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Shigetsugu Hatakeyama - One of the best experts on this subject based on the ideXlab platform.
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TRIM29 regulates the assembly of DNA repair Proteins into damaged chromatin.
Nature communications, 2015Co-Authors: Yasushi Masuda, Hidehisa Takahashi, Shigeo Sato, Chieri Tomomori-sato, Anita Saraf, Michael P. Washburn, Laurence Florens, Ronald C. Conaway, Joan W. Conaway, Shigetsugu HatakeyamaAbstract:Although DNA double-strand break (DSB) repair is mediated by numerous Proteins accumulated at DSB sites, how DNA repair Proteins are assembled into damaged chromatin has not been fully elucidated. Here we show that a member of the Tripartite Motif Protein family, TRIM29, is a histone-binding Protein responsible for DNA damage response (DDR). We found that TRIM29 interacts with BRCA1-associated surveillance complex, cohesion, DNA-PKcs and components of TIP60 complex. The dynamics of the TRIM29-containing complex on H2AX nucleosomes is coordinated by a cross-talk between histone modifications. TRIM29 binds to modified histone H3 and H4 tails in the context of nucleosomes. Furthermore, chromatin binding of TRIM29 is required for the phosphorylation of H2AX and cell viability in response to ionizing radiation. Our results suggest that TRIM29 functions as a scaffold Protein to assemble DNA repair Proteins into chromatin followed by efficient activation of DDR.
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TRIM29 regulates the p63-mediated pathway in cervical cancer cells.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2015Co-Authors: Yasushi Masuda, Hidehisa Takahashi, Shigetsugu HatakeyamaAbstract:Cell invasion and adhesion play an important role in cancer metastasis and are orchestrated by a complicated network of transcription factors including p63. Here, we show that a member of the Tripartite Motif Protein family, TRIM29, is required for regulation of the p63-mediated pathway in cervical cancer cells. TRIM29 knockdown alters the adhesion and invasion activities of cervical cancer cells. TRIM29 knockdown and overexpression cause a significant decrease and increase of TAp63α expression, respectively. TRIM29 knockdown alters the expression pattern of integrins and increases ZEB1 expression. TRIM29 is required for suppression of an increase in the adhesion activity of cells by TAp63α. These findings suggest that TRIM29 regulates the p63-mediated pathway and the behavior of cervical cancer cells.
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TRIM29 as a novel prostate basal cell marker for diagnosis of prostate cancer.
Acta histochemica, 2014Co-Authors: Yukiko Kanno, Masashi Watanabe, Taichi Kimura, Katsuya Nonomura, Shinya Tanaka, Shigetsugu HatakeyamaAbstract:Tripartite Motif Protein 29 (TRIM29) is one of the TRIM family Proteins, some of which function as E3 ubiquitin ligases. In this study, we investigated the usefulness of TRIM29 for diagnosis of prostate cancer. Prostate tissues including carcinoma and non-carcinoma tissues obtained by needle biopsy and radical prostatectomy were used. Immunohistochemistry was performed according to standard procedures using an antibody against TRIM29. Immunohistochemical staining with an antibody against 34βE12, which recognizes cytokeratins 1, 5, 10 and 14, was performed as a control. Basal cells of normal prostatic glands were stained with anti-TRIM29 antibody in all cases, whereas prostate cancer tissues had no or little staining with anti-TRIM29 antibody. TRIM29 is selectively expressed in basal cells of the normal prostate gland, and immunohistochemical staining with anti-TRIM29 antibody showed the same expression pattern as that with 34βE12 in prostate cancer and its benign mimics. Our data indicate that TRIM29 may be useful for distinguishing prostate cancers from benign tissues.
