Renal cell carcinoma risk in type 2 von Hippel–Lindau disease correlates with defects in pVHL stability and HIF-1α interactions

Abstract

The von Hippel-Lindau (VHL) tumor suppressor protein is the substrate binding subunit of the CBC VHL E3 ubiquitin ligase complex. Mutations in the VHL gene cause a variety of tumors with complex genotype/ phenotype correlations. Type 2A and type 2B VHL disease are characterized by a low or high risk of renal cell carcinoma, respectively. To investigate the molecular basis underlying the difference between disease types 2A and 2B, we performed a detailed biochemical analysis of the two most frequent type 2A mutations, Y98 H and Y112 H, in comparison to type 2B mutations in the same residues, Y98N and Y112N. While none of these mutations affected the assembly of CBC VHL complexes, the type 2A mutant proteins exhibited higher stabilities at physiological temperature. Moreover, the type 2A mutant proteins possessed higher binding affinities for the key cellular substrate, hypoxia-inducible transcription factor 1 (HIF-1a). Consistent with these results, type 2A but not type 2B mutant VHL proteins retained significant ubiquitin ligase activity towards HIF-1a in vitro. We propose that this residual ubiquitin ligase activity is sufficient to suppress renal cell carcinogenesis in vivo.

VHL Mutations Linked to Type 2C von Hippel-Lindau Disease Cause Extensive Structural Perturbations in pVHL

“…Protein Purification-Expression and purification of recombinant VCB complex were performed exactly as described (15). For isotopically enriched protein preparations, VCB or ElonginBC complexes were co-expressed in minimal medium containing 15 NH 4 Cl and purified as will be published elsewhere.…”

“…For isotopically enriched protein preparations, VCB or ElonginBC complexes were co-expressed in minimal medium containing 15 NH 4 Cl and purified as will be published elsewhere. 4 Biochemical Analyses-In vitro transcription and translation using pcDNA3-HA-VHL as template and HIF-1␣ peptide binding measurements were performed exactly as described (15).…”

“…For isotopically enriched protein preparations, VCB or ElonginBC complexes were co-expressed in minimal medium containing 15 NH 4 Cl and purified as will be published elsewhere. 4 Biochemical Analyses-In vitro transcription and translation using pcDNA3-HA-VHL as template and HIF-1␣ peptide binding measurements were performed exactly as described (15). Partial tryptic proteolysis was essentially performed as described (15) 4 Immunoprecipitation and Immunoblots-Expression of FLAG-pVHL in 293T cells and immunoprecipitation were performed as described (42).…”

“…Type 1-associated pVHL mutant proteins fail to assemble into a functional CBC VHL E3 ubiquitin ligase complex (14), whereas type 2A-and type 2B-associated pVHL mutant proteins were shown to be differentially affected in HIF-1/2␣ binding (15)(16)(17). Defective HIF-1/2␣ regulation is believed to be sufficient to induce development of hemangioblastomas and to be a key pathogenic event in the development of RCC (reviewed in Ref.…”

Identification of novel VHL targets that are associated with the development of renal cell carcinoma

“…Previously Type 2A mutations (that are not associated with RCC) affecting the pVHL HIF-binding domain were shown to be associated with HIF-dysregulation (Clifford et al, 2001). However the HIF-dysregulation associated with 2A mutations appears to be less severe than that with Type 1 and 2B mutations and Type 2A mutant pVHL retains significant ubiquitin ligase activity towards HIF-1a in vitro (Clifford et al, 2001;Knauth et al, 2006). These observations are consistent with our findings that both HIF-dependent and -independent target genes were associated with RCC susceptibility.…”