Preventing degradation of the VHL tumor suppressor
Inactivation of the gene encoding the von Hippel-Lindau tumor suppressor (VHL) gene causes an inherited syndrome that promotes the development of tumors in various organs. Certain mutations in VHL can affect the folding of the pVHL protein and thus its functionality. In this article, published in the journal PLos Genetics, the scientists identified a chaperone complex called prefoldin that binds to VHL. In the absence of pre-foldin, poorly structured pVHL can aggregate and/or be degraded. The level of expression of PFDN3, a component of the pre-foldin complex, is correlated with the malignancy of kidney tumors.
Mutations in the human von Hippel Lindau gene (VHL) are responsible for VHL disease, an autosomal dominant cancer syndrome that predisposes to a variety of benign and malignant tumors, including clear cell renal carcinoma (ccRCC), hemangioblastoma (central nervous system tumor) or pheochromocytoma (adrenal tumor). In addition, the VHL gene is mutated or absent in 80% of cases of sporadic ccRCC, the most common subtype of kidney cancer.
The VHL gene encodes a small protein of 213 amino acids, pVHL, whose various functions give it tumor suppressor activity. In particular, the pVHL protein belongs to a ubiquitin-ligase-like VBC complex that is responsible for the destruction of protein targets involved in the growth and vascularization of solid tumors. The best known substrate of the BCV complex is HIFa, which controls the response of cells to oxygen levels.
Approximately one-third of the mutations that inactivate pVHL are misdirected mutations producing a complete but non-functional protein. Some of these mutations lie outside the known functional domains of pVHL and their impact on its function remains to be elucidated. The researchers hypothesize that some of these mutations affect the optimal folding of pHLP and promote its degradation.
Proteins called "molecular chaperones" facilitate folding and prevent the aggregation of polypeptides in the dense environment of the cytoplasm by protecting the hydrophobic areas on the surface of unfolded proteins. Misfolded proteins are detected by "protein quality control" pathways that target them to the protein degradation machinery to prevent the formation of non-functional proteins or cytotoxic aggregates. The Prefoldin Complex is a hexameric co-chaperon conserved from archae to eukaryotes. It plays an important role in the protein quality control system. The eukaryotic prefoldin complex consists of two alpha subunits (PFDN3, PFDN5) and four beta subunits (PFDN1, PFDN2, PFDN4 and PFDN6). Once the pre-folded complex is assembled, its structure is reminiscent of a jellyfish with "tentacles" (the ends of the subunits) to capture newly synthesized unfolded proteins. The Prefoldin Complex transfers these unfolded proteins (e.g., actin and tubulin) to the CCT/TRiC chaperonin complex to help fold them and prevent their aggregation.
In Schizosaccharomyces pombe yeast, only two subunits of the pre-foldin complex (Pfd3 and Pfd5) have been characterized so far. The researchers had recently observed that Pfd3 controls the level of expression of the exogenously expressed human pVHL protein in S. pombe. In this work, they show that pVHL associates with the 6 subunits of the pre-foldin complex. In S. pombe yeast, the chaperone function of the pre-foldin complex is retained for tubulin and mutants of all subunits of this complex show microtubule assembly defects. Using a conditional expression system, the researchers show that the entire prefoldine complex protects pVHL from aggregation, improves its solubility and refolding. Expression of the human PFDN3 subunit in a mutant deleted from the yeast (pfd3D) gene restores the solubility of pVHL, which an inactive PFDN3 (deficient in chaperone function) does not do. This indicates that this chaperone function is retained from yeast to man. In human cells, the decrease in the expression of the whole pre-foldin complex by "RNA silencing" also affects the organization of the microtubule network and reduces pVHL expression levels. The region encoded by VHL at the junction of exon2 and exon3, rich in hydrophobic amino acids, is crucial for the binding of the pre-foldin complex. In the TCGA ("The Cancer Genome Atlas") databases for ccRCC, a low level of expression of pDNFP3 correlates with significantly reduced survival in patients with certain VHL mutations.
These data suggest that the interaction of the pre-foldin complex with pVHL is a key step in the process of folding pVHL to achieve a native conformation that allows it to perform its cellular functions. Thus, the function of the pre-foldin complex may have an impact on the protein homeostasis of pHLP and this could influence the progression of pHLP-associated diseases in patients.
A) The prefoldin complex (blue) binds the tumor suppressor protein pVHL (green) to allow its folding and functionalization via another complex, the CCT/TRiC complex. Without binding to the prefoldin complex, the unfolded pVHL protein is degraded by the cells.
B) Prefoldin complex and pVHL protein are co-localized (red dots) in cultured human kidney cells (blue, cell nuclei).
C) The fluorescent pVHL protein is misfolded in mutant pfd3D yeast cells to a subunit of the prefoldin complex and forms aggregates.
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Chesnel F, Couturier A, Alusse A, Gagné JP, Poirier GG, Jean D, Boisvert FM, Hascoet P, Paillard L, Arlot-Bonnemains Y, Le Goff X. PLoS Genet. 2020 Nov 2;16(11):e1009183. doi: 10.1371/journal.pgen.1009183