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Tripartite Motif Protein 32 facilitates cell growth and migration via degradation of abl interactor 2
Cancer Research, 2008Co-Authors: Satoshi Kano, Naoto Miyajima, Satoshi Fukuda, Shigetsugu HatakeyamaAbstract:Tripartite Motif Protein 32 (TRIM32) mRNA has been reported to be highly expressed in human head and neck squamous cell carcinoma, but the involvement of TRIM32 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that TRIM32 binds to Abl-interactor 2 (Abi2), which is known as a tumor suppressor and a cell migration inhibitor, and we showed that TRIM32 mediates the ubiquitination of Abi2. Overexpression of TRIM32 promoted degradation of Abi2, resulting in enhancement of cell growth, transforming activity, and cell motility, whereas a dominant-negative mutant of TRIM32 lacking the RING domain inhibited the degradation of Abi2. In addition, we found that TRIM32 suppresses apoptosis induced by cis-diamminedichloroplatinum (II) in HEp2 cell lines. These findings suggest that TRIM32 is a novel oncogene that promotes tumor growth, metastasis, and resistance to anticancer drugs. [Cancer Res 2008;68(14):5572–80]
Leo C James - One of the best experts on this subject based on the ideXlab platform.
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cytosolic fc receptor trim21 inhibits seeded tau aggregation
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: William A Mcewan, Marina Vaysburd, Benjamin Falcon, Dean Clift, Michel Goedert, Adrian L. Oblak, Bernardino Ghetti, Leo C JamesAbstract:Alzheimer’s disease (AD) and other neurodegenerative disorders are associated with the cytoplasmic aggregation of microtubule-associated Protein tau. Recent evidence supports transcellular transfer of tau misfolding (seeding) as the mechanism of spread within an affected brain, a process reminiscent of viral infection. However, whereas microbial pathogens can be recognized as nonself by immune receptors, misfolded Protein assemblies evade detection, as they are host-derived. Here, we show that when misfolded tau assemblies enter the cell, they can be detected and neutralized via a danger response mediated by tau-associated antibodies and the cytosolic Fc receptor Tripartite Motif Protein 21 (TRIM21). We developed fluorescent, morphology-based seeding assays that allow the formation of pathological tau aggregates to be measured in situ within 24 h in the presence of picomolar concentrations of tau seeds. We found that anti-tau antibodies accompany tau seeds into the cell, where they recruit TRIM21 shortly after entry. After binding, TRIM21 neutralizes tau seeds through the activity of the proteasome and the AAA ATPase p97/VCP in a similar manner to infectious viruses. These results establish that intracellular antiviral immunity can be redirected against host-origin endopathogens involved in neurodegeneration.
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intracellular antibody receptor trim21 prevents fatal viral infection
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Marina Vaysburd, Ruth E. Watkinson, Helen Cooper, Martin Reed, Kevin F Oconnell, James Cruickshanks, Jackie Smith, Leo C JamesAbstract:Host species have evolved mechanisms that can inhibit pathogen replication even after a cell has been successfully invaded. Here we show that Tripartite-Motif Protein 21 (TRIM21), a ubiquitously expressed E3 ubiquitin ligase that targets viruses inside the cytosol, protects mice against fatal viral infection. Upon infection with mouse adenovirus-1, naive mice lacking TRIM21 succumb to encephalomyelitis within 7 d. In contrast, wild-type mice rapidly up-regulate TRIM21 and control viremia. Trim21 heterozygous mice have a haploinsufficiency phenotype in which reduced TRIM21 expression leads to a viral load that is higher than wild types but lower than knockouts. TRIM21 is a high-affinity antibody receptor that allows antibodies to operate inside an infected cell. In passive transfer experiments at high viral dose, antisera that fully protects wild-type mice fails to protect most Trim21 knockout animals. These results demonstrate that TRIM21 provides potent antiviral protection and forms an important part of the humoral immune response.
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The V86M mutation in HIV-1 capsid confers resistance to TRIM5α by abrogation of cyclophilin A-dependent restriction and enhancement of viral nuclear import
Retrovirology, 2013Co-Authors: Maxime Veillette, Leo C James, Katsiaryna Bichel, Paulina Pawlica, Stefan M.v. Freund, Mélodie B. Plourde, Quang Toan Pham, Carlos Reyes-moreno, Lionel BerthouxAbstract:Background HIV-1 is inhibited early after entry into cells expressing some simian orthologues of the Tripartite Motif Protein family member TRIM5α. Mutants of the human orthologue (TRIM5αhu) can also provide protection against HIV-1. The host Protein cyclophilin A (CypA) binds incoming HIV-1 capsid (CA) Proteins and enhances early stages of HIV-1 replication by unknown mechanisms. On the other hand, the CA-CypA interaction is known to increase HIV-1 susceptibility to restriction by TRIM5α. Previously, the mutation V86M in the CypA-binding loop of HIV-1 CA was found to be selected upon serial passaging of HIV-1 in cells expressing Rhesus macaque TRIM5α (TRIM5αrh). The objectives of this study were (i) to analyze whether V86M CA allows HIV-1 to escape mutants of TRIM5αhu, and (ii) to characterize the role of CypA in the resistance to TRIM5α conferred by V86M.
