CYYR1 promotes the degradation of the E3 ubiquitin ligase WWP1 and is associated with favorable prognosis in breast cancer.

Ubiquitination plays a crucial role in cellular homeostasis by regulating the degradation, localization, and activity of proteins, ensuring proper cell function and balance. Among E3 ubiquitin ligases, WW domain-containing protein 1 (WWP1) is implicated in cell proliferation, survival, and apoptosis. Notably WWP1 is frequently amplified in breast cancer and associated with poor prognosis. Here, we identify the protein cysteine and tyrosine-rich protein 1 (CYYR1) that had previously no assigned function, as a regulator of WWP1 activity and stability. We show that CYYR1 binds to the WW domains of the E3 ubiquitin ligase WWP1 through its PPxY motifs. This interaction triggers K63-linked autoubiquitination and subsequent degradation of WWP1. We furthermore demonstrate that CYYR1 localizes to late endosomal vesicles and directs polyubiquitinated WWP1 toward lysosomal degradation through binding to ANKyrin repeat domain-containing protein 13 A (ANKRD13A). Moreover, we found that CYYR1 expression attenuates breast cancer cell growth in anchorage-dependent and independent colony formation assays in a PPxY-dependent manner. Finally, we highlight that CYYR1 expression is significantly decreased in breast cancer and is associated with beneficial clinical outcome. Taken together our study suggests tumor suppressor properties for CYYR1 through regulation of WWP1 autoubiquitination and lysosomal degradation.

The UAS thioredoxin-like domain of UBXN7 regulates E3 ubiquitin ligase activity of RNF111/Arkadia.

BACKGROUND: E3 ubiquitin ligases play critical roles in regulating cellular signaling pathways by inducing ubiquitylation of key components. RNF111/Arkadia is a RING E3 ubiquitin ligase that activates TGF-ß signaling by inducing ubiquitylation and proteasomal degradation of the transcriptional repressor SKIL/SnoN. In this study, we have sought to identify novel regulators of the E3 ubiquitin ligase activity of RNF111 by searching for proteins that specifically interacts with its RING domain. RESULTS: We found that UBXN7, a member of the UBA-UBX family, directly interacts with the RING domain of RNF111 or its related E3 RNF165/ARK2C that shares high sequence homology with RNF111. We showed that UBXN7 docks on RNF111 or RNF165 RING domain through its UAS thioredoxin-like domain. Overexpression of UBXN7 or its UAS domain increases endogenous RNF111, while an UBXN7 mutant devoid of UAS domain has no effect. Conversely, depletion of UBXN7 decreases RNF111 protein level. As a consequence, we found that UBXN7 can modulate degradation of the RNF111 substrate SKIL in response to TGF-ß signaling. We further unveiled this mechanism of regulation by showing that docking of the UAS domain of UBXN7 inhibits RNF111 ubiquitylation by preventing interaction of the RING domain with the E2 conjugating enzymes. By analyzing the interactome of the UAS domain of UBXN7, we identified that it also interacts with the RING domain of the E3 TOPORS and similarly regulates its E3 ubiquitin ligase activity by impairing E2 binding. CONCLUSIONS: Taken together, our results demonstrate that UBXN7 acts as a direct regulator for the E3 ubiquitin ligases RNF111, RNF165, and TOPORS and reveal that a thioredoxin-like domain can dock on specific RING domains to regulate their E3 ubiquitin ligase activity.

Quantitative Ubiquitylome Analysis Reveals the Specificity of RNF111/Arkadia E3 Ubiquitin Ligase for its Degradative Substrates SKI and SKIL/SnoN in TGF-ß Signaling Pathway.

RNF111/Arkadia is an E3 ubiquitin ligase that activates the transforming growth factor-ß (TGF-ß) pathway by degrading transcriptional repressors SKIL/SnoN and SKI. Truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently activate TGF-ß signaling are observed in some cancers. In the present study, we sought to perform a comprehensive analysis of RNF111 endogenous substrates upon TGF-ß signaling activation using an integrative proteomic approach. In that aim, we carried out label-free quantitative proteomics after the enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared with U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of its C-terminal RING domain. We compared two methods of enrichment for ubiquitylated proteins before proteomics analysis by mass spectrometry, the diGlycine (diGly) remnant peptide immunoprecipitation with a K-ε-GG antibody, and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody that recognizes all ubiquitin chains and monoubiquitylation. Although we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the ubiquitin pan nanobody method also constitutes a powerful approach because it enabled the detection of 52 potential RNF111 substrates including SKI, SKIL, and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled the identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in the presence of TGF-ß. Our results indicate that lysine 343 localized in the SAND domain of SKIL constitutes a target for RNF111 ubiquitylation and demonstrate that RNF111 E3 ubiquitin ligase function specifically targets SKI and SKIL ubiquitylation and degradation upon TGF-ß pathway activation.

HSP110 T17 simplifies and improves the microsatellite instability testing in patients with colorectal cancer.

BACKGROUND: Every colorectal cancer (CRC) patient should be tested for microsatellite instability (MSI, a marker for defective DNA mismatch repair) as a first screen for Lynch syndrome (LS). In this study, we investigated whether it may be possible to improve the detection of MSI in CRC. We examined whether the HT17 DNA repeat (critical for correct splicing of the chaperone HSP110) might constitute a superior marker for diagnosis of the MSI phenotype in patients with CRC compared with the standard panel of markers (pentaplex). METHODS: The HT17 polymorphism was analysed in germline DNA from 1037 multi-ethnic individuals. We assessed its sensitivity and specificity for detecting MSI in a multicentre, population-based cohort of 685 patients with CRC and an additional series of 70 patients with CRC considered to be at-risk of LS. All cases were screened earlier for MSI using pentaplex markers. Cases showing discordant HT17/pentaplex results were further examined for the expression of mismatch repair proteins. RESULTS: HT17 status was analysed independently and blinded to previous results from pentaplex genotyping. HT17 showed no germline allelic variation outside a very narrow range. Compared with the pentaplex panel, HT17 showed better sensitivity (0.984 (95% CI 0.968 to 0.995) vs 0.951 (95% CI 0.925 to 0.972)) and similar specificity (0.997 (95% CI 0.989 to 1.000) for both) for the detection of MSI. Furthermore, HT17 alone correctly classified samples judged to be uncertain with the pentaplex panel and showed excellent ability to detect MSI in patients with LS. CONCLUSIONS: HT17 simplifies and improves the current standard molecular methods for detecting MSI in CRC.