Sabu, M. | Search bioRxiv

bioRxiv2023

Beta-catenin-mediated activation of Wnt target genes utilizes a biomolecular condensate-dependent mechanism

The Wnt/{beta}-catenin signaling pathway plays numerous, essential roles in animal development and tissue/stem cell maintenance. The activation of genes regulated by Wnt/{beta}-catenin signaling requires the nuclear accumulation of {beta}-catenin, a transcriptional co-activator. {beta}-catenin is recruited to many Wnt-regulated enhancers through direct binding to T-cell factor/Lymphoid enhancer factor (TCF/LEF) family transcription factors. {beta}-catenin has previously been reported to form phase-separated biomolecular condensates (BMCs), which was implicated as a component of {beta}-catenins mechanism of action. This function required aromatic amino acid residues in the intrinsically disordered regions (IDRs) at the N- and C-termini of the protein. In this report, we further explore a role for {beta}-catenin BMCs in Wnt target gene regulation. We find that {beta}-catenin BMCs are miscible with LEF1 BMCs in vitro. We characterized a panel of {beta}-catenin mutants with different combinations of aromatic residue mutations in human cell culture and Drosophila melanogaster. Our data support a model in which aromatic residues across both IDRs contribute to BMC formation in vitro and signaling activity in vivo. Although different Wnt targets have different sensitivities to loss of {beta}-catenins aromatic residues, the activation of every target examined was compromised by aromatic substitution. These mutants are not defective in nuclear import, and residues in the N-terminal IDR with no previously known role in signaling are clearly required for the activation of various Wnt readouts. Consistent with this, deletion of the N-terminal IDR results in a loss of signaling activity, which can be rescued by the addition of heterologous IDRs enriched in aromatic residues. Overall, our work supports a model in which the ability of {beta}-catenin to form biomolecular condensates in the nucleus is tightly linked to its function as a transcriptional co-regulator.

molecular biology↗

bioRxiv2022

ApoE isoforms differentially regulate neuronal membrane proteasomes to shift the threshold for pathological aggregation of endogenous Tau

Neuroproteasomes are a subset of 20S proteasomes that are localized to the neuronal plasma membrane and degrade newly synthesized proteins. To date, the molecular composition of neuroproteasomes is undefined, and moreover, whether neuroproteasomes can influence protein aggregation with relevance to neurodegenerative disorders remains unexplored. Using a Cre-dependent conditional knock-in mouse line to endogenously tag the proteasome, we find that neuroproteasomes co-purify with ApoE, the most significant risk factor for late-onset Alzheimers Disease (AD). We discover that neuroproteasome membrane localization is differentially modulated by ApoE isoforms (E4<E3<E2) in vitro, in vivo, and in human postmortem samples. We synthesized selective, neuroproteasome-specific inhibitors and discovered that neuroproteasome inhibition induces aggregation of endogenous mouse and human Tau, without the need for seeding or pathogenic mutations. Using hApoE-KI/hTau-KI crosses, we find that ApoE isoforms differentially shift the aggregation threshold for Tau. Neuroproteasome inhibition in vivo is sufficient to induce sarkosyl-insoluble and Thioflavin-S positive endogenous Tau aggregates in only three days, which are completely abrogated by co-application of cycloheximide. Newly synthesized Tau levels increase threefold after neuroproteasome inhibition, leading us to posit that newly synthesized Tau is uniquely susceptible to aggregation due to neuroproteasome dysfunction. Overall, our data define neuroproteasomes as a pivotal proteostasis mechanism underlying the formation of endogenous Tau aggregates, which is directly regulated by the largest genetic risk factor for late-onset Alzheimers Disease.

neuroscience↗