Gj Towers - One of the best experts on this subject based on the ideXlab platform.
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Isolation of an active Lv1 gene from cattle indicates that Tripartite Motif Protein-mediated innate immunity to retroviral infection is widespread among mammals
J VIROL, 2006Co-Authors: Gj TowersAbstract:Lv1/TRIM5 alpha (Tripartite Motif 5 alpha) has recently emerged as an important factor influencing species-specific permissivity to retroviral infection in a range of primates, including humans. Old World monkey TRIM5 alpha blocks human immunodeficiency virus type 1 (HIV-1) infectivity, and the human and New World monkey TRIM5 alpha Proteins are inactive against HIV-1 but active against divergent murine (N-tropic marine leukemia virus [MLV-N]) and simian (simian immunodeficiency virus from rhesus macaque [SIVmac]) retroviruses, respectively. Here we demonstrate antiviral activity of the first nonprimate TRIM Protein, from cattle, active against divergent retroviruses, including HIV-1. The number of closely related human TRIM sequences makes assignment of the bovine sequence as a TRIM5 alpha ortholog uncertain, and we therefore refer to it as bovine Lv1. Bovine Lv1 is closely related to primate TRIN15 alpha Proteins in the N-terminal RING and B-box 2 domains but significantly less homologous in the C-terminal B30.2 domain, particularly in the region shown to influence antiviral specificity. Intriguingly, some viruses restricted by bovine Lv1, including HIV-1 and MLV-N, are unable to synthesize viral DNA by reverse transcription, whereas restricted HIV-2 makes normal amounts of DNA. The data support the conclusion that TRIM Protein-mediated restriction of retroviral infection is a more common attribute of mammals than previously appreciated.
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Restriction of retroviruses by TRIM5 alpha
FUTURE VIROL, 2006Co-Authors: Gj TowersAbstract:Despite multiple transfers of primate lentiviruses to humans, the current AIDS pandemic has resulted from a single zoonosis of simian immunodeficiency virus from chimpanzees. The rarity of successful zoonosis is due to effective species barriers that are mediated partly by dominant antiviral factors, termed restriction factors. The Tripartite Motif Protein TRIM5 alpha has emerged as an important restriction factor controlling species-specific retroviral replication. TRIM5 alpha was identified as an antiviral factor, active against HIV-1, in rhesus macaques. Subsequently, it was shown to encode previously described antiviral factors in humans (Ref 1) and monkeys (Lv1). TRIM5 alpha. causes a block to sensitive retroviral infection after viral entry into the target cell and usually before viral DNA synthesis. This review considers the role of TRIM5 alpha as an antiviral Protein in mammals. Recent results from mutational analysis of TRIM5 alpha and their contribution to a mechanistic model for TRIM5 alpha antiviral activity are discussed, as is the future for postentry restriction factors.
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Control of viral infectivity by Tripartite Motif Proteins
HUM GENE THER, 2005Co-Authors: Gj TowersAbstract:It is of great interest to understand the molecular details of the pathways that constitute species barriers to viral infection. The Tripartite Motif Protein TRIM5 alpha has emerged as an important mediator of species-specific retroviral replication and innate immunity. This review considers the role of TRIM5 alpha as an antiviral Protein in mammals. The methods used to identify species-specific restriction to retroviral infection, and the identification of TRIM5 alpha itself, are outlined. TRIM5 alpha mediates an early postentry block to sensitive retroviral infection, usually before viral DNA synthesis. Results from mutational analysis of TRIM5 alpha and their contribution to a mechanistic model for TRIM5 alpha antiviral activity are discussed. The antiviral role of other TRIM Proteins is considered, as is the role of TRIM5 alpha cytoplasmic bodies.
Hee Jeong Kong - One of the best experts on this subject based on the ideXlab platform.
