGME) is an essential part of medical education and increasingly shifts into focus of educational stakeholders. Structured postgraduate medical training programs are required in the U.S. through the "American Council for Graduate Medical Education" (ACGME) guidelines with their six core competencies as common program requirements. The basis for this development was provided in Germany with the implementation of the "Standard Framework for Postgraduate Medical Training" (Musterweiterbildungsordnung) issued by the German Medical Association (Bundesärztekammer). However, implementation has been gradual and program development is often conducted in a time consuming, lengthy and top-down approach without that trainee experiences or needs are being assessed or evaluated for their impact on successful medical training.We demonstrate how application of agile working can enable rapid and efficient creation and implementation of a novel postgraduate training program. The postgraduate training program ped.tracks aims to achieve a high-quality, structured and reliable postgraduate training. Moreover, it provides the opportunity to select a priority on scientific or clinical education. The entire process from the first draft to full release of the program was completed within 8 months through agile working. Our team worked using agile working techniques, creating a trainee- customized and -centred program. We anticipate that the quantity of structured postgraduate training programs will significantly increase in Germany and Europe to improve training quality and employee satisfaction. Therefore, the use of agile methods for the creation and implementation of structured training programs represents a useful approach to support program directors rapidly and effectively in this effort.Die postgraduierte medizinische Ausbildung (PGME) ist ein wesentlicher Bestandteil der medizinischen Ausbildung und rückt zunehmend in den Fokus von Weiterbildungseinrichtungen und -befugten. Strukturierte postgraduierte medizinische Ausbildungsprogramme werden in den USA durch die Richtlinien des "American Council for Graduate Medical Education" (ACGME) mit ihren sechs Kernkompetenzen vorgegeben. Die Grundlage für deren Entwicklung auch in Deutschland wurde mit der Einführung der Musterweiterbildungsordnung der Bundesärztekammer geschaffen. Die Umsetzung erfolgte in vielen Fällen schrittweise und die Entwicklung neuer Weiterbildungsprogramme wird häufig in einem zeitaufwändigen, langwierigen und hierarchisch verlaufenden Ansatz durchgeführt, ohne dass die Erfahrungen oder Bedürfnisse der Auszubildenden im Hinblick auf ihre Auswirkungen zu einer erfolgreichen medizinischen Ausbildung einbezogen werden. Wir zeigen, wie die Nutzung agilen Arbeitens die schnelle und effiziente Erstellung und Implementierung eines neuartigen postgraduierten Ausbildungsprogramms ermöglicht. Das postgraduierte Ausbildungsprogramm ped.tracks zielt auf eine qualitativ hochwertige, strukturierte und zuverlässige postgraduierte Ausbildung ab. Darüber hinaus bietet es die Möglichkeit, einen Schwerpunkt auf die wissenschaftliche oder klinische Ausbildung zu legen. Der gesamte Prozess vom ersten Entwurf bis zur vollständigen Freigabe des Programms wurde innerhalb von 8 Monaten durch agiles Arbeiten abgeschlossen. Unser Team arbeitete mit agilen Arbeitstechniken und entwickelte ein auf die Auszubildenden zugeschnittenes Programm. Wir gehen davon aus, dass der Bedarf an strukturierten Weiterbildungsprogrammen in Deutschland und Europa deutlich zunehmen wird, um die Ausbildungsqualität und die Mitarbeitendenzufriedenheit zu verbessern. Der Einsatz agiler Methoden für die Erstellung und Umsetzung strukturierter Weiterbildungsprogramme stellt dabei einen sinnvollen Ansatz dar, um Weiterbildungsbefugte schnell und effektiv bei diesem Vorhaben zu unterstützen

The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery

Synuclein alpha accumulation mediates podocyte injury in Fabry nephropathy

AbstractCurrent therapies for Fabry disease are based on reversing intra-cellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosome dysfunction. However, their effect in the reversal of endorgan damage, like kidney injury and chronic kidney disease remains unclear. First, ultrastructural analysis of serial human kidney biopsies showed that longterm use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/CAS9-mediated α-Galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified alpha-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.</jats:p