Tetsu Kamitani

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.

NUB1, a NEDD8-interacting Protein, Is Induced by Interferon and Down-regulates the NEDD8 Expression

NEDD8, a ubiquitin-like protein, covalently conjugates to cullin family members. It appears to control vital biological events through its conjugation to cullins. To study how this conjugation pathway is regulated, we performed yeast two-hybrid screening by using NEDD8 as a bait and isolated a cDNA fragment encoding a potent down-regulator of the NEDD8 expression. Here, we report this novel regulator, NUB1 (NEDD8Ultimate Buster-1). NUB1 is composed of 601 residues with a calculated 69.1-kDa molecular mass. It is an interferon-inducible protein and predominantly localized in the nucleus. The NUB1 message is specifically expressed in adult human testis, ovary, heart, and skeletal muscle tissues and is developmentally down-regulated in mouse embryos. In biochemical analysis, we found that NUB1 overexpression leads to severe reduction of NEDD8 monomer and NEDD8 conjugates in cells. This reduction is not due to down-regulation of NEDD8 transcription, but due to post-transcriptional mechanism. As expected from this activity, overexpression of NUB1 had a profound growth-inhibitory effect on U2OS cells. Thus, NUB1 is a strong down-regulator of the NEDD8 expression and appears to play critical roles in regulating biological events, including cell growth.

Accumulation of NEDD8 in neuronal and glial inclusions of neurodegenerative disorders

NEDD8 (neural precursor cell expressed, developmentally down‐regulated 8) is a ubiquitin‐like protein that controls vital biological events through its conjugation to members of the cullin family, which are components of certain ubiquitin E3  ligases.  Recent  studies  have  shown  that  NEDD8 is incorporated into Lewy bodies (LBs) in Parkinsons disease, Mallory bodies in alcoholic liver disease and Rosenthal fibres in astrocytoma. In order to examine whether NEDD8 plays a role in the formation of ubiquitinated inclusions, we performed immunohistochemical staining of brain tissue from patients with various neurodegenerative disorders, using an affinity‐purified polyclonal antibody raised against NEDD8 that did not cross‐react with ubiquitin. In LB disease, NEDD8 immunoreactivity was present in almost all of the LBs and Lewy neurites. Moreover, NEDD8 immunoreactivity was found in a variety of ubiquitinated inclusions, including neuronal and oligodendroglial inclusions in multiple system atrophy, neurofibrillary tangles in Alzheimers disease, ubiquitinated inclusions in motor neurone disease, and intranuclear inclusions in triplet repeat diseases. These findings suggest that NEDD8 is involved in the formation of various ubiquitinated inclusions via the ubiquitin‐proteasome system.

NEDD8 protein is involved in ubiquitinated inclusion bodies.

Proteolysis by the ubiquitin–proteasome system is considered to play a pathological role in several degenerative diseases that involve ubiquitinated inclusion bodies. In recent years, several ubiquitin‐like proteins have been isolated, but it is uncertain whether their roles are associated with protein degradation through the ubiquitin–proteasome system. NEDD8 (neural precursor cell‐expressed and developmentally down‐regulated gene), which consists of 81 amino acid residues, possesses the highest sequence similarity to ubiquitin. Recent studies have indicated that NEDD8 is covalently ligated to cullin family proteins, which are components of certain ubiquitin E3 ligases, by a pathway analogous to that of ubiquitin. Thus, by focusing on the structural and functional association between NEDD8 and ubiquitin, it would be of interest to know whether the NEDD8 system is involved in pathological disorders of the ubiquitin–proteasome system. This study has examined the immunohistochemical distribution of NEDD8 protein by using a highly purified antibody in normal tissues and in tissues known to contain ubiquitinated inclusions. NEDD8 protein expression was widely observed in most types of tissues. Furthermore, accumulation of the NEDD8 protein was commonly observed in ubiquitinated inclusion bodies, including Lewy bodies in Parkinsons disease, Mallory bodies in alcoholic liver disease, and Rosenthal fibres in astrocytoma. Two of ten cases of neurofibrillary tangles and senile plaques from patients with Alzheimers disease showed intense staining for NEDD8 as well as for ubiquitin. These findings suggest the possibility that the NEDD8 system is involved in the metabolism of these inclusion bodies via the ubiquitin–proteasome system. Copyright

Regulation of the NEDD8 Conjugation System by a Splicing Variant, NUB1L

NEDD8 is a ubiquitin-like protein that controls vital biological events through its conjugation to target proteins. We previously identified a negative regulator of the NEDD8 conjugation system, NUB1, which works by recruiting NEDD8 and its conjugates to the proteasome for degradation. Recently, we found its splicing variant, NUB1L. It possesses an insertion of 14 amino acids that codes for a UBA domain. Structural study revealed that NUB1 has a NEDD8-binding site at the C terminus, whereas NUB1L has an additional site at the newly generated UBA domain. Interestingly, the sequence A(X4)L(X10)L(X3)L was conserved in these NEDD8-binding sites among human and other mammals. Mutational studies revealed that at least three Leu residues in the conserved sequence are required for binding with NEDD8. Functional study suggested that the NEDD8-binding ability at the C terminus of NUB1 and NUB1L is mainly involved in the down-regulation of NEDD8, but the NEDD8-binding ability at the UBA2 domain of NUB1L is minimally or not involved at all. The NEDD8-binding ability at the UBA2 domain might be required for an unknown function of NUB1L.

