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Tetsu Kamitani

Publications and source records attributed to Tetsu Kamitani.

18 recordsLinked to original sources
PubMed2006

UnpEL/Usp4 is ubiquitinated by Ro52 and deubiquitinated by itself.

The autoantigen Ro52 is an E3 ubiquitin ligase that can ubiquitinate itself (self-ubiquitination). Recently, we showed that UnpEL/Usp4 is an isopeptidase that can deconjugate ubiquitin from self-ubiquitinated Ro52. Here, we showed that UnpEL is ubiquitinated by Ro52 in cooperation with UbcH5B in vitro. We also showed that UnpEL is ubiquitinated by Ro52 in HEK293T cells. Interestingly, a catalytically inactive UnpEL mutant was strongly ubiquitinated by Ro52 in HEK293T cells. These results suggest that wild-type UnpEL is ubiquitinated by Ro52 and deubiquitinated by itself (self-deubiquitination), while mutant UnpEL is ubiquitinated by Ro52 but not deubiquitinated by itself. In conclusion, Ro52 and UnpEL transregulate each other by ubiquitination and deubiquitination.

Cell Line↗
PubMed2006

Oncogenic protein UnpEL/Usp4 deubiquitinates Ro52 by its isopeptidase activity.

UnpEL (also known as Usp4 or Unph) is an oncogenic protein, because its expression with a strong promoter results in the tumorigenic transformation of NIH3T3 cells injected into nude mice. Although the structure of UnpEL is that of a deubiquitinating enzyme, neither its precise function in mammalian cells nor the mechanism of UnpEL-mediated tumorigenesis is known. Here, we show that UnpEL functions as a deubiquitinating enzyme in human HEK293T cells and its isopeptidase activity deconjugates ubiquitin specifically from a UnpEL-interacting protein Ro52. We further show that UnpEL translocates to the cytoplasmic rod-like structures and colocalizes with Ro52 when Ro52 is overexpressed in HEK293 cells. These results suggest that UnpEL colocalizes with the unubiquitinated form of Ro52 to the cytoplasmic rod-like structures, where it keeps Ro52 unubiquitinated. The continuous deubiquitination of Ro52 might be involved in tumorigenesis.

Cell Line↗
PubMed2006

NUB1 suppresses the formation of Lewy body-like inclusions by proteasomal degradation of synphilin-1.

NUB1 is a potent down-regulator of the ubiquitin-like protein NEDD8, because it targets NEDD8 to the proteasome for proteolytic degradation. From results in this study, we found that NUB1 physically interacts with synphilin-1 through its NEDD8-binding site, implying that NUB1 also targets synphilin-1 to the proteasome for degradation. Synphilin-1 is a major component of inclusion bodies found in the brains of patients with neurodegenerative alpha-synucleinopathies, including Parkinson's disease. In this study, we immunostained sections of brains from patients with Parkinson's disease and other alpha-synucleinopathies and demonstrated that NUB1, as well as synphilin-1, accumulates in the inclusion bodies. To define the role of NUB1 in the formation of these inclusion bodies, we performed a co-transfection assay using cultured HEK293 cells. This assay showed that NUB1 suppresses the formation of synphilin-1-positive inclusions. Further, biochemical assays revealed that NUB1 overexpression leads to the proteasomal degradation of synphilin-1. These results and our previous observations suggest that NUB1 indeed targets synphilin-1 to the proteasome for its efficient degradation, which, because of the resultant reduction in synphilin-1, suppresses the formation of synphilin-1-positive inclusions.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Function and subcellular location of Ro52beta.

Autoantigen Ro52alpha was recently identified as an E3 ubiquitin ligase. Its splicing variant Ro52beta, which lacks a leucine zipper, has not been characterized yet. We therefore characterized Ro52beta in contrast to Ro52alpha. Our biochemical assays revealed that both Ro52alpha and Ro52beta function as E3 ubiquitin ligases and self-ubiquitinate in cooperation with UbcH5B in vitro. In addition, both Ro52alpha and Ro52beta are ubiquitinated when overexpressed with ubiquitin in HEK293T cells, suggesting that both function as E3 ligases and self-ubiquitinate in vivo. However, cytological studies revealed that Ro52alpha mainly localizes to the cytoplasmic rod-like structures, whereas Ro52beta diffusely localizes to both the cytoplasm and the nucleus. Since the leucine zipper plays a role in the homodimerization and heterodimerization of Ro52alpha, the dimerization might be required for the localization of Ro52alpha to the rod-like structures. On the basis of these results, Ro52alpha and Ro52beta appear to ubiquitinate their particular substrates at different locations.

