Biology subjects

Knutson, S. D.

Publications and source records attributed to Knutson, S. D..

4 recordsLinked to original sources

bioRxiv2025

Micromapping (μMap) of HER2 Across Human Breast Cancers: Photocatalytic Proximity Labeling Identifies Primary Resistance Mechanisms and Functional Interactors

Interactions among receptor tyrosine kinases (RTKs), particularly HER2, are critical in driving the growth of certain breast cancer subtypes. However, the mechanisms of HER2 dependency and resistance to targeted therapies remain unclear. Using photocatalytic micromapping ({micro}Map) proximity labeling, we profiled the HER2 interactome across human breast cancer lines. We identify galectin family proteins as uniquely enriched in trastuzumab-resistant models, and show that genetic and pharmacological inhibition of galectins restores trastuzumab sensitivity. Mechanistically, galectin inhibition destabilized HER2 and other RTKs and enhanced antibody-mediated receptor degradation. Galectin inhibition in combination with trastuzumab triggered mitochondrial and endoplasmic reticulum stress pathways, revealing new mechanisms underlying HER2 signaling, dependency, and resistance in breast cancer. We also identified protein tyrosine phosphatase F (PTPRF) as a pan-cancer HER2 interactor, which is broadly upregulated and whose knockdown suppresses proliferation in HER2-low cancers. This work provides an extensive new interactomic resource and underscores the utility of proximity labeling for mapping complex cancer networks and identifying new therapeutic targets.

cancer biology↗

bioRxiv2025

Temporal Microenvironment Mapping (microMap) of Intracellular Trafficking Pathways of Cell-Penetrating Peptides Across the Blood-Brain Barrier

Peptides play critical roles in cellular functions such as signaling and immune regulation, and peptide-based biotherapeutics show great promise for treating various diseases. Among these, cell-penetrating peptides (CPPs) are particularly valuable for drug delivery due to their ability to cross cell membranes. However, the mechanisms underlying CPP-mediated transport, especially across the blood-brain barrier (BBB), remain poorly understood. Mapping intracellular CPP pathways is essential for advancing drug delivery systems, particularly for neurological disorders, as understanding how CPPs navigate the complex environment of the BBB could enable the development of more effective brain-targeted therapies. Here, we leverage a nanoscale proximity labeling technique, termed {micro}Map, to precisely probe the peptide- receptor interactions and intracellular trafficking mechanisms of photocatalyst-tagged CPPs. The unique advantage of the Map platform lies in the ability to control the timing of light exposure, which enables the collection of time-gated data, depending on when the blue light is applied to the cells. By harnessing this spatiotemporal precision, we can uncover key peptide-receptor interactions and cellular processes, setting the stage for new innovations in drug design and brain-targeted therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/633151v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1585958org.highwire.dtl.DTLVardef@151b288org.highwire.dtl.DTLVardef@a03efeorg.highwire.dtl.DTLVardef@87d6a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Spatial visualization of A-to-I Editing in cells using Endonuclease V Immunostaining Assay (EndoVIA)

Adenosine-to-Inosine (A-to-I) editing is one of the most widespread post-transcriptional RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Varying across tissue types, A-to-I editing is essential for numerous biological functions and dysregulation leads to autoimmune and neurological disorders, as well as cancer. Recent evidence has also revealed a link between RNA localization and A-to-I editing, yet understanding of the mechanisms underlying this relationship and its biological impact remains limited. Current methods rely primarily on in vitro characterization of extracted RNA that ultimately erases subcellular localization and cell-to-cell heterogeneity. To address these challenges, we have repurposed Endonuclease V (EndoV), a magnesium dependent ribonuclease that cleaves inosine bases in edited RNA, to selectively bind and detect A-to-I edited RNA in cells. The work herein introduces Endonuclease V Immunostaining Assay (EndoVIA), a workflow that provides spatial visualization of edited transcripts, enables rapid quantification of overall inosine abundance, and maps the landscape of A-to-I editing within the transcriptome at the nanoscopic level.

molecular biology↗

bioRxiv2019

Selective enrichment of A-to-I edited transcripts from cellular RNA using Endonuclease V

Immunoprecipitation enrichment has significantly improved the sensitivity and accuracy of detecting RNA modifications in the transcriptome. However, there are no existing methods for selectively isolating adenosine-to-inosine (A-to-I) edited RNAs. Here we show that Escherichia coli Endonuclease V (eEndoV), an inosine-cleaving enzyme, can be repurposed to bind and isolate A-to-I edited transcripts from cellular RNA through adjustment of cationic conditions. While Mg2+ is required for eEndoV catalysis, it has also been shown that similar levels of Ca2+ instead promote binding of inosine without cleavage. Leveraging these properties, we observe that Ca2+-supplemented eEndoV is highly specific for inosine in RNA and exhibits low nanomolar binding affinity. We then demonstrate EndoVIPER (Endonuclease V inosine precipitation enrichment) as a facile and robust method to isolate A-to-I edited transcripts from cellular RNA. We envision the use of this approach as a straightforward and cost-effective strategy to enrich edited RNAs and detect A-to-I sites with improved sensitivity and fidelity.

molecular biology↗