Structural insights into the role of the WW2 domain on tandem WW-PPxY motif interactions of oxidoreductase WWOX.

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW-peptide interactions is not always intuitive. The WW domain-containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1-WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.

Phosphorylation of the WWOX Protein Regulates Its Interaction with p73.

We describe a molecular characterization of the interaction between the cancer-related proteins WWOX and p73. This interaction is mediated by the first of two WW domains (WW1) of WWOX and a PPXY-motif-containing region in p73. While phosphorylation of Tyr33 of WWOX and association with p73 are known to affect apoptotic activity, the quantitative effect of phosphorylation on this specific interaction is determined here for the first time. Using ITC and fluorescence anisotropy, we measured the binding affinity between WWOX domains and a p73 derived peptide, and showed that this interaction is regulated by Tyr phosphorylation of WW1. Chemical synthesis of the phosphorylated domains of WWOX revealed that the binding affinity of WWOX to p73 is decreased when WWOX is phosphorylated. This result suggests a fine-tuning of binding affinity in a differential, ligand-specific manner: the decrease in binding affinity of WWOX to p73 can free both partners to form new interactions.

A novel missense variant in the SDR domain of the WWOX gene leads to complete loss of WWOX protein with early-onset epileptic encephalopathy and severe developmental delay.

The human WWOX (WW domain-containing oxidoreductase) gene, originally known as a tumor suppressor gene, has been shown to be important for brain function and development. In recent years, mutations in WWOX have been associated with a wide phenotypic spectrum of autosomal recessively inherited neurodevelopmental disorders. Whole exome sequencing was completed followed by Sanger sequencing to verify segregation of the identified variants. Functional WWOX analysis was performed in fibroblasts of one patient. Transcription and translation were assessed by quantitative real-time PCR and Western blotting. We report two related patients who presented with early epilepsy refractory to treatment, progressive microcephaly, profound developmental delay, and brain MRI abnormalities. Additionally, one of the patients showed bilateral optic atrophy. Whole exome sequencing revealed homozygosity for a novel missense variant affecting the evolutionary conserved amino acid Gln230 in the catalytic short-chain dehydrogenase/reductase (SDR) domain of WWOX in both girls. Functional studies showed normal levels of WWOX transcripts but absence of WWOX protein. To our knowledge, our patients are the first individuals presenting the more severe end of the phenotypic spectrum of WWOX deficiency, although they were only affected by a single missense variant of WWOX. This could be explained by the functional data indicating an impaired translation or premature degradation of the WWOX protein.

RHBDD2­WWOX protein interaction during proliferative and differentiated stages in normal and breast cancer cells.

Rhomboid pseudoproteases are catalytically inactive members of the rhomboid superfamily that modulate the traffic, turnover and activity of their target proteins. Rhomboid domain containing 2 (RHBDD2) is a rhomboid family member overexpressed during mammary gland development and advanced stages of breast cancer. Interactome profiling studies have identified RHBDD2 as a novel binding partner of WW domain­containing oxidoreductase (WWOX) protein. The present study characterized the RHBDD2­WWOX interaction in proliferating and differentiated stages of normal mammary and breast cancer cells by co­immunoprecipitation and confocal microscopy. Normal breast and proliferating cancer cells showed significantly increased RHBDD2 mRNA levels compared with their differentiated counterparts. WWOX mRNA was primarily expressed in differentiated cells. WWOX co­precipitated with RHBDD2, indicating that endogenous RHBDD2 and WWOX were physically associated in normal and breast cancer proliferating cells compared with the differentiated stage. Co­localization assays corroborated the co­immunoprecipitation results, demonstrating the RHBDD2­WWOX protein interaction in normal and proliferating breast cancer cells. RHBDD2 harbors a conserved LPPY motif at the C­terminus region that directly interacted with the WW domains of WWOX. Since WWOX serves as an inhibitor of the TGFß/SMAD3 signaling pathway in breast cells, modulation of SMAD3 target genes was analyzed in proliferating and differentiated mammary cells and in RHBDD2 silencing assays. Increased expression levels of SMAD3­regulated genes were detected in proliferating cells compared with their differentiated counterparts. Follistatin and angiopoietin­like 4 mRNA was significantly downregulated in RHBDD2 transiently silenced cells compared with scrambled control small interfering RNA. Based on these results, WWOX was suggested to be a novel RHBDD2 target protein involved in the modulation of breast epithelial cell proliferation and differentiation.