Alternative Splicing↗
PubMed2005

Autoantigen Ro52 is an E3 ubiquitin ligase.

Anti-Ro/SSA antibodies are classic autoantibodies commonly found in patients with Sjögren's syndrome, a chronic autoimmune disease characterized by dryness of the eyes and mouth. The autoantibodies recognize a RING-finger protein, Ro52, whose function is still unknown. Since many RING-finger proteins have been identified as E3 ubiquitin ligases, this study was designed to determine whether Ro52 functions as an E3 ubiquitin ligase. For this purpose, recombinant Ro52 was purified from bacterial lysate and used to investigate its activity of E3 ubiquitin ligase in vitro. Its enzymatic activity was also tested in HEK293T cells using wild-type Ro52 and its RING-finger mutant. Our results indicated that Ro52 ubiquitinates itself in cooperation with E2 ubiquitin-conjugating enzyme UbcH5B, thereby validating that Ro52 is a RING-finger-type E3 ubiquitin ligase. Importantly, this ubiquitin modification is predominantly monoubiquitination, which does not target Ro52 to the proteasome for degradation.

Amino Acid Sequence↗
PubMed2005

Interaction of NUB1 with the proteasome subunit S5a.

NUB1 interacts with a ubiquitin-like protein NEDD8 to target the NEDD8 monomer and neddylated proteins to the proteasome for degradation. Therefore, NUB1 is thought to be a potent downregulator of NEDD8 conjugation system. Since NUB1 possesses a UBL domain, which was previously shown to be an S5a-interacting motif in RAD23/HHR23, we initially hypothesized that NUB1 interacts with the S5a subunit of the proteasome through its UBL domain. To examine this, we performed an in vitro GST pull-down assay and a yeast two-hybrid assay. Unexpectedly, our studies revealed that NUB1 directly interacts with the S5a subunit through its C-terminal region between amino acid residues 536 and 584, not through its UBL domain. Although the UBL domain was not an S5a-interacting motif in NUB1, our further studies revealed that the UBL domain is required for the function of NUB1.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The transcription factor Sox9 is degraded by the ubiquitin-proteasome system and stabilized by a mutation in a ubiquitin-target site.

Sox9 is a transcription factor that is critical for chondrogenesis, testis determination, and development of several other organs in vertebrates. Thus the levels of Sox9 protein and its activity may be tightly regulated. Here we show that inhibitors of the 26S proteasome increase both the levels of Sox9 protein and its transcriptional activity measured with Col2a1 promoter/enhancer construct in RCS cells and C3H10T1/2 cells. Indeed, in intact cells ubiquitination assays indicate that Sox9 is multiply ubiquitinated. The K398A mutation, which was introduced in a potential ubiquitin-binding site, increases the stability of Sox9 protein and its transcriptional activity of Col2a1, Col11a2, and AMH promoter/enhancer constructs without affecting the subcellular localization and the DNA binding efficiency of Sox9. Pulse-chase experiments show that the increased Sox9 levels resulting from treatment with the MG132 proteasome inhibitor or from the K398A mutation produce stabilization of the protein. Our in vitro studies indicate that the ubiquitin-proteasome proteolytic system degrades Sox9 and regulates its transcriptional activity.

Animals↗
PubMed2004

Abolished interaction of NUB1 with mutant AIPL1 involved in Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is often considered the most severe inherited retinopathy, and AIPL1 was the fourth gene identified as associated with LCA. Although the function of AIPL1 is unknown, it has been reported to interact with NUB1. Here, we searched for a NUB1-binding site on AIPL1 and located it between amino acid residues 181 and 330 in AIPL1. Importantly, many LCA-associated mutations of AIPL1 have been found at this site. Hence, we hypothesized that the interaction between NUB1 and AIPL1 is affected in patients with LCA. To test this possibility, we used three different assays to investigate the interaction between NUB1 and the AIPL1 mutants associated with LCA. Some of the AIPL1 mutants did not interact with NUB1, suggesting that abolishment of this interaction is involved in the pathogenesis of LCA. Other AIPL1 mutants, however, did interact with NUB1, suggesting that other molecules are also involved in the pathogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2004

NUB1-mediated targeting of the ubiquitin precursor UbC1 for its C-terminal hydrolysis.

