Biology subjects

Hew, B. E.

Publications and source records attributed to Hew, B. E..

3 recordsLinked to original sources

bioRxiv2026

The exocyst is an insulin-sensitive regulator of amyloid precursor protein trafficking and amyloid-beta generation in neurons

Intracellular trafficking of amyloid precursor protein (APP) critically influences amyloidogenic processing, yet the mechanisms regulating this pathway remain incompletely defined. The exocyst is a highly conserved, insulin-responsive, eight-protein Rab effector complex that directs intracellular transport vesicle targeting and docking. We identified APP in a proteomics screen of neuronal cell surface proteins altered after chemical inhibition of exocyst activity. In SH-SY5Y cells expressing a mutant APP that enhances amyloidogenic processing, RNAi-mediated silencing of exocyst subunits significantly decreased sAPP and A{beta} secretion, leading to significant intracellular APP accumulation. We found high-resolution co-localization of APP with exocyst subunits in soma and neurites of differentiated human SH-SY5Y neurons and mouse primary hippocampal neurons, and live-cell TIRF microscopy identified highly coordinated movement between fluorescently-tagged exocyst and APP proteins. These interactions were confirmed in these cells and in mouse brain histological sections by proximity ligation assays (PLAs) demonstrating close (<40nm) APP-EXOC5 association. To examine if exocyst activity in neurons is regulated by insulin, as it is in adipocytes and muscle, we generated a SH-SY5Y cell line with pHluorin-tagged GLUT4. Inhibition of the exocyst prevented exocytosis of GLUT4 to the plasma membrane in response to insulin. Additionally, using PLAs in mouse primary hippocampal neurons and SH-SY5Y neurons, we found that GLUT4-EXOC5 associations were increased by insulin signaling, but APP-EXOC5 associations were markedly reduced, indicating insulin-dependent retargeting of the exocyst complex away from APP+ vesicles towards GLUT+ vesicles. All together, these data identify the exocyst as a novel insulin-regulated mediator of neuronal APP trafficking and A{beta} secretion. In BriefWe show that the insulin-responsive exocyst regulates amyloidogenic processing of APP in neurons and that insulin signaling shifts the exocyst away from APP trafficking to promote the translocation of GLUT4-containing vesicles to the plasma membrane of neurons.

neuroscience↗

bioRxiv2025

Gene-sized DNA insertion at genomic safe harbors in human cells using a site-directed transposase

Achieving precise and efficient integration of gene-sized DNA sequences into the human genome remains a major obstacle to gene therapy. Existing approaches depend on double-strand DNA breaks, which can lead to unintended genome alterations. Many monogenic diseases arise from diverse patient-specific mutations, making individualized correction impractical and underscoring the need for universal full-gene replacement strategies. We developed INsertion by Targeted Anchoring and Conditional Transposition (INTACT) to enable targeted insertion at genomic safe harbor loci. We engineered a mammalian transposase with mutations in its DNA-binding domain to reduce off-target integration. Site specificity was then restored by linking programmable sequence-specific DNA-binding proteins to the transposase. Systematic optimization of INTACT revealed key determinants of precision, including non-covalent linkage between the transposase and DNA-binding protein, strict spacing between the binding site and the TTAA insertion sequence, and linkage of the DNA-binding protein to an internal position within the transposase. On-target insertion was achieved across multiple loci, with optimized INTACT averaging 1.2 targeted insertions per cell. An off-target assay confirmed that DNA-binding domain mutations substantially reduced unwanted integration events to near-background levels. Our site-directed transposase enables precise, efficient genomic insertion of >4kb DNA without double-strand breaks, offering a powerful new tool for genome engineering.

molecular biology↗

bioRxiv2024

Directed evolution of hyperactive integrases for site specific insertion of transgenes

The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites. By targeting att sites to the genome using technologies such as prime editing, integrases can target safe loci while avoiding double-strand breaks. We developed a method of phage-assisted continuous evolution we call IntePACE, that we used to rapidly perform hundreds of rounds of mutagenesis to systematically improve activity of PhiC31 and Bxb1 serine integrases. Novel hyperactive mutants were generated by combining synergistic mutations resulting in integration of a multi-gene cargo at rates as high as 80% of target chromosomes. Hyperactive integrases inserted a 15.7 kb therapeutic DNA cargo containing Von Willebrand Factor. This technology could accelerate gene delivery therapeutics and our directed evolution strategy can easily be adapted to improve novel integrases from nature. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/598370v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@438582org.highwire.dtl.DTLVardef@f242f9org.highwire.dtl.DTLVardef@10ae9eborg.highwire.dtl.DTLVardef@183543f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