学位論文内容の要旨
博士の専攻分野の名称

博士（医 学）

氏 名

鈴木 正宣

学 位 論 文 題 名TRIM39 negatively regulates NFκB signals through stabilization of Cactin(TRIM39 は Cactin の安定化を介して、NFκB シグナルを負に制御する。)
Background and Objectives
NF B functions as an important regulator for cell survival, immunity, inflammation, carcinogenesis andorganogenesis. In this NF B signaling pathway, ubiquitination plays important roles in regulation of theactivation of NF B. Skp1-Cul1-F-box protein complex (SCF)-type ubiquitin ligase, SCF -TrCP interacts
with and ubiquitinates I B protein phosphorylated by IKK. Furthermore, the activation of IKK requires
K63-linked polyubiquitin [2,6]. It has also been reported that linear ubiquitin chains are necessary for
NF B activation [7]. These findings indicate that activation of the NF B signaling pathway greatlydepends on several steps of ubiquitination.
Ubiquitination is an important posttranslational modification used by eukaryotic cells. The conjugation ofubiquitin to the target protein is catalyzed by several components, including ubiquitin-activating enzyme(E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3).
E3 ubiquitin ligases are the most directly responsible for recognition of the substrate in the ubiquitin
conjugation system. E3 ubiquitin ligases have been classified into three groups. Tripartite motif-containing(TRIM) proteins are defined as proteins that consist of a RING domain, one or two B-boxes, a coiled-coilmotif, and carboxyl-terminal unique domains. TRIM family proteins are involved in various biologicalprocesses, and their alterations therefore often lead to diverse clinical disorders, such as developmentaldisorders, neurodegenerative diseases, vulnerability to viral infection, and carcinogenesis. It has beenreported that approximately half of the TRIM proteins are involved in inflammatory signaling pathways.
TRIM39 is a one of the TRIM family proteins and has an E3 activity, leading to the degradation of some
proteins, while TRIM39 has been reported to stabilize some proteins. The TRIM39 gene is located in theMHC region within chromosome 6p21–23, in which there are six other TRIM genes (TRIM10, TRIM15,TRIM26, TRIM27, TRIM31 and TRIM38) and many genes that are related to immune response. It has beenreported that TRIM39 is involved in the regulation of type I interferon response. In addition, a single
nucleotide polymorphism (SNP) on exon 9 of TRIM39 has been identified as one of the genetic factors in
Behcet's disease by multiple logistic regression analysis. These findings suggest that TRIM39 is related toinflammatory signaling pathways. However, the role of TRIM39 in inflammatory signaling pathways has
not been fully elucidated.
In this study, with the aim of elucidating the molecular function of TRIM39 in inflammatory signaling
pathways, we performed yeast two-hybrid screening using TRIM39 as bait, and examined how TRIM39
affects the identified protein and functions in inflammatory signaling pathways.
Materials and Methods
1. Cloning of cDNAs and plasmid construction
Human TRIM39 and Cactin cDNAs were amplified from a human B cell cDNA library by polymerase
chain reaction (PCR). The ampliﬁed fragments were subcloned into expression vectors for eukaryotic cells,
for a yeast two-hybrid system, and for a baculovirus protein expression system.
2. Yeast two-hybrid screening
To screen for proteins that interact with TRIM39, the yeast strain L40 was transformed with a B-cellMatchmaker cDNA library using the lithium acetate method.
3. Transfection, immunoprecipitation and immunoblot analysis
HEK293T cells were transfected by the calcium phosphate method. The cell lysates were centrifuged andthe resulting supernatant was incubated with an antibody. After addition of protein A-Sepharose, each of themixtures rotated. The resin was washed, and then boiled in SDS sample buffer. Immunoblot analysis wasperformed to detect immune complexes.
4. Generation of Recombinant Protein.
His6-tagged TRIM39 and Cactin were expressed in the Sf9 insect cell line by a baculovirus protein
expression system. The recombinant His6-tagged proteins were purified by using ProBond metal affinity
beads.

