Untersuchungen an einem zellfreien Proteinsynthesesystem basierend auf S30 Extrakten von Escherichia coli

Die zellfreie Proteinsynthese hat sich in den letzten Jahren zu einer wichtigen Methode der Proteinexpression entwickelt, die viele Vorteile gegenüber der in vivo Expression von Proteinen bietet. Ein Grund, warum die zellfreie Proteinsynthese die in vivo Expression noch nicht als Standardmethode zur Herstellung rekombinanter Proteine abgelöst hat, ist in der vergleichsweise geringen Produktivität dieser Systeme zu finden. Intensive Bemühungen der letzten Jahre konnten zwar die Ausbeute der zellfreien Proteinsynthese stark erhöhen, dennoch sind die erzielten Mengen noch weit von dem entfernt, was theoretisch möglich sein könnte. Aus diesem Grund wurden im Rahmen dieser Arbeit verschiedene Ansätze verfolgt, die Ausbeute der zellfreien Proteinsynthese, am Beispiel eines Systems, das auf S30 Extrakten von Escherichia coli basiert, zu erhöhen. Darüber hinaus sollte dieses System als Teil eines von der GENEART AG entwickelten in vitro Evolutionssystems eingesetzt werden. Zu Beginn wurde ein Protokoll zur Herstellung der S30 Extrakte und zur Durchführung von zellfreien Proteinsynthesereaktionen optimiert, um ein solides und modernes Testsystem für weitere Versuche zur Verfügung zu haben. Dabei konnte sowohl der Zellaufschluss durch Sonifikation, als auch die Präinkubationsmethode als kritische Einflussfaktoren der Syntheseleistung identifiziert werden. Auch zeigte sich die Verwendung eines RNase E-defizienten E. coli Stammes für die Herstellung der Extrakte, sowie die Erhöhung der Konzentration an DTT in der Synthesereaktion als förderlich für die erzielten Proteinausbeuten. Insgesamt führten die Optimierungen zu der Etablierung eines Systems, dessen Produktivität nun sowohl mit den Systemen anderer Autoren, als auch mit kommerziell erhältlichen vergleichbar ist. Das optimierte zellfreie Proteinsynthesesystem sollte als Teil eines in vitro Evolutionssystems eingesetzt werden, wobei unter Anwesenheit der S30 Extrakte die Produktbildung einer reversen Transkriptase, die essentiell für eine Amplifikationsreaktion dieses Systems war, inhibiert wurde. Intensive Untersuchungen führten zu dem Schluss, dass zu viele Proteine in den S30 Extrakten, wie beispielsweise RNasen und DNA Polymerasen, dafür verantwortlich sind. Der Einsatz isolierter Ribosomen unterschiedlicher Reinheit war nicht möglich, da Translationsaktivität und Produktbildung der reversen Transkriptase nicht vereinbar schienen. Im Zusammenhang der Erhöhung der Produktivität konnte in dieser Arbeit gezeigt werden, dass die Translationsmaschinerie in den S30 Extrakten optimal rekonstituiert ist. Dazu testete man alle Faktoren des Translationsapparates einzeln auf ihren Einfluss auf die Synthese eines Testproteins. Es wirkten sich jedoch nur die Elongationsfaktoren Tu und Ts positiv aus, wobei die Erhöhung der Syntheseleistung mit 13% bzw. 6% jedoch zu gering ausfiel, um von limitierenden Faktoren zu sprechen. Darüber hinaus wurden auch zellfreie Proteinsynthesereaktionen mit geringeren Konzentrationen an Translationsfaktoren durchgeführt. Jedoch konnte auch hier keine Steigerung der Syntheseleistung erzielt werden. Dies, zusammen mit den obigen Ergebnissen, erlaubt die Aussage der optimalen Rekonstitution der Translationsmaschinerie in S30 Extrakten von Escherichia coli. Desweiteren behandelte diese Arbeit das Problem der Sekundärstrukturbildung der mRNA, das mehrere Autoren als Grund für eine limitierte Proteinausbeute sehen. Durch den Einsatz der verschiedensten RNA Chaperone der unterschiedlichsten Klassen wurde versucht, auf die Translation inhibitorisch wirkende Sekundärstrukturen der mRNAs aufzulösen und diese Limitation zu überwinden, wodurch jedoch keine Steigerung der Syntheseleistung erreicht werden konnte. Die erstmalige Verwendung einer miRNA in einem zellfreien System, die durch Hybridisierung mit der 5‘ untranslatierten Region der mRNA regulatorische Sequenzen freilegen sollte, führte zu einer etwa 13%igen Steigerung der Syntheseleistung. Die größten Steigerungen der Syntheseleistung des zellfreien Systems aus E. coli erreichte man in dieser Arbeit durch die Erniedrigung der Reaktionstemperatur, wodurch unerwünschte Nebenreaktionen reduziert werden sollten. Dabei war der Einsatz der 5‘ untranslatierten Region eines Kälteschockgens nötig, um bei niedrigeren Temperaturen eine effiziente Proteinsynthese zu gewährleisten. Die Auswirkungen dieser 5‘ untranslatierten Region auf die zellfreie Expression mehrerer Proteine wurde getestet, wobei bei 60% sowohl eine erhöhte Ausbeute, beispielsweise 20% im Falle der Chloramphenicol Acetyltransferase, als auch eine einheitliche optimale Expressionstemperatur von 25°-30° C festgestellt werden konnte. Zusammenfassend konnten in dieser Arbeit mehrere vielversprechende Ansätze identifiziert werden, die Syntheseleisung von zellfreien Proteinsynthesesystemen aus E. coli zu verbessern, sowie einige bedeutende Hinweise zum besseren Verständnis dieser Methode geliefert werden

Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation

Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson’s Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson’s disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.Instituto de Investigaciones Bioquímicas de La Plat

Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons. A cross-ancestry genome-wide association meta-analysis of amyotrophic lateral sclerosis (ALS) including 29,612 patients with ALS and 122,656 controls identifies 15 risk loci with distinct genetic architectures and neuron-specific biology

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons. A cross-ancestry genome-wide association meta-analysis of amyotrophic lateral sclerosis (ALS) including 29,612 patients with ALS and 122,656 controls identifies 15 risk loci with distinct genetic architectures and neuron-specific biology

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology

A cross-ancestry genome-wide association meta-analysis of amyotrophic lateral sclerosis (ALS) including 29,612 patients with ALS and 122,656 controls identifies 15 risk loci with distinct genetic architectures and neuron-specific biology. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons.peer-reviewe

Downstream Effects of Mutations in <i>SOD1</i> and <i>TARDBP</i> Converge on Gene Expression Impairment in Patient-Derived Motor Neurons

Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disease marked by death of motor neurons (MNs) present in the spinal cord, brain stem and motor cortex. Despite extensive research, the reason for neurodegeneration is still not understood. To generate novel hypotheses of putative underlying molecular mechanisms, we used human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs) from SOD1- and TARDBP (TDP-43 protein)-mutant-ALS patients and healthy controls to perform high-throughput RNA-sequencing (RNA-Seq). An integrated bioinformatics approach was employed to identify differentially expressed genes (DEGs) and key pathways underlying these familial forms of the disease (fALS). In TDP43-ALS, we found dysregulation of transcripts encoding components of the transcriptional machinery and transcripts involved in splicing regulation were particularly affected. In contrast, less is known about the role of SOD1 in RNA metabolism in motor neurons. Here, we found that many transcripts relevant for mitochondrial function were specifically altered in SOD1-ALS, indicating that transcriptional signatures and expression patterns can vary significantly depending on the causal gene that is mutated. Surprisingly, however, we identified a clear downregulation of genes involved in protein translation in SOD1-ALS suggesting that ALS-causing SOD1 mutations shift cellular RNA abundance profiles to cause neural dysfunction. Altogether, we provided here an extensive profiling of mRNA expression in two ALS models at the cellular level, corroborating the major role of RNA metabolism and gene expression as a common pathomechanism in ALS

Commentary : Effects of ALS-associated TANK binding kinase 1 mutations on protein-protein interactions and kinase activity

A Commentary on Effects of ALS-associated TANK binding kinase 1 mutations on protein-protein interactions and kinase activity, by Ye, J., Cheung, J., Gerbino, V., Ahlsén, G., Zimanyi, C., Hirsh, D., et al. (2019). Proc. Natl. Acad. Sci. U.S.A. 116, 24517–24526. doi: 10.1073/pnas.1915732116</p

FUS mutations dominate TBK1 mutations in FUS/TBK1 double-mutant ALS/FTD pedigrees

Mutations in FUS and TBK1 often cause aggressive early-onset amyotrophic lateral sclerosis (ALS) or a late-onset ALS and/or frontotemporal dementia (FTD) phenotype, respectively. Co-occurrence of mutations in two or more Mendelian ALS/FTD genes has been repeatedly reported. However, little is known how two pathogenic ALS/FTD mutations in the same patient interact to shape the final phenotype. We screened 28 ALS patients with a known FUS mutation by whole-exome sequencing and targeted evaluation for mutations in other known ALS genes followed by genotype–phenotype correlation analysis of FUS/TBK1 double-mutant patients. We report on new and summarize previously published FUS and TBK1 double-mutant ALS/FTD patients and their families. We found that, within a family, mutations in FUS cause ALS while TBK1 single mutations are observed in FTD patients. FUS/TBK1 double mutations manifested as ALS and without a manifest difference regarding age at onset and disease duration when compared to FUS single-mutant individuals. In conclusion, TBK1 and FUS variants do not seem to interact in a simple additive way. Rather, the phenotype of FUS/TBK1 double-mutant patients appears to be dominated by the FUS mutation