Protein Misfolding as an Underlying Molecular Defect in Mucopolysaccharidosis III Type C

Mucopolysaccharidosis type IIIC or Sanfilippo syndrome type C (MPS IIIC, MIM #252930) is an autosomal recessive disorder caused by deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT, EC 2.3.1.78), which catalyses transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by α-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death causing neurodegeneration and is accompanied by mild visceral and skeletal abnormalities, including coarse facies and joint stiffness. Surprisingly, the majority of MPS IIIC patients carrying missense mutations are as severely affected as those with splicing errors, frame shifts or nonsense mutations resulting in the complete absence of HGSNAT protein

N-acétyltransférase lysosomale : organisation, fonctionnement et défauts moléculaires chez les patients atteints du syndrome de Sanfilippo type C

L’acétylation des résidus de glucosamine terminaux par la N-acétyltransférase lysosomale (HGSNAT) est une étape essentielle de la dégradation catabolique de l’héparan sulfate. Des défauts dans cette réaction causent une maladie de surcharge lysosomale autosomale récessive rare : le désordre de Sanfilippo type C (SFC). À ce jour, 54 mutations ont été rapportées chez des patients SFC, incluant 13 mutations des sites d’épissage, 11 insertions et délétions, 8 mutations non-sens, 18 mutations faux-sens et 4 polymorphismes, avec différentes manifestations phénotypiques. Nous avons identifié 10 d’entre elles et effectué une étude exhaustive portant sur l’éventail des mutations SFC, leur distribution dans la population de patients, ainsi que leur impact potentiel sur la structure de la HGSNAT. Les erreurs d’épissage, les mutations non-sens, les insertions et les délétions devraient toutes entraîner un ARN non fonctionnel qui est rapidement dégradé par des mécanismes de contrôle qualité cellulaire. Les 4 polymorphismes identifiés sont des changements d'acides aminés qui ne modifient pas l'activité enzymatique, la glycosylation ou la localisation et n'ont donc pas de signification au niveau clinique. Au niveau des enzymes, les polymorphismes sont des changements d’acides aminés qui n’affectent pas la fonction, mais dans un contexte d’acides nucléiques ils peuvent être considérés comme des mutations faux-sens. Les dix-huit mutations faux-sens qui ont été exprimées ont produit des protéines inactives, en raison d'erreurs dans leur repliement. Ceci expliquerait donc la progression sévère de la maladie chez les personnes porteuses de ces mutations. Les protéines mutantes mal repliées sont anormalement glycosylées et conservées dans le réticulum endoplasmique. La thérapie par amélioration de l’activité enzymatique par des chaperonnes est une option thérapeutique potentielle, spécifiquement conçue pour exploiter l'activité enzymatique résiduelle de mutants mal repliés, afin d’éliminer les substrats stockés. Nous avons démontré que le traitement de plusieurs lignées de fibroblastes de patients SFC avec le chlorhydrate de glucosamine, un inhibiteur spécifique de la HGSNAT, a partiellement restauré l’activité de l'enzyme mutante, fournissant une preuve de l’utilité future de la thérapie par des chaperonnes dans le traitement de la maladie de SFC.The acetylation of terminal glucosamine residues by lysosomal N-acetyltransferase (HGSNAT) is an essential part of the catabolic breakdown of heparan sulfate. Defects in this reaction result in the rare autosomal recessive lysosomal storage disorder Sanfilippo syndrome type C (SFC). To date 54 mutations in SFC patients have been reported including 13 splice-site mutations, 11 insertions and deletions, 8 nonsense, 18 missense and 4 polymorphisms, with different phenotypic manifestations. We have identified 10 of them and conducted a comprehensive review discussing the spectrum of Sanfilippo C mutations, their distribution within the patient population as well as how the mutations could potentially affect the structure of HGSNAT. Splicing errors, nonsense mutations, insertions and deletions were all predicted to result in non-functional RNA which is rapidly degraded by cellular quality control mechanisms. The 4 identified polymorphisms resulted in amino acid changes which did not affect the enzyme activity, glycosylation or targeting and were therefore not clinically significant. Polymorphisms, in the context of enzymes are amino acid changes not affecting function, but in the context of nucleic acids can still be considered as missense mutations. Eighteen missense mutations were expressed and shown be inactive due to errors in protein folding providing an explanation for the severe disease progression seen in individuals with these mutations. Misfolded mutants were abnormally glycosylated and retained in the endoplasmic reticulum. Enzyme enhancement/chaperone therapy is a potential treatment option specifically designed to exploit the residual enzyme activity of misfolded mutants in order to clear stored substrates. We demonstrated that treatment of several fibroblast lines of SFC patients with a specific inhibitor of HGSNAT; glucosamine-hydrochloride partially rescued mutant enzyme activity providing a proof of principle for the future use of chaperone therapeutics in the treatment of SFC

Permutation Entropy as a Conceptual Model to Analyse Brain Activity in Sleep

Sleep stage classification is a widely discussed topic, due to its importance in the diagnosis of sleep disorders, e.g. insomnia. Analysis of the brain activity during sleep is necessary to gain further insight into the processing that occurs in our brains. We want to use permutation entropy as a model for this analysis. Therefore, the signal processing in terms of electroencephalography is described. This results in a time discrete signal, that can be further processed by applying the method of permutation entropy, which is a modification of the Shannon entropy as a measure of information processing. The method is applied to 18 data sets, nine electroencephalography measurements of patients suffering from insomnia and nine of people without a sleep disorder. A strong correlation between the permutation entropy value and the sleep stages was found during the simulation runs. The results are analysed and presented using boxplot diagrams of the permutation entropy over the sleep stages. Furthermore, it is investigated that there is a steady decrease in the value when the patient is in a deeper sleep. This suggests that the method is a good parameter for sleep stage classification. Finally, we propose an extension of the conceptual model to other pathological conditions and also to the analysis of brain activity during surgery.Comment: 14 pages, 5 figures, included in abstract volume of the 11th EUROSIM Congress on Modelling and Simulatio

