Protein-protein interactions in human pluripotent stem cell-derived neural stem cells and their neuronal progeny

Abstract

While most approaches in cell-based disease modeling are focused on the effects of defined mutations on the molecular or cellular phenotype, the assessment of underlying alterations in the interactomes of disease-relevant proteins has faced several technical challenges. First, experiments were typically conducted using overexpression paradigms resulting in unphysiologically high protein levels and thus promoting unspecific interactions. Second, such studies have been relying mostly on transformed cell lines, which enable mass production of transgenic cells but do not exhibit a tissue-specific proteomic environment. For that reason, the present study aimed at addressing these issues by bacterial artificial chromosome (BAC)-based expression of tagged proteins in pluripotent stem cell-derived long-term neuroepithelial like stem cells (lt-NES cells), a stable and robust cell population, which generates authentic human neurons with high fidelity. Tagged proteins were found to be expressed at endogenous levels, and fluorescence in situ hybridisation (FISH) analyses revealed an average integration rate of one copy per genome for the majority of cell lines analyzed. Correct compartmentalization and size of the tagged proteins could be confirmed by high-resolution confocal and live cell imaging as well as Western immunoblotting analysis, respectively. Employing this approach, multiple cell lines were generated harboring tagged proteins associated with human developmental disorders, cancer and neurodegeneration. Representatives of these groups include Proliferating Cell Nuclear Antigen (PCNA), Aurora Kinase A (AURKA), Cyclin-Dependent Kinase 2-Associated Protein 1 (CDK2AP1), Set Domain-Containing Protein 1B (SETD1B), RuvB-Like 2 (RUVBL2), the Methyl CpG Binding Protein 2 (MECP2) and the Alzheimer’s disease-associated proteins Nicastrin (NCSTN) and Valosin-Containing Protein (VCP). Using a label-free, quantitative affinity purification-mass spectrometry approach, numerous novel interaction partner candidates of these proteins were identified. Direct comparison of protein-specific interactomes of proliferating lt-NES cells and their neuronal progeny further revealed changes in the composition of several chromatin-remodeling complexes, suggesting that this system is sufficiently sensitive and specific to identify the dynamic differential recruitment of individual proteins as a response to developmental switches. In a proof-of-concept study, the approach of BAC-mediated expression of tagged proteins with a subsequent analysis of interacting proteins was successfully transferred to induced pluripotent stem cell (iPS)-derived lt-NES cells in order to enable PPI analyses in the context of complex diseases in following studies.<br /> Finally, an adeno-associated virus based approach for epitope tagging of endogenous genes in iPS-derived lt-NES cells from a patient suffering from Machado-Joseph disease allowed the generation of cell pools exhibiting both the diseased and healthy isoform of N-terminal FLAG-tagged Ataxin-3. The present work demonstrates a successful establishment of two different methods for protein tagging in somatic cell populations that subsequently can be employed for a multitude of analytical techniques including fluorescent microscopic visualization of protein localization, dynamics of protein recruitment or the detection of PPI.