Xu Lixin, Yang Lihong, Moitra Prasun K, Hashimoto Keiko, Rallabhandi Prasad, Kaul Sunil, Meroni Germana, Jensen Jane P, Weissman Allan M, D'Arpa Peter
Department of Biochemistry and Molecular Biology, F. Edward Hèbert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA.
Exp Cell Res. 2003 Aug 1;288(1):84-93. doi: 10.1016/s0014-4827(03)00187-3.
We previously identified BTBD1 and BTBD2 as novel topoisomerase I-interacting proteins that share 80% amino acid identity. Here we report the characterization of their subcellular localization. In a number of mouse and human cells, BTBD1 and BTBD2 (BTBD1/2) colocalized to punctate or elongated cytoplasmic bodies (< 5 microm long and several per cell) that were larger and more elongated in cancer cell lines than in fibroblasts and myoblasts. A search for potential colocalizing proteins identified TRIM family members that localize to morphologically similar cytoplasmic bodies, which were then tested for colocalization with BTBD1/2. TRIM5delta, expressed as a GFP fusion, colocalized with BTBD1/2 immunostaining and appeared to serve as a scaffold for the assembly of endogenous BTBD1/2 proteins. TRIM family members contain a RING domain, B-box(es), and coiled-coil regions, which have a characteristic order and spacing (RBCC domain). RING-dependent ubiquitin ligase activity and multimerization via the coiled-coil region may be defining properties of the RBCC/TRIM protein family. We found that TRIM5delta with a deleted coiled-coil region or a mutated RING domain failed to colocalize with BTBD1/2. Additionally, TRIM5delta ubiquitylated itself in a RING finger- and UbcH5B-dependent manner. BTBD1/2 each contain a PHR-similarity region, repeated twice on the putative ubiquitin ligases PAM, highwire and RPM-1, which also contain a RING and B-box. Thus, four protein modules found on each of these putative ubiquitin ligases, a RING, a B-box and two PHR repeats, are present on BTBD1/2 and TRIM5delta that are colocalized to cytoplasmic bodies.
我们之前鉴定出BTBD1和BTBD2是新型的与拓扑异构酶I相互作用的蛋白质，它们具有80%的氨基酸同一性。在此，我们报告它们亚细胞定位的特征。在许多小鼠和人类细胞中，BTBD1和BTBD2（BTBD1/2）共定位于点状或细长的细胞质小体（长度小于5微米，每个细胞有几个），这些小体在癌细胞系中比在成纤维细胞和成肌细胞中更大且更细长。对潜在共定位蛋白的搜索鉴定出定位于形态相似的细胞质小体的TRIM家族成员，然后测试它们与BTBD1/2的共定位情况。以GFP融合蛋白形式表达的TRIM5δ与BTBD1/2免疫染色共定位，并且似乎作为内源性BTBD1/2蛋白组装的支架。TRIM家族成员包含一个RING结构域、B盒和卷曲螺旋区域，它们具有特征性的顺序和间隔（RBCC结构域）。RING依赖性泛素连接酶活性以及通过卷曲螺旋区域的多聚化可能是RBCC/TRIM蛋白家族的决定性特性。我们发现，缺失卷曲螺旋区域或RING结构域发生突变的TRIM5δ无法与BTBD1/2共定位。此外，TRIM5δ以RING指结构和UbcH5B依赖性方式自身泛素化。BTBD1/2各自包含一个PHR相似区域，在推定的泛素连接酶PAM、highwire和RPM-1上重复两次，这些酶也包含一个RING和一个B盒。因此，在这些推定的泛素连接酶中的每一个上发现的四个蛋白质模块，即一个RING、一个B盒和两个PHR重复序列，存在于共定位于细胞质小体的BTBD1/2和TRIM5δ上。