5. In vitro binding assay
The mixtures of purified recombinant proteins were incubated with the antibody. After addition of protein
A-Sepharose, each of the mixtures rotated. The resin was washed and then boiled in SDS sample buffer.
Immunoblot analysis was performed to detect immune complexes.
6. RNA interference
In some experiments, siRNA were transfected into HEK293T cells or HeLa cells with Lipofectamine
RNAiMAX.
7. Protein stability assay with cycloheximide.
HEK293T cells were transfected with siRNA specific for human TRIM39 (siTRIM39). Forty-eight hr
after transfection, the cells were cultured in the presence of cycloheximide for the indicated times. Cell
lysates were subjected to immunoblot analysis.
8. Real-time PCR
Total RNA was isolated from HEK293T or HeLa cells, followed by reverse transcription. The resulting
cDNA was subjected to real-time PCR. The average threshold cycle (Ct) was determined from independent
experiments.
9. Dual-luciferase assayExpression vectors, reporter plasmids and the internal control plasmid coding Renilla luciferase weretransfected into the cells. Forty-eight hr after transfection, cells were incubated with TNFα or with PolyICfor 6 or 12 hr, and assayed by the Dual-Luciferase Reporter Assay System.
ResultTo clarify the function of TRIM39, we isolated TRIM39-interacting proteins from a human B cell cDNA
library by yeast two-hybrid screens. Using TRIM39 as a bait, we obtained human Cactin as candidate. Toexamine whether TRIM39 interacts with Cactin in mammalian cells, we performed an in vivo binding assay,
which showed that TRIM39 specifically bound to Cactin in mammalian cells. Next, to examine whether
TRIM39 directly binds to Cactin, we performed an in vitro binding assay between TRIM39 and Cactin. The
result showed there was direct interaction between TRIM39 and Cactin.
We speculated that TRIM39 also affected the stability of Cactin. Protein stability analysis showed thatknockdown of TRIM39 caused a decrease of endogenous Cactin, suggesting that TRIM39 upregulates thestability of Cactin protein.
To confirm that Cactin regulates NFκB mediated transcription, we performed a luciferase reporter assay,
which showed that Cactin significantly suppressed NFκB-mediated transcriptional activity. We also foundthat the mRNA expression level and protein level of Cactin was significantly increased by TNFα treatment.
These findings indicated that Cactin also provides a negative feedback loop in the NFκB pathway.
Finally, we examined whether TRIM39 acts as a regulator of transcription including the NFκB andToll-like receptor (TLR). Luciferase reporter assays revealed that knockdown of TRIM39 significantly
promoted NFκB- and ISRE-mediated transcriptional activity induced by TNFα and PolyIC. Quantitative
real-time PCR revealed that the mRNA expression levels of IL-6 and IL-8 were significantly increased inHeLa cells in which TRIM39 had been knocked down. These findings suggest that TRIM39 negatively
regulated NFκB- and TLR-mediated transcriptional activity. Furthermore, we also showed by luciferase
assay that overexpression of Cactin suppressed the activity of NFκB even in cells in which TRIM39 hadbeen knocked down, which indicated that the stabilization of Cactin by TRIM39 negatively regulates NFκBactivity.
Discussion
In this study, we identified Cactin as TRIM39-interacting proteins. Cactin was identified as a novel
protein interacting with Cactus, the Drosophila ortholog of IκBα, which is important for negative regulationof the NFκB signaling pathway. Human Cactin has been reported to interact with IκBL (Inhibitor-κB-like),which is one of negative regulators of the NF B signaling pathway. The human IκBL is localized in theMHC class III region of chromosome 6, in which the TRIM39 gene is also localized.
According to a previous report, the SNP on exon 9 of TRIM39 is involved in Behcet's disease.
Furthermore, the polymorphism of the IκBL gene locus, which is known to interact with Cactin, isassociated with several inflammatory diseases such as, rheumatoid arthritis, Takayasu’s arteritis, ulcerativecolitis, systemic lupus erythematosus, Sjogren’s syndrome, type 1 diabetes, and multiple sclerosis. Thesefindings imply that the dysfunction of TRIM39, Cactin, and IκBL may be involved in the etiology ofinflammatory diseases.
Conclusion
In conclusion, we revealed that TRIM39 regulated NFκB- and TLR-mediated transcriptional activity
through stabilization of Cactin, which provided the negative feedback loop in the NFκB pathway. However,
it is still unclear how TRIM39 regulates NFκB activity with Cactin. Further study on these proteins willlead not only to the elucidation of their molecular function but also to therapeutic benefits for these
inflammatory diseases.