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Isabella is a rising senior studying chemistry with a concentration in biochemistry at the New Mexico Institute of Mining and Technology.

Protein ubiquitylation is a critical regulatory mechanism that governs processes such as proteasomal degradation, signal transduction, and cell cycle control. Disruptions in ubiquitylation pathways contribute to diverse diseases, including many cancers.

The BSRP program was truly a transformative experience for me. I felt deeply supported and believed in. Despite my fears for the future, the people running the program, my mentor, the cohort, and everyone involved made me feel brave enough to take a leap of faith into my dream of being part of the scientific world. I can’t imagine a more life-changing program.Although the human genome encodes over 600 E3 ubiquitin ligases, the specific substrates of most remain unknown. To overcome this challenge, the Sellers lab developed a ubiquitin-specific proximity labeling method named E3-Substrate Tagging by Ubiquitin Biotinylation (E-STUB) that selectively biotinylates substrates ubiquitylated near a specific E3 ligase, allowing robust identification of E3-substrate pairs. To enable E-STUB studies of the Cullin 4-DDB1 (CRL4) E3 ligases, a family of ligase implicated in various diseases and targeted protein degradation, I engineered and validated DCAF mutants that lack the ability to bind DDB1, thereby disrupting the formation of the E3 ligase complex and eliminating its ability to recruit substrates for ubiquitylation. These mutants will serve as critical controls to distinguish specific substrate labeling from background signal in E-STUB assays. My work integrates computational modeling of DCAF-DDB1 interactions using AlphaFold3 to identify binding interfaces, construct engineering via Gibson Assembly, and assessment of DCAF-DDB1 interactions through co-immunoprecipitation and Western blotting. By establishing these critical controls, this project will enable E-STUB studies of the CRL4 substrate networks. Ultimately, these insights may support the development of therapeutic strategies targeting this family of enzymes.

Project: Engineering Mutant DCAFs to Define CRL4 Ligase Substrate Specificity Using E-STUB Ubiquitomic

Mentor: Hubert Huang, Cancer Program