NEDD8 is a ubiquitin-like protein that controls vital biological events through its conjugation to target proteins. Previously, we identified a negative regulator of the NEDD8 conjugation system, NEDD8 ultimate buster-1 (NUB1), that recruits NEDD8 and its conjugates to the proteasome for degradation. Recently, we performed yeast two-hybrid screening with NUB1 as bait and isolated a ubiquitin precursor UbC1 that is composed of nine tandem repeats of a ubiquitin unit through alpha-peptide bonds. Interestingly, NUB1 interacted with UbC1 through its UBA domain. Further study revealed that the UBA domain interacted with alpha-peptide bond-linked polyubiquitin, but not with isopeptide bond-linked polyubiquitin, indicating that the UBA domain of NUB1 is a specific acceptor for the linear ubiquitin precursor. A functional study revealed that an unidentified protein that was immunoprecipitated with NUB1 served as a ubiquitin C-terminal hydrolase for UbC1. Thus, NUB1 seems to form a protein complex with the unidentified ubiquitin C-terminal hydrolase and recruit UbC1 to this complex. This might allow the ubiquitin C-terminal hydrolase to hydrolyze UbC1, in order to generate ubiquitin monomers. Northern blot analysis showed that the mRNAs of both NUB1 and UbC1 were enriched in the testis. Furthermore, in situ hybridization showed that both mRNAs were strongly expressed in seminiferous tubules of the testis. These results may imply that the UbC1 hydrolysis mediated by NUB1 is involved in cellular functions in the seminiferous tubules such as spermatogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2003

pVHL-independent ubiquitination of HIF1alpha and its stabilization by cobalt ion.

Recent studies have shown that hypoxia-inducible factor1alpha (HIF1alpha) is ubiquitinated by an E3-ligase complex containing von Hippel-Lindau gene product (pVHL) after which it is targeted for proteasomal degradation. In this study, we showed that HIF1alpha was stabilized in the pVHL-deficient cell line 786-0 treated with a proteasome inhibitor or Co(2+). This suggests that HIF1alpha is also ubiquitinated by a pVHL-independent pathway and that its stability is regulated by Co(2+). Indeed, using the COS cell expression system, we confirmed that HIF1alpha is ubiquitinated at the N-terminal region by a pVHL-independent pathway and that its degradation is inhibited by Co(2+). We also demonstrated that Co(2+) binds to both PAS domains in the N-terminal region of HIF1alpha. These observations imply that the stability of HIF1alpha is regulated by an additional pathway through the cobalt binding of PAS domains.

Animals↗
PubMed2003

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.

Adaptor Proteins, Signal Transducing↗
PubMed2003

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 Parkinson's 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 Alzheimer's 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.

Alzheimer Disease↗
PubMed2003

Role of the NF-kappaB signaling pathway and kappaB cis-regulatory elements on the IRF-1 and iNOS promoter regions in mycobacterial lipoarabinomannan induction of nitric oxide.

Nitric oxide (NO(.)) produced by inducible nitric oxide synthase (iNOS) is an important host defense molecule against Mycobacterium tuberculosis in mononuclear phagocytes. The objective of this study was to determine the role of the IkappaBalpha kinase-nuclear factor kappaB (IKK-NF-kappaB) signaling pathway in the induction of iNOS and NO(.) by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM) in mouse macrophages costimulated with gamma interferon (IFN-gamma). NF-kappaB was activated by ManLAM as shown by electrophoretic mobility shift assay, by immunofluorescence of translocated NF-kappaB in intact cells, and by a reporter gene driven by four NF-kappaB-binding elements. Transduction of an IkappaBalpha mutant (Ser32/36Ala) significantly inhibited NO(.) expression induced by IFN-gamma plus ManLAM. An activated SCF complex, a heterotetramer (Skp1, Cul-1, beta-TrCP [F-box protein], and ROC1) involved with ubiquitination, is also required for iNOS-NO(.) induction. Two NF-kappaB-binding sites (kappaBI and kappaBII) present on the 5'-flanking region of the iNOS promoter bound ManLAM-induced NF-kappaB similarly. By use of reporter constructs in which one or both sites are mutated, both NF-kappaB-binding positions were essential in iNOS induction by IFN-gamma plus ManLAM. IFN-gamma-induced activation of the IRF-1 transcriptional complex is a necessary component in host defense against tuberculosis. Although the 5'-flanking region of the IRF-1 promoter contains an NF-kappaB-binding site and ManLAM-induced NF-kappaB also binds to this site, ManLAM was unable to induce IRF-1 expression. The influence of mitogen-activated protein kinases on IFN-gamma plus ManLAM induction of iNOS-NO(.) is not due to any effects on ManLAM induction of NF-kappaB.

5' Flanking Region↗
PubMed2002

The inherited blindness associated protein AIPL1 interacts with the cell cycle regulator protein NUB1.