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molecular characterization of rhodeus uyekii Tripartite Motif Protein 1 trim1 involved in ifn γ lps induced nf κb signaling
Fish & Shellfish Immunology, 2018Co-Authors: Julan Kim, Ju-won Kim, Dong-gyun Kim, Bo-hye Nam, Young-ok Kim, Jung Youn Park, Hee Jeong KongAbstract:Abstract The Tripartite Motif-containing (TRIM) Proteins are involved in a wide range of cellular processes, and the role of TRIM1 in immunity has been explored. However, fundamental studies on fish TRIM1 are lacking. In this study, we cloned and characterized TRIM1 cDNA from the Korean rose bitterling, Rhodeus uyekii (RuTRIM1). Two RuTRIM1 isoforms (RuTRIM1-X1 and RuTRIM1-X2) were identified. The coding sequence (CDS) of RuTRIM1-X1 comprised 2157 bp encoding a 718-aa Protein, and the CDS of RuTRIM1-X2 comprised 1929 bp encoding a 642-aa Protein. Both RuTRIM1 isoforms contained a RING finger domain, B-box 1, B-box 2, coiled-coil domain, COS box, FN3 Motif, and PRY/SPRY domain. The deduced RuTRIM1-X1 and RuTRIM1-X2 Proteins had high amino acid identity (76.27–98.89%) with orthologs from various other species, and a phylogenetic tree was constructed. RuTRIM1-X1 and RuTRIM1-X2 mRNA were expressed in all tissues examined, with the highest expression levels detected in the hepatopancreas. During early development, RuTRIM1-X1 and RuTRIM1-X2 mRNA levels changed differently from the gastrula period to the first feeding stage. An in vivo ubiquitination assay showed that RuTRIM1 exhibited RING-dependent E3 ubiquitin ligase activity, mainly by comparing RuTRIM1-X2 to RuTRIM1-X1. The subcellular localization of the two RuTRIM1 Protein isoforms was characterized, revealing that they formed aggregates in cytoplasmic bodies in Raw264.7 cells. Interferon-γ/lipopolysaccharide-induced nuclear factor-κB signaling was negatively regulated by RuTRIM1-X1 and RuTRIM1-X2, and the negative effect was reversed in RING deletion mutants. To our knowledge, this is the first study to characterize fish TRIM1, which may play a role in the inflammatory response.
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Molecular characterization of Rhodeus uyekii Tripartite Motif Protein 1 (TRIM1) involved in IFN-γ/LPS-induced NF-κB signaling.
Fish & shellfish immunology, 2018Co-Authors: Julan Kim, Ju-won Kim, Dong-gyun Kim, Bo-hye Nam, Young-ok Kim, Jung Youn Park, Hee Jeong KongAbstract:Abstract The Tripartite Motif-containing (TRIM) Proteins are involved in a wide range of cellular processes, and the role of TRIM1 in immunity has been explored. However, fundamental studies on fish TRIM1 are lacking. In this study, we cloned and characterized TRIM1 cDNA from the Korean rose bitterling, Rhodeus uyekii (RuTRIM1). Two RuTRIM1 isoforms (RuTRIM1-X1 and RuTRIM1-X2) were identified. The coding sequence (CDS) of RuTRIM1-X1 comprised 2157 bp encoding a 718-aa Protein, and the CDS of RuTRIM1-X2 comprised 1929 bp encoding a 642-aa Protein. Both RuTRIM1 isoforms contained a RING finger domain, B-box 1, B-box 2, coiled-coil domain, COS box, FN3 Motif, and PRY/SPRY domain. The deduced RuTRIM1-X1 and RuTRIM1-X2 Proteins had high amino acid identity (76.27–98.89%) with orthologs from various other species, and a phylogenetic tree was constructed. RuTRIM1-X1 and RuTRIM1-X2 mRNA were expressed in all tissues examined, with the highest expression levels detected in the hepatopancreas. During early development, RuTRIM1-X1 and RuTRIM1-X2 mRNA levels changed differently from the gastrula period to the first feeding stage. An in vivo ubiquitination assay showed that RuTRIM1 exhibited RING-dependent E3 ubiquitin ligase activity, mainly by comparing RuTRIM1-X2 to RuTRIM1-X1. The subcellular localization of the two RuTRIM1 Protein isoforms was characterized, revealing that they formed aggregates in cytoplasmic bodies in Raw264.7 cells. Interferon-γ/lipopolysaccharide-induced nuclear factor-κB signaling was negatively regulated by RuTRIM1-X1 and RuTRIM1-X2, and the negative effect was reversed in RING deletion mutants. To our knowledge, this is the first study to characterize fish TRIM1, which may play a role in the inflammatory response.
