Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages
Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages
复制标题
DOI：
10.1074/jbc.m609659200
复制
发表时间：
2007-06-15
影响因子：
4.8
通讯作者：
Goldberg, Alfred L.
中科院分区：
文献类型：
--
作者：
Kim, Hyoung Tae;Kim, Kwang Pyo;Goldberg, Alfred L.
一般认为，特异性泛素连接酶（E3）将蛋白质底物连接到含有单一类型异肽键的聚泛素链上，并且由通过Lys（48）而不是通过Lys（63）的键组成的链是蛋白酶体降解的靶蛋白。然而，当我们对由不同纯化的E3和Ub缀合酶（E2）形成的泛素（Ub）链的类型进行系统分析时，我们发现，使用Ub突变体和质谱，U-box E3，CHIP，和环指E3，MuRF 1和Mdm 2，与E2，UbcH 5，形成一种新型的Ub链，包含所有七种可能的键，但主要是Lys（48）、Lys（63）和Lys（11）键。此外，这些异质链含有叉（分叉），其中两个Ub分子在前一个Ub分子上的Lys（6）+ Lys（11）、Lys（27）+ Lys（29）或Lys（29）+ Lys（33）处连接到相邻的赖氨酸。然而，HECT结构域E3 s、E6 AP和Nedd 4与相同的E2、UbcH 5仅形成同质链，即Lys 48链（E6 AP）或Lys（63）链（Nedd 4）。此外，与其他家族的E2，CHIP和MuRF 1合成均匀的Ub链的基板上。利用UbcH 13/Uev 1a和UbcH 1（E2- 25 K），MuRF 1在底物上分别合成了Lys（48）链和Lys（63）链。当肌钙蛋白I通过MuRF 1连接到Lys（48）-Ub链上，或者令人惊讶地连接到Lys（63）-Ub链上时，肌钙蛋白I被纯26 S蛋白酶体迅速降解。然而，当与混合叉状链连接时，肌钙蛋白I的降解相当差，并且其polyUb链，尤其是叉状连接，被蛋白酶体相关的异肽酶缓慢分解。因为这些环指和U盒E3与UbcH 5靶向蛋白质在体内降解，但赖氨酸（63）链不，细胞可能含有额外的因子，防止形成这种不可降解的Ub-缀合物，并保护连接到赖氨酸（63）-Ub链的蛋白质免受蛋白酶体降解.
It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys(48), but not through Lys(63), target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys(48), Lys(63), and Lys(11) linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys(6) + Lys(11), Lys(27) + Lys(29), or Lys(29) + Lys(33) on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys48 chains (E6AP) or Lys(63) chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/ Uev1a, they attach Lys(63) chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys(48) chains on the substrate.We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys(48)-Ub chain or, surprisingly, to a Lys(63)-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome- associated isopeptidases. Because these Ring finger and U- box E3s with UbcH5 target proteins for degradation in vivo, but Lys(63) chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub- conjugates and that protect proteins linked to Lys(63)-Ub chains from proteasomal degradation.