Evaluierung des flexiblen Lernbedarfs bei Studierenden – Implikationen für die Praxis

In der vorliegenden Evaluationsstudie wurden Studierende und Lehrende einer österreichischen Universität befragt, in welchen Bereichen der Lehre sich Studierende eine stärkere Flexibilisierung wünschen und inwieweit sich diese Wünsche von der Einschätzung Lehrender unterscheiden. Die Ergebnisse zeigen, dass Studierende und Lehrende in ihren Präferenzen zur Lehrveranstaltungsgestaltung zum großen Teil übereinstimmen, sich aber auch in einzelnen Bereichen unterscheiden. Insbesondere hinsichtlich einzelner Aspekte von Zeit, Inhalt, Lernressourcen und Support, Beurteilung und Bereitstellung schätzen Studierende Flexibilität bedeutsamer ein als Lehrende. Es zeigen sich hier bedeutsame statistische Effekte

Fairness als zentrale Herausforderung moderner Aufnahmeverfahren

In der Praxis wird zumeist angenommen, dass die Ergebnisse in einem Aufnahmeverfahren individuelle Unterschiede in den zu erfassenden Fähigkeiten fair messen. Der vorliegende Beitrag gibt einen Überblick über Modelle zur Fairness von Aufnahmeverfahren und deren Überprüfung in der Praxis anhand von Beispielen zum Aufnahmeverfahren für Medizinische Studiengänge in Österreich (MedAT). Der Beitrag arbeitet zudem auch den engen Zusammenhang zwischen Fairness und anderen Gütekriterien heraus und veranschaulicht die Vorteile einer stark theoriegeleiteten automatischen Itemgenerierung (AIG), um auf die erhöhten Qualitätsanforderungen bei modernen Aufnahmeverfahren reagieren zu können

Exploring the transition to the digital age in higher education teaching

The global Covid 19-pandemic led to many challenges for higher education (HE) teachers. However, it also provided  an opportunity to not only re-evaluate a previously  unquestioned teaching culture, but also to fundamentally  question the capabilities of HE institutions and teachers to  implement digitally supported education at the  organizational, teaching, and learning levels. In this study,  we analyzed data from 1,339 HE teachers’ conclusions for  post-pandemic teaching terms of instructional course  quality. Our findings shed light on HE teachers’ mindsets  toward traditional face-to-face and online teaching, as well  as (im)probable changes in academic teaching culture. Our  results offer a “bottom-up” basis for recommendations on  how institutions can support their faculty for digital  transformations in HE based on teachers’ expertise and  perceptions of the advantages and hindrances of HE online  teaching

Characterization of the Biosynthesis, Processing and Kinetic Mechanism of Action of the Enzyme Deficient in Mucopolysaccharidosis IIIC

Heparin acetyl-CoA:alpha-glucosaminide N-acetyltransferase (N-acetyltransferase, EC 2.3.1.78) is an integral lysosomal membrane protein containing 11 transmembrane domains, encoded by the HGSNAT gene. Deficiencies of N-acetyltransferase lead to mucopolysaccharidosis IIIC. We demonstrate that contrary to a previous report, the N-acetyltransferase signal peptide is co-translationally cleaved and that this event is required for its intracellular transport to the lysosome. While we confirm that the N-acetyltransferase precursor polypeptide is processed in the lysosome into a small amino-terminal alpha- and a larger ß- chain, we further characterize this event by identifying the mature amino-terminus of each chain. We also demonstrate this processing step(s) is not, as previously reported, needed to produce a functional transferase, i.e., the precursor is active. We next optimize the biochemical assay procedure so that it remains linear as N-acetyltransferase is purified or protein-extracts containing N-acetyltransferase are diluted, by the inclusion of negatively charged lipids. We then use this assay to demonstrate that the purified single N-acetyltransferase protein is both necessary and sufficient to express transferase activity, and that N-acetyltransferase functions as a monomer. Finally, the kinetic mechanism of action of purified N-acetyltransferase was evaluated and found to be a random sequential mechanism involving the formation of a ternary complex with its two substrates; i.e., N-acetyltransferase does not operate through a ping-pong mechanism as previously reported. We confirm this conclusion by demonstrating experimentally that no acetylated enzyme intermediate is formed during the reaction

A Drosophila-centric view of protein tyrosine phosphatases

AbstractMost of our knowledge on protein tyrosine phosphatases (PTPs) is derived from human pathologies and mouse knockout models. These models largely correlate well with human disease phenotypes, but can be ambiguous due to compensatory mechanisms introduced by paralogous genes. Here we present the analysis of the PTP complement of the fruit fly and the complementary view that PTP studies in Drosophila will accelerate our understanding of PTPs in physiological and pathological conditions. With only 44 PTP genes, Drosophila represents a streamlined version of the human complement. Our integrated analysis places the Drosophila PTPs into evolutionary and functional contexts, thereby providing a platform for the exploitation of the fly for PTP research and the transfer of knowledge onto other model systems