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Molecular characterization of Tripartite Motif Protein 25 (TRIM25) involved in ERα-mediated transcription in the Korean rose bitterling Rhodeus uyekii.
Comparative biochemistry and physiology. Part B Biochemistry & molecular biology, 2012Co-Authors: Hee Jeong Kong, Ye Ji Lee, Jihye Shin, Hyun Kook Cho, Woo-jin Kim, Hyung Soo Kim, Jaehun Cheong, Young Chang Sohn, Sang-jun Lee, Bong-seok KimAbstract:Abstract Tripartite Motif-containing 25 (TRIM25), also known as estrogen-responsive finger Protein (EFP), plays an essential role in cell proliferation and innate immunity. In the present study, we isolated and characterized the TRIM25 cDNA of the Korean rose bitterling Rhodeus uyekii , designated RuTRIM25. It encodes an open reading frame of 669 amino acids containing an N-terminal RBCC Motif composed of a RING domain, two B boxes, and a coiled-coil domain and a C-terminal B30.2 (PRY/SPRY) domain. RuTRIM25 shows strong homology (79.7%) to zebrafish TRIM25 and shared 32.4–28.8% homology with TRIM25 from other species, including mammals. RuTRIM25 mRNA was expressed ubiquitously. It was highly expressed in the ovary, spleen, and liver and moderately in the stomach and intestine of normal Korean rose bitterling. The intracellular localization of RuTRIM25 in HEK293T cells was diffusely localized in the cytoplasm and its RING domain deletion mutant (RuTRIM25ΔR) was detected diffusely with some aggregates in the cytoplasm. RuTRIM25, but not RuTRIM25ΔR, is ubiquitinated in vivo . Ectopic expression of RuTRIM25 synergistically activated the estrogen receptor (ER)-mediated luciferase reporter activity in a dose-dependent manner in HEK293T cells. Together, these results suggest that the RuTRIM25 regulates the ER-mediated transcription in fish similarly to its mammalian counterpart.
Kiyohiko Hatake - One of the best experts on this subject based on the ideXlab platform.
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Abstract B68: Ubiquitin E3 ligase, Tripartite Motif Protein 68 (TRIM68) inhibits TCP‐1β function and can be a target of imatinib‐resistance
Drug Resistance and Modifiers, 2009Co-Authors: Ryoko Kuniyoshi, Yasuhito Terui, Yuji Mishima, Satoshi Matsusaka, Kiyohiko HatakeAbstract:Background: Resistance to imatinib mesylate is a major problem in chronic myeloid leukaemia (CML) treatment. Most of the studies about the resistance have focused on point mutations in BCR/ABL. However, other mechanisms of resistance that do not imply mutations in BCR/ABL have been also described. The identification of new Proteins induced by imatinib may lead to find the novel potent molecular targets in imatinib‐resistant CML. Methods: K562 cells were treated with or without 1 µM imatinib for 24 hours, and then differential display between them was performed. TRIM68 expression was examined by RT‐PCR, and in vivo ubiquitination or sumoylation assay was performed by transfection experiment and Western blot analysis. The substrates for TRIM68 were analyzed by mass spectorometry. Results: From the results of RNA differential display, we found that the expression of TRIM68 mRNA was increased when the K562 cells were treated with 1 µM imatinib for 24 hours. TRIM68 Protein possesses a RING finger domain at its N‐terminal site. Since many RING‐finger Proteins have been identified as E3 ligases for ubiquitination or sumoylation, we examined whether TRIM68 functions as an E3 ligase for ubiquitination or sumoylation. To examine the function of TRIM68 as an E3 ligase, wild type TRIM68 and a RING domain deletion mutant of TRIM68 (TRIM68/ΔR) genes were constructed into a mammalian expression vector and they were transfected into MCF7 cells. TRIM68 had auto‐ubiquitination activity but not autosumoylation activity in vivo assays. This suggests that TRIM68 is could be an ubiquitin E3 ligase but not sumo E3 ligase. Moreover, wild type TRIM68 promoted the whole ubiqutination in the cells, whereas TRIM68/ΔR prevented the ubiquitination inside of the cells. To identify the TRIM68‐interacting Proteins, we transfected FLAG‐tagged wild type TRIM68 gene or B30.2/SPRY domain of TRIM68 gene into MCF7 cells, and immunoprecipitation with FLAG‐M2 agarose was performed and mass