Insight into non-canonical pVHL functions

One of the most frequent key factors driving tumorigenesis is the functional impairment of tumor suppressor proteins. Mutations of the von Hippel-Lindau tumor suppressor protein (pVHL) are causative of the von-Hippel Lindau syndrome, a hereditary predisposition to develop cancers. The protein exists in two main isoforms: the full-length pVHL30 and a shorter pVHL19 lacking the N-terminal portion. Both these proteins exert their function as oncosuppressor by regulating the hypoxia-inducible factor 1-alpha (HIF-1a) stability. Besides its well-characterized role in HIF-1α regulation, pVHL is proposed as a multifunctional adaptor protein involved in multiple different cellular processes. These additional functions are collectively referred as non-canonical pVHL functions. Aim of this thesis is the further characterization of these HIF-independent functions. A multidisciplinary strategy combining experimental and bioinformatics approaches was used to address three different biological questions: the relationship between pVHL and cell-cycle regulation, the role of pVHL30 in testis-tissue and the existence of a functional link between pVHL30 and the androgen receptor (AR). My work identified novel protein-protein interactions that further link pVHL to cell-cycle control. In particular, I identified a new direct interaction between pVHL and the Cip/Kip protein family (i.e. p21,p27,p57), mediated by the pVHL30 β-domain and a COOD-like motif located into the linear CDKN1s N-terminal domain. In parallel, my efforts in dissecting pVHL also demonstrated pVHL30 to bind the human Mouse double minute 2 homolog (MDM2), an E3-ubiquitin ligase involved in the degradation of the Tumor protein p53 (p53). This interaction was found to be pVHL30-specific and mediated by its N-terminal tail, with the shorter pVHL19 lacking this binding property. The molecular dissection distinguished the pVHL30 B-domain and the N-terminal tail as the regions interacting with a long disordered portion located at the MDM2 C-terminus. On note, the association was found to be mediated by two specific intrinsically disordered portions confirming the pVHL ability to recognize and bind linear motifs. Then using a Y2H genome-wide screening approach I investigated the unknown pVHL30 interactome in testis-tissue. This analysis identified 260 positive clones further classified into 2 main groups addressing 61 human proteins and 118 non coding sequences, respectively. Among the first group I found 6 already known pVHL30 interactors (e.g. Elongin C) that confirmed the approach reliability, whereas the remaining 55 novel interactors were never associated to pVHL30 so far. These testis-specific interactors include proteins involved in relevant cellular pathways such as cell-cycle regulation, DNA damage repair, apoptosis and cytoskeleton regulation. On the other hand, a preliminary protein-protein interacting network generated using these data showed limited internal connections at the pathway level, suggesting a broader involvement of pVHL30 in other pathways not yet identified. Finally considering the relevance of AR in testis-tissue, I investigated the functional relationship between pVHL30 and this protein. Multiple cellular biology techniques were employed to identify a physical interaction between pVHL30 and AR wild-type (AR24Q) or poly-Q expanded (AR65Q). The expansion of the AR poly-Q stretch is linked to protein dysfunction that leads to pathological conditions. My findings demonstrated pVHL30 to inhibit the AR transcriptional activity as well as influencing AR protein stability by accelerating its turnover. In summary, findings reported in this thesis identify 4 novel general interactors that point to pVHL30 involvement in cell-cycle regulation, while 55 new interactors were found to be testis-specific. Moreover, important pieces of evidence shedding light on the functional relationship between pVHL and AR are also discussed. Collectively taken, all of these evidence suggest pVHL30 to have isoform-specific functions as well as putative testis-specific and sex-linked functions.