Kunikazu Tanji

Ubiquitination of E3 ubiquitin ligase TRIM5α and its potential role

HIV‐1 efficiently infects susceptible cells and causes AIDS in humans. Although HIV can also enter the cells of Old World monkeys, it encounters a block before reverse transcription. Data have shown that this species‐specific restriction is mediated by tripartite motif (TRIM)5α, whose molecular function is still undefined. Here, we show that TRIM5α functions as a RING‐finger‐type E3 ubiquitin ligase both in vitro and in vivo and ubiquitinates itself in cooperation with the E2 ubiquitin‐conjugating enzyme UbcH5B. In addition to the self‐ubiquitination, we show that TRIM5α is ubiquitinated by another E3 ubiquitin ligase, Ro52, and deubiquitinated by YopJ, one of the pathogenic proteins derived from Yersinia species. Thus, the ubiquitination of TRIM5α is catalyzed by itself and Ro52 and downregulated by YopJ. Unexpectedly, although TRIM5α is ubiquitinated, our results have revealed that the proteasome inhibitors MG115 and MG132 do not stabilize it in HeLa cells, suggesting that the ubiquitination of TRIM5α does not lead to proteasomal degradation. Importantly, TRIM5α is clearly conjugated by a single ubiquitin molecule (monoubiquitination). Our monoubiquitin‐fusion assay suggests that monoubiquitination is a signal for TRIM5α to translocate from cytoplasmic bodies to the cytoplasm.

Dynamic movements of Ro52 cytoplasmic bodies along microtubules

The RING-finger protein Ro52/TRIM21 is known as an autoantigen and is recognized by anti-Ro/SSA antibodies, which are commonly found in patients with Sjögren’s syndrome and systemic lupus erythematosus. Recently, Ro52 has been shown to localize to distinct structures called cytoplasmic bodies and function as an E3 ubiquitin ligase. However, the Ro52 cytoplasmic bodies have not been well characterized. In this study, we investigated the Ro52 cytoplasmic bodies using fluorescence microscopy. This analysis revealed that the Ro52 cytoplasmic bodies are diffusely located in the cytoplasm and exist independently of TRIM5α cytoplasmic bodies. Our results further showed that the Ro52 cytoplasmic bodies are not stained with MitoTracker dye and are not colocalized with the proteasome subunit Rpt5, the caveolae component caveolin-1, the endosome markers (EEA1, Rab5, and Rab7), and the lysosome marker LAMP2. These results indicate that the Ro52 cytoplasmic bodies are not mitochondria, proteasome-enriched structures, caveolae, endosomes, or lysosomes. Importantly, the Ro52 cytoplasmic bodies are highly motile and are located along the microtubule network. These results suggest that the Ro52 cytoplasmic bodies are unidentified structures that are transported along the microtubule network.

Chapter 27 – The Role of Atg8 Homologue in Lewy Body Disease

Lewy body disease (LBD) consists of Parkinson’s disease and dementia with Lewy bodies, and is pathologically characterized by the presence of cytoplasmic inclusions such as Lewy bodies. Formation of cytoplasmic inclusions is regarded as one of the pathophysiological events related to protein degradation systems – the ubiquitin–proteasome and autophagy–lysosome systems. Animal experiments clearly showed that alteration of these systems results in behavioral defects such as neurodegeneration and formation of ubiquitin-positive cytoplasmic inclusions. Our previous study has shown that autophagosomal components in LBD are qualitatively and quantitatively different from those in controls. These findings suggest that the autophagic process is impaired through alteration of autophagosomal components in LBD. Based on these results, we further discuss the capacity of autophagy to prevent LBD.