Biology subjects

Bowen, C. A.

Publications and source records attributed to Bowen, C. A..

3 recordsLinked to original sources

bioRxiv2025

Proximity labeling of the Tau repeat domain enriches RNA-binding proteins that are altered in Alzheimer's disease and related tauopathies

In Alzheimers disease (AD) and other tauopathies, tau dissociates from microtubules and forms toxic aggregates that contribute to neurodegeneration. Although some of the pathological interactions of tau have been identified from postmortem brain tissue, these studies are limited by their inability to capture transient interactions. To investigate the interactome of aggregate-prone fragments of tau, we applied an in vitro proximity labeling technique using split TurboID biotin ligase (sTurbo) fused with the tau microtubule repeat domain (TauRD), a core region implicated in tau aggregation. We characterized sTurbo TauRD co-expression, robust enzyme activity and nuclear and cytoplasmic localization in a human cell line. Following enrichment of biotinylated proteins and mass spectrometry, we identified over 700 TauRD interactors. Gene ontology analysis of enriched TauRD interactors highlighted processes often dysregulated in tauopathies, including spliceosome complexes, RNA-binding proteins (RBPs), and nuclear speckles. The disease relevance of these interactors was supported by integrating recombinant TauRD interactome data with human AD tau interactome datasets and protein co-expression networks from individuals with AD and related tauopathies. This revealed an overlap with the TauRD interactome and several modules enriched with RBPs and increased in AD and Progressive Supranuclear Palsy (PSP). These findings emphasize the importance of nuclear pathways in tau pathology, such as RNA splicing and nuclear-cytoplasmic transport and establish the sTurbo TauRD system as a valuable tool for exploring the tau interactome.

neuroscience↗

bioRxiv2025

Identification of novel Kv1.3 channel-interacting proteins using proximity labelling in T-cells

Potassium channels regulate membrane potential, calcium flux, cellular activation and effector functions of adaptive and innate immune cells. The voltage-activated Kv1.3 channel is an important regulator of T cell-mediated autoimmunity and microglia-mediated neuroinflammation. Kv1.3 channels, via protein-protein interactions, are localized with key immune proteins and pathways, enabling functional coupling between K+ efflux and immune mechanisms. To gain insights into proteins and pathways that interact with Kv1.3 channels, we applied a proximity-labeling proteomics approach to characterize protein interactors of the Kv1.3 channel in activated T-cells. Biotin ligase TurboID was fused to either N or C termini of Kv1.3, stably expressed in Jurkat T cells and biotinylated proteins in proximity to Kv1.3 were enriched and quantified by mass spectrometry. We identified over 1,800 Kv1.3 interactors including known interactors (beta-integrins, Stat1) although majority were novel. We found that the N-terminus of Kv1.3 preferentially interacts with protein synthesis and protein trafficking machinery, while the C-terminus interacts with immune signaling and cell junction proteins. T- cell Kv1.3 interactors included 335 cell surface, T-cell receptor complex, mitochondrial, calcium and cytokine-mediated signaling pathway and lymphocyte migration proteins. 178 Kv1.3 interactors in T-cells also represent genetic risk factors of T cell-mediated autoimmunity, including STIM1, which was further validated using co-immunoprecipitation. Our studies reveal novel proteins and molecular pathways that interact with Kv1.3 channels in adaptive (T-cell) and innate immune (microglia), providing a foundation for how Kv1.3 channels may regulate immune mechanisms in autoimmune and neurological diseases.

cell biology↗

bioRxiv2024

Proximity labeling proteomics reveals Kv1.3 potassium channel immune interactors in microglia.

Microglia are the resident immune cells of the brain and regulate the brains inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a state referred to as disease associated microglia (DAM). DAM express higher levels of proinflammatory signaling, like STAT1 and TLR2, and show transitions in mitochondrial activity toward a more glycolytic response. Inhibition of Kv1.3 decreases the proinflammatory signature of DAM, though how Kv1.3 influences the response is unknown. Our goal was to establish the potential proteins interacting with Kv1.3 during the TLR4-mendiated transition to DAM. We utilized TurboID, a biotin ligase, fused to Kv1.3 to evaluate the potential interacting proteins with Kv1.3 via mass spectrometry in BV-2 microglia during an immune response. Electrophysiology, western blots, and flow cytometry were used to evaluate Kv1.3 channel presence and TurboID biotinylation activity. We hypothesized that Kv1.3 contains domain-specific interactors that vary during an TLR4-induced inflammatory response, some of which are dependent on the PDZ-binding domain on the C-terminus. We determined that the N-terminus of Kv1.3 is responsible for trafficking Kv1.3 to the cell surface and mitochondria (e.g. NUNDC, TIMM50). The C-terminus interacts with immune signaling proteins in an LPS-induced inflammatory response (e.g. STAT1, TLR2, and C3). There are 70 proteins that rely on the c-terminal PDZ-binding domain to interact with Kv1.3 (i.e. ND3, Snx3, and Sun1). Overall, we highlight that the Kv1.3 potassium channel functions beyond outward flux of potassium in an inflammatory context and contributes to activity of key immune signaling proteins, such as STAT1 and C3. MAIN POINTSKv1.3 channels are highly abundant in pro-inflammatory microglia in neurological diseases. Kv1.3 channels may regulate microglial functions by interacting with other proteins via its N and C terminal domains. Using proximity-based proteomics, we identified several novel proteins that interact with the N and C terminus of Kv1.3 channels, some of which are domain-specific. Kv1.3 channels in microglia interact with many immune signaling proteins, including Tlr2, Stat1 and integrins. Under homeostatic conditions, the N-terminus of Kv1.3 interacts with proteins involved in protein trafficking, to the cell surface and mitochondria. The PDZ-binding region was an important determinant of the C terminal interactome. During an LPS-induced inflammatory response, the C-terminus of Kv1.3 uniquely interacts with immune and signaling proteins of disease relevance, including STAT1

neuroscience↗