Author(s): So Maezawa * , Rie Fukushima, Toyofumi Matsushita, Tomoyoshi Kato, Yoshiki Takagaki, Yoshihiro Nishiyama, Sachiko Ando, Takuro Matsumoto, Kousuke Kouda, Takahide Hayano, Masahiro Suzuki, Kotaro Koiwai, Osamu Koiwai
Terminal deoxynucleotidyltransferase (TdT) is a unique DNA polymerase that catalyzes DNA polymerization in the absence of a DNA template . Genetic rearrangement of immunoglobulin (Ig) and T-cell receptor (TcR) genes enhances their diversity, and extra nucleotides (N region) are inserted during rearrangement at the V-J, V-D, and D-J junctions by TdT , . After synthesis of the N region, TdT in pro-B- or pro- and pre-T-cells rapidly disappears. It may be degraded by ubiquitylation in lymphoid cells .
The ubiquitylation system is an essential regulator of multiple cellular processes that marks proteins for proteasome-mediated degradation, receptor internalization, endocytic trafficking , , histone modification , vesicular trafficking , DNA repair , , viral budding , , or transcriptional regulation , . Ubiquitin (Ub), a 76-aa polypeptide, is attached to the [epsilon]-amino group of lysine residue in target proteins by multienzymes. A Ub-activating enzyme (E1) initially activates Ub ATP-dependently. The activated Ub is then transferred to the cysteine in the active site of a Ub-conjugating enzyme (E2) by a transesterification reaction. E2s are grouped into four classes (class I, II, III and IV) that contain, respectively, a catalytic core domain (UBC) composed of ~150 aa, a UBC and a COOH-terminal extension, a UBC and an NH2 -terminal extension, and a UBC and both of NH2 - and COOH-terminal extensions. The third component, a Ub protein ligase (E3), cooperates with E2 to transfer Ub to the substrate.
E3s are grouped into two classes . One class, RING finger proteins, directly transfers Ub from E2 to substrates. E3s in the second class (homologous to E6-AP COOH terminus [HECT] domain proteins) contain a cysteine in the active site; these enzymes first accept Ub from E2 and then transfer it to the substrate. After transfer of the first Ub to a lysine in the target protein, subsequent Ubs are added sequentially. When the Ub polymer is synthesized using K48 of Ub to attach each component, the poly-Ub functions to signal delivery of the protein to the 26 S proteasome for degradation. When the Ub components are polymerized on other Ub lysines (e.g., K63), the target protein is not degraded. Mono-ubiquitylation of a protein can also serve as a marker for DNA repair .
Protein ubiquitylation is balanced by a set of de-ubiquitylating isopeptidases that release Ub from ubiquitylated proteins . Protein ubiquitylation in eukaryotes is regulated by several E2s and E3s. In humans, more than thirty E2s and hundreds of putative E3 ligases have been identified. In addition, different E2s can interact with a common E3, and a single E2 can function together with a variety of E3s including the RING finger and the HECT domain types , , .
We recently reported that BPOZ-2 functions as a substrate-specific adaptor for Cul3-based E3 ligase and that TdT is a substrate of the BPOZ-2/Cul3 complex ....