Improved Immunodetection of Endogenous α-Synuclein

α-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative α-synucleinopathies such as Parkinsons disease. Despite extensive research, however, its precise function remains unclear partly because of a difficulty in immunoblotting detection of endogenous α-synuclein. This difficulty has largely restricted the progress for α-synucleinopathy research. Here, we report that α-synuclein monomers tend to easily detach from blotted membranes, resulting in no or very poor detection. To prevent this detachment, a mild fixation of blotted membranes with paraformaldehyde was applied to the immunoblotting method. Amazingly, this fixation led to clear and strong detection of endogenous α-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous α-synuclein in various human cell lines, including SH-SY5Y, HEK293, HL60, HeLa, K562, A375, and Daoy, and a mouse cell line B16 as well as in several mouse tissues such as the spleen and kidney. Moreover, it should be noted that we could clearly detect endogenous α-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous α-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous α-synuclein, thereby facilitating α-synuclein research.

Parkinson's Disease-Related Protein, α-Synuclein, in Malignant Melanoma

Background Melanoma is the major cause of skin cancer death worldwide. Parkinsons disease is a neurodegenerative disorder that is caused by mutation of α-synuclein or other genes. Importantly, epidemiological studies have reported co-occurrence of melanoma and Parkinsons disease, suggesting that these two diseases could share common genetic components. Methodology/Principal Findings Recently, we found that human melanoma cell lines highly express α-synuclein, whereas the protein is undetectable in the non-melanoma cancer cell lines tested. To investigate the expression of α-synuclein in human melanoma tissues, we immunostained sections of melanoma, nevus, non-melanocytic cutaneous carcinoma, and normal skin. α-Synuclein was positively detected in 86% of the primary and 85% of the metastatic melanoma sections, as well as in 89% of nevus sections. However, α-synuclein was undetectable in non-melanocytic cutaneous carcinoma and normal skin. Conclusions/Significance The Parkinsons disease-related protein, α-synuclein, is expressed in both malignant and benign melanocytic lesions, such as melanomas and nevi. Although α-synuclein cannot be used to distinguish between malignant and benign melanocytic skin lesions, it might be a useful biomarker for the diagnosis of metastatic melanoma.

TRIM9, a novel brain-specific E3 ubiquitin ligase, is repressed in the brain of Parkinson’s disease and dementia with Lewy bodies

TRIM family proteins are involved in a broad range of biological processes, and their alteration results in many diverse pathological conditions found in genetic diseases, viral infections, and cancers. However, the spatial and temporal expression and function of TRIM9, one of TRIM family proteins, remain obscure. Our results here showed that TRIM9 protein is mainly expressed in the cerebral cortex, and functions as an E3 ubiquitin ligase collaborating with an E2 ubiquitin conjugating enzyme UbcH5b. Immunohistochemical examination revealed that TRIM9 is localized to the neurons in the normal mouse and human brain and that TRIM9 immunoreactivity is severely decreased in the affected brain areas in Parkinsons disease and dementia with Lewy bodies. This repressed level of TRIM9 protein was supported by immunoblotting analysis. Intriguingly, cortical and brainstem-type Lewy bodies were immunopositive for TRIM9. These results suggest that TRIM9 plays an important role in the regulation of neuronal functions and participates in pathological process of Lewy body disease through its ligase activity.

Downregulation of active IKKβ by Ro52-mediated autophagy

Upon activation, NF-kappaB translocates into the nucleus and initiates many biological events. This NF-kappaB signaling is mainly induced by the protein kinase IKK beta. Early in this signaling pathway, IKK beta is phosphorylated for activation by several factors, such as pro-inflammatory cytokines and the Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1). In cells expressing Tax protein, IKK beta is persistently phosphorylated, which chronically activates NF-kappaB signaling. But the active IKK beta is conjugated with a monoubiquitin by the E3 ubiquitin ligase Ro52, and the IKK beta-induced NF-kappaB signaling is downregulated. However, the mechanism of the downregulation has been unknown. Here, we show that Ro52-mediated monoubiquitination is involved in the subcellular translocation of active IKK beta to autophagosomes. Furthermore, using reporter assays, we show that Ro52 suppresses IKK beta-induced NF-kappaB signaling and that this suppression is blocked by an autophagy inhibitor. These results suggest that Ro52-mediated monoubiquitination plays a critical role in the downregulation of active IKK beta through autophagy.

Inhibition of NEDD8-conjugation pathway by novel molecules: Potential approaches to anticancer therapy

Cancer cells can survive through the upregulation of cell cycle and the escape from apoptosis induced by numerous cellular stresses. In the normal cells, these biological cascades depend on scheduled proteolytic degradation of regulatory proteins via the ubiquitin–proteasome pathway. Therefore, interruption of regulated proteolytic pathways leads to abnormal cell‐proliferation. Ubiquitin ligases called SCF complex (consisting of Skp‐1, cullin, and F‐box protein) or CRL (cullin‐RING ubiquitin ligase) are predominant in a family of E3 ubiquitin ligases that control a final step in ubiquitination of diverse substrates. To a great extent, the ubiquitin ligase activity of the SCF complex requires the conjugation of NEDD8 to cullins, i.e. scaffold proteins. This review is anticipated to review the downregulation system of NEDD8 conjugation by several factors including a chemical compound such as MLN4924 and protein molecules (e.g. COP9 signalosome, inactive mutant of Ubc12, and NUB1/NUB1L). Since the downregulation of NEDD8 conjugation affects cell‐cycle progression by inhibiting the ligase activity of SCF complexes, such knowledge in the NEDD8‐conjugation pathway will contribute to the more magnificent therapies that selectively suppress tumorigenesis.