In conclusion we propose that UBE T FANCT mutations define
In conclusion, we propose that UBE2T (FANCT) mutations define a FA subtype. This is also a rare example of a mutated E2 enzyme causing an inherited human disorder, like UBE2A. The p.Gln2Glu substitution is probably hypomorphic, as indicated by the fact that a siUBE2T knockdown made AP65P-hTERT rhodamine 123 more sensitive to MMC and completely eliminated the trace FANCD2 monoubiquitination that could still be observed in the siLuc control knockdown cells (Figures 3B and 3C). Finally, it is interesting to note a recent report suggesting that UBE2T functions with an unknown E3 in nucleotide excision repair. It is common for an E2 to function with a set of E3 ligases, since far fewer E2s (∼38) are encoded in the genome than E3 ligases (600–1,000). UBE2T might have a partner other than FANCL, such as BRCA1 or other E3s, as has been suggested by yeast two-hybrid assays,24, 25 raising the possibility that UBE2T might have a function outside the FA pathway. Although a siUBE2T knockdown in AP65P-hTERT modestly sensitized cells to UV (Figure 3C), we detected only a marginal impact of UBE2T lentiviral transduction on UV survival (Figure 3D). These results suggest that the p.Gln4Glu substitution is a separation of function alteration that specifically reduces UBE2T function in the FA pathway but not in UV resistance. In line with this, neither of our FA-T-affected individuals experienced any photosensitivity. It thus remains unclear whether or how complete loss of UBE2T function would impact human phenotypes.
Acknowledgments The use of FANCT as an alias for UBE2T was approved by the HUGO Gene Nomenclature Committee. We would like to thank the individuals PNGS-252 and -255 and their family members for making this work possible. We also thank Dr. Masao S. Sasaki (Professor Emeritus, Kyoto University) for his long-standing effort to collect Japanese FA samples, including AP65P fibroblasts; Dr. James Hejna (Graduate School of Biostudies, Kyoto University) for critical reading of the manuscript and English editing; Dr. Takayuki Yamashita (Gunma University) for GM6914 cells; Dr. Hiroyuki Miyoshi (RIKEN, currently at Keio University) and RIKEN Bio-resource Center (Tsukuba, Ibaragi, Japan) for a lentivirus construct (CSII-CMV-MCS-IRES-Bsd) and the packaging system; Dr. Settara C. Chandrasekharappa (NIH) for advice on the CGH array; Dr. Yoko Katsuki for advice on immunofluorescence; Ms. Tomoko Hirayama (JCRB) for a protocol for karyotyping in fibroblasts; Ms. Fumiko Tsuchida, Chinatsu Ohki, Akiko Watanabe, and Mao Hisano for expert technical help; and Drs. Toshiyasu Taniguchi and Agata Smogorzewska for advice on anti-FANCD2/FANCI immunoblotting. The AP65P cell line (KURB1562) was kindly provided by JCRB Cell Bank, National Institute of Biomedical Innovation (Saito, Ibaraki, Osaka). This work was supported in part by grants from the Ministry of Health, Labor and Welfare of Japan.
Introduction Ubiquitylation is a posttranslational modification that regulates many events in eukaryotic cells. Most famously, ubiquitylation is involved in directing proteins for degradation by the 26S proteasome (Varshavsky, 2012). Attachment of the 8.5kDa protein ubiquitin to target proteins involves three classes of enzymes: (i) ubiquitin activating E1 enzymes that activate ubiquitin in an ATP-dependent process; (ii) ubiquitin conjugating (UBC) E2 enzymes, which are charged with ubiquitin by the E1; and (iii) the ubiquitin E3 ligases, which provide specificity for the final transfer of ubiquitin to a substrate Lys or N-terminal Met residue (Pickart, 2001). Of the >600 E3 ligases encoded in the human genome most can be classified into one of two families based on the presence of either, a homologous to the E6AP carboxyl terminus (HECT) domain, or a really interesting new gene (RING) domain. The HECT family of ligases is involved in catalysis as they receive ubiquitin from the E2 and transfer it to the substrate (Kee & Huibregtse, 2007). In contrast, the RING family bring the ubiquitin conjugated E2 enzyme (E2~Ub) and the substrate together, allowing transfer of ubiquitin directly from the E2 to the substrate (Deshaies & Joazeiro, 2009). However, recently the RING-in-between-RING (RBR) E3 proteins have been shown to utilise a hybrid RING/HECT mechanism (Wenzel, Lissounov, Brzovic, & Klevit, 2011). These proteins depend on a RING domain for E2~Ub recruitment, but ubiquitin is transferred to a cysteine in the E3 before attachment to the substrate.