spectrometric analysis was performed. As the results, we identified the members of molecular chaperone T‐complex polypeptide 1 (TCP‐1) complex, TCP‐1β and heat shock Protein 70 interacting with TRIM68 at the B30.2/SPRY domain. Then, we examined whether TCP‐1β is one of the substrates for TRIM68‐ related ubiqutination. TCP‐1β was ubiquitinated by wild type TRIM68, but not by TRIM68/ΔR. Furthermore, the ubiquitination of TCP‐1β was accumulated by the treatment with a proteasome inhibitor MG132. These suggested that TCP‐1β is one of the substrates for TRIM68. Conclusion: We found that TRIM68 is induced by the treatment with imatinib and functions as an ubiquitin E3 ligase. Furthermore, we identified that TCP‐1β is a substrate of TRIM68. TRIM68 may inhibit the function of TCP‐1β as a chaperone by ubiquitination and proteasome‐mediated degradation. TRIM68 could be a new target in the imatinib‐resistant CML. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B68.
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abstract b68 ubiquitin e3 ligase Tripartite Motif Protein 68 trim68 inhibits tcp 1β function and can be a target of imatinib resistance
Molecular Cancer Therapeutics, 2009Co-Authors: Ryoko Kuniyoshi, Yasuhito Terui, Yuji Mishima, Satoshi Matsusaka, Kiyohiko HatakeAbstract:Background: Resistance to imatinib mesylate is a major problem in chronic myeloid leukaemia (CML) treatment. Most of the studies about the resistance have focused on point mutations in BCR/ABL. However, other mechanisms of resistance that do not imply mutations in BCR/ABL have been also described. The identification of new Proteins induced by imatinib may lead to find the novel potent molecular targets in imatinib‐resistant CML. Methods: K562 cells were treated with or without 1 µM imatinib for 24 hours, and then differential display between them was performed. TRIM68 expression was examined by RT‐PCR, and in vivo ubiquitination or sumoylation assay was performed by transfection experiment and Western blot analysis. The substrates for TRIM68 were analyzed by mass spectorometry. Results: From the results of RNA differential display, we found that the expression of TRIM68 mRNA was increased when the K562 cells were treated with 1 µM imatinib for 24 hours. TRIM68 Protein possesses a RING finger domain at its N‐terminal site. Since many RING‐finger Proteins have been identified as E3 ligases for ubiquitination or sumoylation, we examined whether TRIM68 functions as an E3 ligase for ubiquitination or sumoylation. To examine the function of TRIM68 as an E3 ligase, wild type TRIM68 and a RING domain deletion mutant of TRIM68 (TRIM68/ΔR) genes were constructed into a mammalian expression vector and they were transfected into MCF7 cells. TRIM68 had auto‐ubiquitination activity but not autosumoylation activity in vivo assays. This suggests that TRIM68 is could be an ubiquitin E3 ligase but not sumo E3 ligase. Moreover, wild type TRIM68 promoted the whole ubiqutination in the cells, whereas TRIM68/ΔR prevented the ubiquitination inside of the cells. To identify the TRIM68‐interacting Proteins, we transfected FLAG‐tagged wild type TRIM68 gene or B30.2/SPRY domain of TRIM68 gene into MCF7 cells, and immunoprecipitation with FLAG‐M2 agarose was performed and mass spectrometric analysis was performed. As the results, we identified the members of molecular chaperone T‐complex polypeptide 1 (TCP‐1) complex, TCP‐1β and heat shock Protein 70 interacting with TRIM68 at the B30.2/SPRY domain. Then, we examined whether TCP‐1β is one of the substrates for TRIM68‐ related ubiqutination. TCP‐1β was ubiquitinated by wild type TRIM68, but not by TRIM68/ΔR. Furthermore, the ubiquitination of TCP‐1β was accumulated by the treatment with a proteasome inhibitor MG132. These suggested that TCP‐1β is one of the substrates for TRIM68. Conclusion: We found that TRIM68 is induced by the treatment with imatinib and functions as an ubiquitin E3 ligase. Furthermore, we identified that TCP‐1β is a substrate of TRIM68. TRIM68 may inhibit the function of TCP‐1β as a chaperone by ubiquitination and proteasome‐mediated degradation. TRIM68 could be a new target in the imatinib‐resistant CML. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B68.