Mutations in the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) gene have been found in patients with Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. To determine the normal function of AIPL1 and to better understand how mutations in this gene cause disease, we performed a yeast two-hybrid screen to identify AIPL1-interacting proteins in the retina. One of the identified interacting proteins corresponds to NUB1 (NEDD8 Ultimate Buster 1), which is thought to control many biological events, especially cell cycle progression, by downregulating NEDD8 expression. The AIPL1-NUB1 interaction was verified by co-immunoprecipitation studies in Y79 retinoblastoma cells, demonstrating that this interaction occurs within cells that share a number of features with retinal progenitor cells. Furthermore, we examined the localization of the AIPL1 protein within developing and adult retinas, and found that AIPL1 is present in the developing photoreceptor layer of the human retina and within the photoreceptors of the adult retina. Similar to AIPL1, NUB1 is also expressed in the developing and adult retina. Therefore, it is possible that the early-onset form of retinal degeneration seen in LCA patients with AIPL1 mutations may be due to a defect in the regulation of cell cycle progression during photoreceptor maturation. These data raise the possibility that AIPL1 is important for appropriate photoreceptor formation during development and/or survival following differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Ubiquitination of Ro52 autoantigen.

Anti-Ro/SSA antibodies are antinuclear antibodies most commonly found in patients with Sjögren's syndrome, a chronic autoimmune disease characterized by dryness of the eyes and mouth. The autoantibodies recognize a RING-finger protein, Ro52/SSA (52 kDa), whose function is still unknown. In this study, the ubiquitination of Ro52 was investigated. We found that Ro52 was strongly conjugated by a single molecule of ubiquitin in cells. Although the biological relevance of this mono-ubiquitination was not defined, the function of Ro52 might be modified by the mono-ubiquitination. We also found that Ro52 was conjugated with poly-ubiquitin chain in cells (poly-ubiquitination), suggesting that Ro52 may be downregulated by the ubiquitin-proteasome pathway in vivo. Interestingly, sera from patients with Sjögren's syndrome showed heterogeneity in their reactivity to poly-ubiquitinated Ro52, probably because of their differing antigenic determinants. This heterogeneity of the reactivity might be associated with the varying clinical features found in patients with Sjögren's syndrome.

Animals↗
PubMed2002

Cobalt- and nickel-binding property of cullin-2.

Treatment with divalent metal ions such as cobalt (Co(2+)) or nickel (Ni(2+)) result in the stabilization of hypoxia-inducible factor-1alpha (HIF1alpha). Recently, HIF1alpha was shown to be ubiquitinated by an E3-ligase complex and be subsequently targeted for proteasomal degradation. In this study, we demonstrated that Co(2+) and Ni(2+) specifically bind to cullin-2. Mutant analysis revealed that cullin-2 possesses at least three sites for the binding. Furthermore, fluorescence spectroscopy revealed that only Co(2+) and Ni(2+) have the binding activity to cullin-2, but other metal ions, including Cu(2+), Ca(2+), Mg(2+), Mn(2+), and Zn(2+), did not. Finally, we found that Co(2+) and Ni(2+) do not bind to any components of the E3-ligase other than cullin-2, suggesting that cullin-2 is a key target of Co(2+) and Ni(2+). Interestingly, Co(2+) did not affect the complex formation of the ligase, suggesting that the metal binding to cullin-2 affects the function, but not the assembly of the E3-ligase.

Adenoviridae↗
PubMed1994

Chromosomal assignment of genes involved in glycosylphosphatidylinositol anchor biosynthesis: implications for the pathogenesis of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder that affects both sexes equally. The biochemical defect in PNH resides in the incomplete enzymatic assembly of glycosylphosphatidylinositol (GPI) anchors used for surface protein attachment. In all PNH patients tested to date, the biosynthetic defect occurs at the addition of N-acetyl-glucosamine to the phosphatidylinositol molecule (class A defect). A human cDNA, Piga, that repairs cell lines with the class A GPI-anchor biosynthetic defect has been recently cloned. Mapping of Piga to the X chromosome suggests that a single acquired mutation within Piga could alter GPI-anchor synthesis and result in PNH. However, this finding does not explain why all PNH patients have the class A defect. In the current study, the chromosomal assignment of Piga, as well as of Pigf and Pigh, two additional genes involved in GPI-anchor biosynthesis, has been established using a mouse interspecific backcross mapping technique. In contrast to Piga, both human and mouse Pigf and Pigh genes map to autosomes. The location of Pigf and Pigh suggests that mutations on both alleles of these autosomal genes would be necessary to produce PNH. This helps to explain the predominant class A defect in PNH.

Animals↗