Several neurodevelopmental disorders have been linked to neuronal hyperexcitability due to chloride (Cl-) imbalance in neurons. The K+-Cl- cotransporter 2 (KCC2) is required for Cl- homeostasis maintenance but its dysregulation is involved in the pathogenesis of multiple diseases. To date, there are no effective pharmacological treatments targeting KCC2, however the identification of key KCC2-interacting protein partners suggests a new opportunity to regulate neuronal excitability through complexes stabilization. This work aims at biochemically and structurally characterize KCC2 in complex with its partners: the neuropilin and tolloid like-2 protein (NETO2), required for KCC2 expression and localization, and the kainate-sensitive glutamate receptor (GluK2), which positively regulates KCC2 expression and functionality. The atomic-level description of the interactome will allow a mechanistic understanding of neuronal GABAergic inhibition, besides key protein-protein interfaces (PPIs) identification offering potential mechanisms for complex stabilization and for proper KCC2 abundance in neurons. In this work for the first time, we reconstitute the KCC2-NETO2 and KCC2-GluK2 complexes in vitro. The selection of the best expression constructs for recombinant protein production and purification is done by DNA:PEI (polyethylenimine) small-scale transient transfections of suspension human embryonic kidney (HEK293F) cell lines analyzed through fluorescent size exclusion chromatography and western blot. The selected DNA constructs are used to produce recombinant bacmids with which Spodoptera frugiperda insect cell line (Sf9) is transfected to generate recombinant baculoviruses used to coinfect HEK293F cells with greater efficiency. After evaluating the conditions for optimal viral infection efficiency, pull-down assays are done to validate KCC2-NETO2 and KCC2-GluK2 complexes stability during the purification steps. Lastly, KCC2-NETO2 copurification tests allowed to identify the best pipeline to isolate the complex. However, increasing purified protein yields is necessary for further characterization experiments. To achieve this, a new approach will use a multicistronic vector with a self-cleaving 2A peptide between the genes of interest to ensure a better multigene delivery, eventually boosting protein production yields. Besides, a preliminary description of dimeric KCC2 in complex with two NETO2 subunits has been obtained thanks to accurate protein structure prediction through AlphaFold3 server. The NETO2 residues involved in the predicted interaction with KCC2 and in the experimentally determined interaction with GluK2 are almost the same, suggesting their regulatory role. Concurrently, TWIK-related acid sensitive potassium channel (TASK-3) is responsible for leak potassium (K+) currents in neurons and its regulation depends on the interaction with KCC2, thereby alterations influence neuronal excitability. To date, the atomic-level understanding of TASK-3 architecture is lacking, limiting the molecular comprehension of neuronal hyperexcitability in pathological conditions and the development of therapies targeting KCC2-TASK-3 PPIs to restore the resting membrane potential. Here, the selected TASK-3 construct is expressed in Pichia pastoris yeast cultures and further purified in lipid nanodiscs and with its antibody Fab fragment. Purified samples are used to prepare grids for cryo-electron microscopy data acquisition followed by micrographs 2D classification. However, to define the atomic-level architecture of the channel, a more potent cryo-electron microscopy is needed to obtain more detailed micrographs for developing a reliable TASK-3 structural model. These results will provide atomic-based insights into neuronal hyperexcitability understanding and will set the basis for TASK-3-KCC2 complex architecture determination.
Diversi disturbi del neurosviluppo sono collegati all’ipereccitabilità neuronale dovuta allo squilibrio del cloro (Cl-) nei neuroni. Il cotrasportatore K+-Cl- (KCC2) è essenziale per l’omeostasi del Cl-, ma la sua deregolazione è coinvolta nella patogenesi di diverse malattie. Ad oggi mancano trattamenti farmacologici mirati al KCC2; tuttavia, identificare i partner proteici di KCC2 offre l'opportunità di regolare l'eccitabilità neuronale stabilizzando il complesso macromolecolare. Questo lavoro mira a una caratterizzazione biochimica e strutturale del cotrasportatore KCC2 in complesso con i suoi partner: la proteina neuropilina e tolloid-like-2 (NETO2), necessaria per la regolazione di KCC2, e il recettore del glutammato sensibile al kainato (GluK2), che regola positivamente l’espressione e la funzionalità di KCC2. La caratterizzazione molecolare dell’interattoma fornirà una comprensione meccanicistica dell'inibizione GABAergica e identificherà le principali interfacce proteina-proteina (PPIs) che offrono un potenziale meccanismo per stabilizzare i complessi e garantire la presenza di KCC2 nei neuroni. In questo lavoro per la prima volta abbiamo ricostituito i complessi proteici ricombinanti KCC2-NETO2 e KCC2-GluK2 in vitro. La selezione dei migliori costrutti genici di espressione avviene tramite trasfezioni transitorie su piccola scala con DNA:PEI (polietilenimmina) della linea cellulare in sospensione derivante da rene embrionale umano (HEK293F) e analizzate con cromatografia ad esclusione molecolare fluorescente e western blot. I selezionati sono usati per produrre il bacmide ricombinante con cui la linea cellulare di insetto Spodoptera frugiperda (Sf9) è stata trasfettata per generare un baculovirus ricombinante usato per coinfettare le cellule HEK293F con maggiore efficienza. Dopo l’ottimizzazione dell’infezione virale, si eseguono test di pull-down per validare la stabilità dei complessi durante la purificazione. Infine, test di copurificazione di KCC2-NETO2 hanno permesso di identificare la migliore procedura per isolare il complesso. Tuttavia, occorre