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Ubiquitin E3 Ligase, Tripartite Motif Protein 68 (TRIM68) Inhibits TCP-1 b Function by Proteasome-Mediated Degradation and May Overcome Imatinib-Resistance.
Blood, 2009Co-Authors: Yasuhito Terui, Ryoko Kuniyoshi, Yuji Mishima, Yuko Mishima, Kiyohiko HatakeAbstract:Abstract 3789 Poster Board III-725 [Background] Imatinib mesylate is effective therapy against Philadelphia chromosome-positive leukemia, but the resistance develops in all phases of the disease. The identification of new Proteins induced by imatinib may lead to find the novel potent molecular targets in imatinib-resistant CML. [Methods] K562 cells were treated with or without 1 mM imatinib for 24 hours, and then differential display between them was performed. TRIM68 expression was examined by RT-PCR, and in vivo ubiquitination or sumoylation assay was performed by transfection experiment and Western blot analysis. The substrates for TRIM68 were analyzed by mass spectorometry. [Results] As the results of RNA differential display, we found that the expression of TRIM68 mRNA was increased when the K562 cells were treated with 1 mM imatinib for 24 hours. TRIM68 Protein possesses a RING finger domain at its N-terminal site. Since many RING-finger Proteins have been identified as E3 ligases for ubiquitination or sumoylation (Meroni G, Diez-Roux G. TRIM/RBCC, a novel class of esingle Protein RING finger9 E3 ubiquitin ligases. Bioessays 2005; 27: 1147-57.), we examined whether TRIM68 functions as an E3 ligase for ubiquitination or sumoylation. To examine the function of TRIM68 as an E3 ligase, wild type TRIM68 and a RING domain deletion mutant of TRIM68 (TRIM68/¢R) genes were constructed into a mammalian expression vector and they were transfected into MCF7 cells. TRIM68 had auto-ubiquitination activity but not auto-sumoylation activity on the in vivo assays, suggesting that TRIM68 can be an ubiquitin E3 ligase but not sumo ligase. Moreover, wild type TRIM68 promoted the whole ubiqutination in the cells, whereas TRIM68/¢R prevented the ubiquitination inside of the cells. To identify the TRIM68-interacting Proteins, we transfected FLAG-tagged wild type TRIM68 gene or B30.2/SPRY domain of TRIM68 gene into MCF7 cells, and immunoprecipitation with FLAG-M2 agarose was performed and mass spectrometric analysis was performed. As the results, we revealed that the members of molecular chaperone T-complex polypeptide 1 (TCP-1) complex, TCP-1 b and heat shock Protein 70 (HSP70) interacted with TRIM68 at the B30.2/SPRY domain. Then, we examined whether TCP-1 b is one of the substrates for TRIM68-related ubiqutination. TCP-1 b was ubiquitinated by wild type TRIM68, but not by TRIM68/¢R. Furthermore, the ubiquitination of TCP-1 b was accumulated by the treatment with a proteasome inhibitor MG132. These suggested that TCP-1 b is one of the substrates for TRIM68. [Conclusions] We found that TRIM68 is induced by the treatment with imatinib and functions as an ubiquitin E3 ligase. Furthermore, we identified that TCP-1 b is a substrate of TRIM68. TRIM68 may inhibit the function of TCP-1 b as a chaperone by ubiquitination and proteasome-mediated degradation. TRIM68 is possible for a new target in the imatinib-resistant CML. Disclosures: No relevant conflicts of interest to declare.
