Sumoylation inhibits α-synuclein aggregation and toxicity

Sumoylation of α-synuclein decreases its rate of aggregation and its deleterious effects in vitro and in vivo

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

SUMOylierung verändert die Toxizität und Fibrillenbildung von α-Synuklein

SUMOylierung ist eine posttranslationale Modifikation, bei der in einem hochdynamischen Prozess das kleine ubiquitinähnliche Protein SUMO (small ubiquitin-like modifier protein) kovalent an ein Substrat gebunden wird und dessen spezifische Eigenschaften wie die Aktivität, strukturelle Konformation oder Lokalisation verändert. Posttranslationale Modifikationen wie die SUMOylierung sind essentiell für Signaltransduktionswege im zentralen Nervensystem (ZNS). Ihre präzise Kontrolle ist von zentraler Bedeutung für die Lebensfähigkeit, Funktion und Konnektivität von Neuronen.In den letzten Jahren wurden einige neuronspezifische SUMO-Substrate entdeckt, wobei sehr wenig über die Rolle der SUMOylierung bei neuronalem Zelltod und Überleben bekannt ist. Wir zeigen in vitro und in verschiedenen Zelllinien, dass alpha-Synuklein von SUMO1 und SUMO2 kovalent gebunden wird. Darüber hinaus konnten wir in vivo SUMOyliertes alpha-Synuklein im Gehirn von Mäusen nachweisen.Die massenspektrometrische Analyse von in vitro SUMOyliertem alpha-Synuklein ergab, dass 11 Lysinreste als SUMO-Bindungsstellen dienen. In detaillierteren Mutagenese-Experimenten wurden K96 und K102 als maßgebliche alternative SUMOylierungsstellen in alpha-Synuklein identifiziert.In dieser Arbeit wird gezeigt, dass die kovalente Bindung von SUMO die alpha-Synuklein-induzierte Toxizität und Aggregation reguliert. Die SUMO-Bindung an alpha-Synuklein führte in vitro zur Inhibierung der Amyloid-Fibrillenbildung. Die Mutation der zwei maßgeblich SUMOylierten Lysinreste verschärfte die alpha-Synuklein-induzierte Zytotoxizität im Parkinsonmodel der Ratte. Dieses Ergebnis demonstriert, dass die endogene SUMOylierung an den Akzeptorstellen K96/K102 von alpha-Synuklein dessen Neurotoxizität auf dopaminerge Neuronen in der SNpc in vivo umfassend reduziert.In Übereinstimmung mit den in vivo im Parkinsonmodel erhaltenen Ergebnissen zeigten in vitro-Versuche in HEK-Zellen, dass die Reduzierung von SUMOyliertem alpha-Synuklein zu einer erhöhten Aggregationsbildung und Zelltodrate führte.Die vorliegende Arbeit präsentiert die SUMOylierung als neuartigen Faktor bei der Modulation der für Parkinson relevanten charakteristischen Eigenschaften von alpha-Synuklein, der Neurotoxizität und Fibrillenbildung, und weist auf eine mögliche krankheitsrelevante Funktion der SUMOylierung im ZNS hin

Chemical genetic approach identifies microtubule affinity-regulating kinase 1 as a leucine-rich repeat kinase 2 substrate

Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are the most common cause of autosomal dominant forms of Parkinson’s disease. LRRK2 is a modular, multi-domain protein containing two enzymatic domains, including a kinase domain, as well as several protein-protein interaction domains, pointing to a role in cellular signaling, however knowledge of LRRK2’s upstream regulators and downstream effectors is incomplete. In this study, we used a chemical genetics approach to identify LRRK2 substrates from mouse brain. To this end, we first generated a catalytically active LRRK2 protein capable of employing bulky bio-orthogonal ATPγS analogues (M1947A/G2019S). This engineered, bioactive and functional LRRK2 kinase was used to thiophosphorylate putative substrates from brain extracts of LRRK2 KO mice. Isolation of thiophosphorylated peptides led to the identification of putative LRRK2 substrates. Several of these are involved in the regulation of microtubule (MT) dynamics including MAP/Microtubule Affinity-Regulating Kinase 1 (MARK1). MARK1 is a serine/threonine kinase known to phosphorylate microtubule-binding proteins such as Tau, MAP2 and MAP4 at KXGS motifs leading to MT destabilization. In vitro kinase assays and metabolic labelling experiments in living cells confirmed MARK1 as a LRRK2 substrate. In addition, we show that LRRK2 and MARK1 are interacting in eukaryotic cells. Taken together, we used a chemical genetic approach to identify LRRK2 substrates in the context of a complex cellular environment and have identified and validated MARK1 as a substrate. Our findings contribute to the identification of physiological LRRK2 substrates, and point to a potential mechanism explaining the reported effects of LRRK2 on neurite morpholog

Chemical genetic approach identifies microtubule affinity-regulating kinase 1 as a leucine-rich repeat kinase 2 substrate

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant forms of Parkinson's disease. LRRK2 is a modular, multidomain protein containing 2 enzymatic domains, including a kinase domain, as well as several protein-protein interaction domains, pointing to a role in cellular signaling. Although enormous efforts have been made, the exact pathophysiologic mechanisms of LRRK2 are still not completely known. In this study, we used a chemical genetics approach to identify LRRK2 substrates from mouse brain. This approach allows the identification of substrates of 1 particular kinase in a complex cellular environment. Several of the identified peptides are involved in the regulation of microtubule (MT) dynamics, including microtubule-associating protein (MAP)/microtubule affinity-regulating kinase 1 (MARK1). MARK1 is a serine/threonine kinase known to phosphorylate MT-binding proteins such as Tau, MAP2, and MAP4 at KXGS motifs leading to MT destabilization. In vitro kinase assays and metabolic-labeling experiments in living cells confirmed MARK1 as an LRRK2 substrate. Moreover, we also showed that LRRK2 and MARK1 are interacting in eukaryotic cells. Our findings contribute to the identification of physiologic LRRK2 substrates and point to a potential mechanism explaining the reported effects of LRRK2 on neurite morphology.status: publishe

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.peerReviewe