aumentare la resa della purificazione per i successivi esperimenti di caratterizzazione. A questo scopo, un vettore multicistronico con un peptide 2A auto-scindente tra i geni di interesse sarà usato per garantire un migliore trasferimento multigenico, aumentando così i rendimenti di produzione. Inoltre, una descrizione preliminare del dimero di KCC2 in complesso con due subunità di NETO2 è stata ottenuta grazie ad un'accurata previsione della struttura proteica tramite il server AlphaFold3. I residui di NETO2 coinvolti nell'interazione prevista con KCC2 e nell'interazione determinata sperimentalmente con GluK2 sono quasi sempre gli stessi, evidenziando il loro ruolo regolatorio. Parallelamente, il canale del potassio (K+) sensibile all'acido correlato a TWIK (TASK-3) regola le correnti di perdita di K+ nei neuroni e la sua interazione con KCC2 influenza l'eccitabilità neuronale. Ad oggi, manca la descrizione dell'architettura di TASK-3, e ciò limita la comprensione molecolare dell'ipereccitabilità neuronale patologica e lo sviluppo di terapie mirate alle PPIs di KCC2-TASK-3 utili a ripristinare il potenziale di membrana a riposo. Il costrutto TASK-3 selezionato è espresso in colture di lievito di Pichia pastoris e purificato in nanodiscs lipidici e con il frammento Fab del suo anticorpo. I campioni purificati sono utilizzati per preparare le grids per l'acquisizione dei dati al microscopio crioelettronico seguita dalla classificazione 2D delle micrografie. Tuttavia, per definire l'architettura a livello atomico del canale, è necessario un microscopio più potente per ottenere micrografie più dettagliate che consentano lo sviluppo di un corretto modello strutturale di TASK-3. Questi risultati forniranno una comprensione molecolare dell'ipereccitabilità neuronale ponendo le basi per la definizione dell'architettura di TASK-3-KCC2.
Approcci biochimici e strutturali per la caratterizzazione del trasportatore neuronale KCC2 e dei suoi partner proteici
GUZZELONI, SOFIA DILETTA
2023/2024
Abstract
Several neurodevelopmental disorders have been linked to neuronal hyperexcitability due to chloride (Cl-) imbalance in neurons. The K+-Cl- cotransporter 2 (KCC2) is required for Cl- homeostasis maintenance but its dysregulation is involved in the pathogenesis of multiple diseases. To date, there are no effective pharmacological treatments targeting KCC2, however the identification of key KCC2-interacting protein partners suggests a new opportunity to regulate neuronal excitability through complexes stabilization. This work aims at biochemically and structurally characterize KCC2 in complex with its partners: the neuropilin and tolloid like-2 protein (NETO2), required for KCC2 expression and localization, and the kainate-sensitive glutamate receptor (GluK2), which positively regulates KCC2 expression and functionality. The atomic-level description of the interactome will allow a mechanistic understanding of neuronal GABAergic inhibition, besides key protein-protein interfaces (PPIs) identification offering potential mechanisms for complex stabilization and for proper KCC2 abundance in neurons. In this work for the first time, we reconstitute the KCC2-NETO2 and KCC2-GluK2 complexes in vitro. The selection of the best expression constructs for recombinant protein production and purification is done by DNA:PEI (polyethylenimine) small-scale transient transfections of suspension human embryonic kidney (HEK293F) cell lines analyzed through fluorescent size exclusion chromatography and western blot. The selected DNA constructs are used to produce recombinant bacmids with which Spodoptera frugiperda insect cell line (Sf9) is transfected to generate recombinant baculoviruses used to coinfect HEK293F cells with greater efficiency. After evaluating the conditions for optimal viral infection efficiency, pull-down assays are done to validate KCC2-NETO2 and KCC2-GluK2 complexes stability during the purification steps. Lastly, KCC2-NETO2 copurification tests allowed to identify the best pipeline to isolate the complex. However, increasing purified protein yields is necessary for further characterization experiments. To achieve this, a new approach will use a multicistronic vector with a self-cleaving 2A peptide between the genes of interest to ensure a better multigene delivery, eventually boosting protein production yields. Besides, a preliminary description of dimeric KCC2 in complex with two NETO2 subunits has been obtained thanks to accurate protein structure prediction through AlphaFold3 server. The NETO2 residues involved in the predicted interaction with KCC2 and in the experimentally determined interaction with GluK2 are almost the same, suggesting their regulatory role. Concurrently, TWIK-related acid sensitive potassium channel (TASK-3) is responsible for leak potassium (K+) currents in neurons and its regulation depends on the interaction with KCC2, thereby alterations influence neuronal excitability. To date, the atomic-level understanding of TASK-3 architecture is lacking, limiting the molecular comprehension of neuronal hyperexcitability in pathological conditions and the development of therapies targeting KCC2-TASK-3 PPIs to restore the resting membrane potential. Here, the selected TASK-3 construct is expressed in Pichia pastoris yeast cultures and further purified in lipid nanodiscs and with its antibody Fab fragment. Purified samples are used to prepare grids for cryo-electron microscopy data acquisition followed by micrographs 2D classification. However, to define the atomic-level architecture of the channel, a more potent cryo-electron microscopy is needed to obtain more detailed micrographs for developing a reliable TASK-3 structural model. These results will provide atomic-based insights into neuronal hyperexcitability understanding and will set the basis for TASK-3-KCC2 complex architecture determination.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/28274