Biology subjects

Imler, E.

Publications and source records attributed to Imler, E..

3 recordsLinked to original sources

bioRxiv2025

Discovery of a VHL molecular glue degrader of GEMIN3 by Picowell RNA-seq

Targeted protein degradation (TPD) is an emerging therapeutic modality in which small molecules are used to recruit targets to the natural protein degradation machinery of the cell. Molecular glue degraders (MGD) are monovalent small molecules that accomplish this by redirecting E3 ubiquitin ligases to target proteins, offering the potential to degrade previously unliganded and "undruggable" proteins in cancer, neurodegenerative, and other diseases. While attractive due to their drug-like properties, MGDs are exceptionally hard to discover and have largely been identified serendipitously. The Von Hippel-Lindau (VHL) E3 ligase is the second most widely used effector for TPD, though current VHL-based degraders are primarily large heterobifunctional PROTACs (proteolysis-targeting chimeras) designed using target-based ligands. Here, we have instead pursued target-agnostic discovery of VHL MGDs leveraging proprietary ultra-miniaturized microfluidics devices (Picowells) to facilitate unbiased RNA-seq screening of a biased E3-focused library. This resulted in dGEM3, a novel VHL molecular glue that targets the survival of motor neuron (SMN) complex member GEMIN3 for degradation. Through a combination of cellular, biochemical, and biophysical assays, we have characterized the GEMIN3 degron within its helicase ATP-binding domain, and how the kinetics of ternary complex formation impact degradation. These findings provide insights on the re-programmability of VHL for novel targets using drug-like molecular glues.

biochemistry↗

bioRxiv2019

A Drosophila model of neuronal ceroid lipofuscinosis CLN4 reveals a hypermorphic gain of function mechanism

The autosomal dominant neuronal ceroid lipofuscinoses (NCL) CLN4 is caused by mutations in the synaptic vesicle (SV) protein CSP, which is a critical co-chaperone of Hsc70 protecting synapses from activity-dependent degeneration. We developed the first animal models of CLN4 by expressing either CLN4 mutant human CSP (hCSP) or Drosophila CSP (dCSP) in fly neurons. Similar to patients, CLN4 mutations induced excessive oligomerization of mutant hCSP and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most CLN4 mutant hCSP abnormally accumulated in axons and somata, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructurally, abnormal multi-laminar membrane structures were frequently observed in axons and somata next to degenerative abnormalities. The lethality, oligomerization and prelysosomal accumulation induced by CLN4 mutations was attenuated by reducing wild type (WT) dCSP levels and enhanced by increasing WT dCSP or hCSP levels, which indicates that both CLN4 alleles resemble dominant hypermorphic gain of function mutations. Furthermore, reducing the gene dosage of Hsc70 also attenuated CLN4 phenotypes. Taken together, we suggest that CLN4 alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking.

neuroscience↗

bioRxiv2018

Extraction-Free Whole Transcriptome Gene Expression Analysis of FFPE Sections and Histology-Directed Subareas of Tissue

We describe the use of a ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq, to profile formalin-fixed paraffin-embedded (FFPE) tissue, including H&E stained FFPE tissue, by directly lysing tissue scraped from slides without extracting RNA or converting the RNA to cDNA. The correlation of measured gene expression changes in unfixed and fixed samples using blocks prepared from a pellet of a single cell type was R2 = 0.97, demonstrating that no significant artifacts were introduced by fixation. Fixed and fresh samples prepared in an equivalent manner produced comparable sequencing depth results (+/-20%), with similar %CV (11.5 and 12.7%, respectively), indicating no significant loss of measurable RNA due to fixation. The sensitivity of the TempO-Seq assay was the same whether the tissue section was fixed or not. The assay performance was equivalent for human, mouse, or rat whole transcriptome. The results from 10 mm2 and 2 mm2 areas of tissue obtained from 5 m thick sections were equivalent, thus demonstrating high sensitivity and ability to profile focal areas of histology within a section. Replicate reproducibility of separate areas of tissue ranged from R2 = 0.83 (lung) to 0.96 (liver) depending on the tissue type, with an average correlation of R2 = 0.90 across nine tissue types. The average %CVs were 16.8% for genes expressed at greater than 200 counts, and 20.3% for genes greater than 50 counts. Tissue specific differences in gene expression were identified and agreed with the literature. There was negligible impact on assay performance using FFPE tissues that had been archived for up to 30 years. Similarly, there was negligible impact of H&E staining, facilitating accurate visualization for scraping and assay of small focal areas of specific histology within a section.

genomics↗