ABS 053: Investigating the lysine and chaperone requirements for ∆exon2 VHL quality control​

Prokriti Projukti ¹ & Amie J. McClellan ¹

¹ Division of Science and Mathematics, Bennington College, Bennington, VT 05201​

Van Wickle (2025) Volume 1, ABS 053

Introduction: ∆exon2 VHL, a naturally occurring disease-causing variant of the von Hippel-Lindau (VHL) tumor suppressor protein, lacks a critical chaperone binding site; the mutant protein is misfolded, unstable, and degraded via the ubiquitin-proteasome system in both mammalian cells and a yeast model system. This study aims to investigate the impact of lysine removal on ∆exon2 VHL quality control, specifically, whether and how its degradation pathway and chaperone requirements are affected. Steady-state degradation assays in wild-type (WT) and proteasome-mutant Saccharomyces cerevisiae demonstrate that both lysine-containing and lysine-free ∆exon2 VHL exhibit proteasome-dependent degradation, suggesting the possibility of non-canonical ubiquitination. Ongoing experiments are investigating whether ∆exon2 VHL ± lysines exhibits similar co-chaperone requirements, namely for Sti1p/HOP and Sse1p/Hsp110, as previously shown for WT VHL ± lysines. Altogether, this research will advance our understanding of lysine-independent protein ubiquitination and degradation and the potential contributions of molecular chaperones to the quality control of lysine-less proteins.

Methods: Synthetically-generated yeast codon-optimized ∆exon2 VHL constructs (Genscript.com) containing or lacking (mutated to arginine) endogenous lysines were cloned into yeast expression vectors under a galactose-inducible promoter. Transformed WT and pre1-1 (proteasome mutant) strains were cultured in synthetic dropout medium with galactose induction at 30 °C. For steady-state degradation assays, overnight SD galactose cultures of WT and pre1-1 yeast expressing ∆exon2 VHL ± lysines were shifted to YPD to cease expression; samples were harvested at 0, 15, 30, and 45 min and immediately vortexed with 0.1 M sodium azide to halt proteolysis. Cells were lysed by glass-bead disruption in lysis buffer (50 mM Tris-HCl, pH 7.4; 0.5% SDS; 1 mM EDTA) supplemented with 1 mM DTT, 1 mM PMSF, and pepstatin-A; lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-VHL antibodies. Co-chaperone requirements were assessed in Δsti1 and Δsse1 deletion strains under identical conditions.

Results: Steady-state assays revealed that the degradation of ∆exon2 VHL, with or without its endogenous lysines, is proteasome-dependent; both were markedly stabilized in pre1-1 yeast compared to the isogenic WT strain. Deletion of STI1 similarly stabilized ∆exon2 VHL and ∆exon2 VHL∆K in yeast, demonstrating that the Hsp70/Hsp90 co‐chaperone Sti1p is required for efficient degradation of these mutant VHL proteins, as previously described for WT VHL and observed for the L158P VHL point mutant.

Discussion: These findings demonstrate that ∆exon2 VHL undergoes proteasome-dependent degradation even in the absence of lysine residues and that the Hsp70/Hsp90 co‐chaperone Sti1p is required for this process. This implicates non‐canonical ubiquitination pathways and chaperone‐mediated quality control in the handling of disease‐associated misfolded proteins. Our work advances the understanding of VHL tumor suppressor stability and suggests that ubiquitination on non‐lysine residues may be a broadly used protein quality-control mechanism. Future studies will aim to identify the nature of non-lysine ubiquitin attachment sites, define involved E3 ligase(s), and evaluate these mechanisms in mammalian cells to inform potential therapeutic strategies.