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Biomedical subjects

Noriyuki Matsuda

Publications and source records attributed to Noriyuki Matsuda.

9 recordsLinked to original sources
PubMed2007

Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity.

Although cullin-1 neddylation is crucial for the activation of SCF ubiquitin E3 ligases, the underlying mechanisms for NEDD8-mediated activation of SCF remain unclear. Here we demonstrate by NMR and mutational studies that NEDD8 binds the ubiquitin E2 (UBC4), but not NEDD8 E2 (UBC12). Our data imply that NEDD8 forms an active platform on the SCF complex for selective recruitment of ubiquitin-charged E2s in collaboration with RBX1, and thereby upregulates the E3 activity.

Carrier Proteins↗
PubMed2005

Diverse effects of pathogenic mutations of Parkin that catalyze multiple monoubiquitylation in vitro.

Mutational dysfunction of PARKIN gene, which encodes a double RING finger protein and has ubiquitin ligase E3 activity, is the major cause of autosomal recessive juvenile Parkinsonism. Although many studies explored the functions of Parkin, its biochemical character is poorly understood. To address this issue, we established an E3 assay system using maltose-binding protein-fused Parkin purified from Escherichia coli. Using this recombinant Parkin, we found that not the front but the rear RING finger motif is responsible for the E3 activity of Parkin, and it catalyzes multiple monoubiquitylation. Intriguingly, for autosomal recessive juvenile Parkinsonism-causing mutations of Parkin, whereas there was loss of E3 activity in the rear RING domain, other pathogenic mutants still exhibited E3 activity equivalent to that of the wild-type Parkin. The evidence presented allows us to reconsider the function of Parkin-catalyzed ubiquitylation and to conclude that autosomal recessive juvenile Parkinsonism is not solely attributable to catalytic impairment of the E3 activity of Parkin.

Amino Acid Motifs↗
PubMed2005

UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex.

The xeroderma pigmentosum group C (XPC) protein complex plays a key role in recognizing DNA damage throughout the genome for mammalian nucleotide excision repair (NER). Ultraviolet light (UV)-damaged DNA binding protein (UV-DDB) is another complex that appears to be involved in the recognition of NER-inducing damage, although the precise role it plays and its relationship to XPC remain to be elucidated. Here we show that XPC undergoes reversible ubiquitylation upon UV irradiation of cells and that this depends on the presence of functional UV-DDB activity. XPC and UV-DDB were demonstrated to interact physically, and both are polyubiquitylated by the recombinant UV-DDB-ubiquitin ligase complex. The polyubiquitylation altered the DNA binding properties of XPC and UV-DDB and appeared to be required for cell-free NER of UV-induced (6-4) photoproducts specifically when UV-DDB was bound to the lesion. Our results strongly suggest that ubiquitylation plays a critical role in the transfer of the UV-induced lesion from UV-DDB to XPC.

Cell Line↗
PubMed2005

DDB2, the xeroderma pigmentosum group E gene product, is directly ubiquitylated by Cullin 4A-based ubiquitin ligase complex.

Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to UV irradiation and high incidence of skin cancer caused by inherited defects in DNA repair. Mutational malfunction of damaged-DNA binding protein 2 (DDB2) causes the XP complementation group E (XP-E). DDB2 together with DDB1 comprises a heterodimer called DDB complex, which is involved in damaged-DNA binding and nucleotide excision repair. Interestingly, by screening for a cellular protein(s) that interacts with Cullin 4A (Cul4A), a key component of the ubiquitin ligase complex, we identified DDB1. Immunoprecipitation confirmed that Cul4A interacts with DDB1 and also associates with DDB2. To date, it has been reported that DDB2 is rapidly degraded after UV irradiation and that overproduction of Cul4A stimulates the ubiquitylation of DDB2 in the cells. However, as biochemical analysis using pure Cul4A-containing E3 is missing, it is still unknown whether the Cul4A complex directly ubiquitylates DDB2 or not. We thus purified the Cul4A-containing E3 complex to near homogeneity and attempted to ubiquitylate DDB2 in vitro. The ubiquitylation of DDB2 was reconstituted using this pure E3 complex, indicating that DDB-Cul4A E3 complex in itself can ubiquitylate DDB2 directly. We also showed that an amino acid substitution, K244E, in DDB2 derived from a XP-E patient did not affect its ubiquitylation.

Amino Acid Substitution↗
PubMed2003

A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes.

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).

Amino Acid Sequence↗
PubMed2003

Ubiquitin ligase activities of Bombyx mori nucleopolyhedrovirus RING finger proteins.

The genome of Bombyx mori nucleopolyhedrovirus (BmNPV) is predicted to contain six RING finger proteins: IAP1, ORF35, IAP2, CG30, IE2, and PE38. Several other members of the RING finger family have recently been shown to have the ubiquitin-ligase (E3) activity. We thus examined whether BmNPV RING finger proteins have the E3 activity. In vitro ubiquitination assay with the rabbit reticulocyte lysates and BmNPV RING finger proteins fused with maltose-binding protein (MBP) showed that four of them (IAP2, IE2, PE38, and CG30) were polyubiquitinated in the presence of zinc ion. Furthermore, MBP-IAP2, MBP-IE2, and MBP-PE38 were able to reconstitute ubiquitination activity in cooperation with the Ubc4/5 subfamily of ubiquitin-conjugating enzymes. Mutational analysis also showed that ubiquitination activity of MBP-IAP2, MBP-IE2, and MBP-PE38 were dependent on their RING finger motif. Therefore, these results suggest that IAP2, IE2, and PE38 may function as E3 enzymes during BmNPV infection.

Animals↗
PubMed2002

EL5, a rice N-acetylchitooligosaccharide elicitor-responsive RING-H2 finger protein, is a ubiquitin ligase which functions in vitro in co-operation with an elicitor-responsive ubiquitin-conjugating enzyme, OsUBC5b.

EL5, a rice gene responsive to N-acetylchitooligosaccharide elicitor, encodes a RING-H2 finger protein with structural features common to the plant-specific ATL family. We show that the fusion protein of EL5 with maltose binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and the Ubc4/5 subfamily of the ubiquitin-conjugating enzyme (E2). EL5 possesses the activity to catalyse the transfer of ubiquitin to the MBP moiety, and the RING-H2 finger motif of EL5 is necessary for this activity. Thus, we concluded that EL5 represents a ubiquitin ligase (E3). We also show that two rice E2s (OsUBC5a, OsUBC5b) of the Ubc4/5 subfamily function as E2 which catalyses EL5-mediated ubiquitination, and OsUBC5b was induced by elicitor, as well as EL5. These results strongly suggest that EL5 and OsUBC5b have roles in plant defense response through the turnover of protein(s) via the ubiquitin/proteasome system.

Amino Acid Sequence↗