Studies on the molecular mechanisms of transcriptional regulation of human immunodeficiency virus (HIV)

人类免疫缺陷病毒（HIV）转录调控分子机制研究

• 批准号: 18390143
• 负责人: OKAMOTO Takashi
• 金额: $ 9.67万
• 依托单位: Nagoya City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390143/
• 关键词: Human Immunodeficiencv Virus; Transcriptional regulation; Viral latency; NF-kB; Signal transduction; Tat; 3D structure; AKIP1; Tat; 計算化学; 立体構造; AP-4

In this study project I have been attempting to achieve the following goals:(1) To elucidate molecular mechanisms by which gene expression of HIV is controlled, which includes the following subprojects:(1-1) Actions of cellular transcriptional activator, namely NF-kB by identifying protein partners that interact with NF-kB major subunit p65(1-2) Mechanisms of NF-kB activation through intracellular signaling cascades(1-3) Elucidation of cellular transcriptional repressor to understand the mechanism in maintaining the viral latency in the infected cells(1-4) Analysis of molecular actions of viral transcriptional activator Tat by elucidating the 3D-struvture of functional tri-molecular complex comprising Tat-TAR (RNA target)-Cyclin T1 (a major subunit of P-TEFb, positive transcriptional elongation factor using computational chemistry.(1-5) Effects of novel chemical inhibitors for histone deacetylase (HDAC) on the HIV viral replication in the latently infected cells.(2) To develop novel st … More rategy in blocking HEV with NF-kB and Tat as target molecules. Within these two years, we have achieved the following:(1-1) -> We have identified two novel cellular proteins that interact with the p65 subunit of NF-kB, namely AKIP1(Gao, et. al., J Biol Chem 283:7834-7843, 2008) and FKBP4 (in preparation). These proteins were initially identified by yeast two-hybrid screen and the protein-protein interaction was confirmed both in vitro and in vivo (live cells). In particular, the AKIP1-p65 interaction gave a clue to solve the long-standing question regarding the effects of cAMP-PKA-signaling on the NF-kB activation signaling. We found that AKIP1 plays a deterministic role. When AKIP1 is absent, the cAMP-PKA signaling inhibits the NF-kB activation. However, when AKIP1 is abundantly present in cells, the cAMP-PKA signaling augments the NF-kB activation signaling through actively transferring NF-kB protein to the nuclei and facilitating the Ser 276 phosphorylation of p65 mediated by PKAc. With regard to RAI that we have reported in 1999 (J Biol Chem), we found its novel action in regulating trophoblast differentiation (Minekawa et al. Endocrinology 148: 5803-5810, 2007).(1-2) ->We found that the cAMP-PKA signal has dual roles in controlling the cellular threshold in activating NF-kB and thus latently infected BIV provirus as described in (1-1)(1-3) ->We have identified a cellular transcriptional repressor protein AP-4 that involves the maintenance of latent HIV provirus (Imai et at, J Biol Chem 281,12495-12505, 2006). We found that AP-4 constitutively binds to the target site located near the TATAbox of LTR within the HIV 1 proviral DNA and inhibits viral transcription through blocking the binding of TBP to the TATAbox and bringing the HDAC proteins to close chromatin structures. In fact, in cells where HN-1 is latently infected, AP-4 is found bound to the target site near the TATAbox of HIV-1 LTR and immediately released from the HIV LTR upon stimulation such as by TNF signaling and NF-kB activation.(1-4) ->We have elucidated the tri-moleculer structure of Tat-TAR-Cyclin T1 in silico using the published coordinates of Tat, TAR and Cyclin T1 and a novel molecular docking software(Tomoda et al. Cancer Sci, 2008, in press). The contact amino acids were mutated by mutagenesis to confirm the complex structure. However, in order to further solidify the data, we are still analyzing the details of contact surfaces. We have also initiated the molecular design of possible Tat inhibitors and have identified several such candidates. The actual inhibitory actions of these compounds are still in progress.(1-5) ->We found some novel HDAC inhibitors can efficientry activate the latent HIV-1 independently from NF-kB. The paper has been submitted to a journal.(2) ->We published some additional papers that describe the actions and identification of novel compounds that inhibit NF-kB(Victoriano et al, Antimicr. Agents Chemother 50:547-555, 2006; Tanaka, et al Eur J Pharm 565:212-219, 2007). Less

在这项研究项目中，我一直在尝试实现以下目标：（1）阐明控制HIV的基因表达的分子机制，其中包括以下亚参数：（1-1）细胞转录激活剂的作用，即通过与NF-KB大型亚基P65（1-2）相互作用的NF-KB识别NF-KB的作用，即NF-KB的作用。级联（1-3）阐明细胞转录表示，以了解感染细胞中病毒潜伏期的机制（1-4）通过阐明病毒转录激活剂TAT的分子作用，通过阐明了功能性三型复杂型TAT-TAT TAT-TAT-TAT（RNA TARGONT）的3D-ruvuter（RNA）（RNA）的3D-构型（RNA （1-5）新型化学抑制剂对组蛋白脱乙酰基酶（HDAC）对潜在受感染细胞中HIV病毒复制的影响（2）在用NF-KB和TAT作为靶分子的阻断HEV中发展新型ST…更多的ST…更多。在这两年内，我们达到了以下几点：（1-1） - >我们已经确定了与NF-KB的p65亚基相互作用的两种新型细胞蛋白，即AKIP1（Gao等，et。Et。Et。，J Biol Chem 283：7834-7843，2008）和FKBP4（在制备中）。这些蛋白质最初是通过酵母两杂化筛选鉴定的，并且在体外和体内都证实了蛋白质 - 蛋白质相互作用（活细胞）。特别是，AKIP1-P65相互作用提供了一个线索，以解决有关CAMP-PKA信号对NF-KB激活信号的影响的长期问题。我们发现AKIP1起着确定性的作用。当不存在AKIP1时，CAMP-PKA信号传导会抑制NF-KB激活。但是，当细胞中明显存在AKIP1时，CAMP-PKA信号传导通过主动将NF-KB蛋白转移到核并支撑PKAC介导的P65的Ser 276磷酸化中来增强NF-KB激活信号传导。 With regard to RAI that we have reported in 1999 (J Biol Chem), we found its novel action in determining trophoblast differentiation (Minekawa et al. Endocrinology 148: 5803-5810, 2007).(1-2) ->We have identified a cellular transcriptional replica protein AP-4 that involves the maintenance of latent HIV provirus (Imai et at, J Biol Chem 281,12495-12505，2006）。我们发现，AP-4与位于HIV 1前LTR的Tatabox附近的目标位点结合，并通过阻断TBP与TATABOX与TATABOX结合并将HDAC蛋白结合到亲密铬蛋白结构而抑制病毒转录。实际上，在HN-1受到潜在感染的细胞中，发现AP-4与HIV-1 LTR的Tatabox附近的目标部位结合，并立即从HIV LTR中释放出刺激，例如通过TNF信号传导和NF-KB激活。（1-4） - >>>我们已经阐明了使用TAT-TAT-TAT-CYCLIN TAT-CYCLIN TICOCLIN TARICCLIN tARICCLIN tATIN和TAT的TRI-MOLECOLITER TATAR-MELATIAN和NF-KB。 Cyclin T1和一种新型的分子对接软件（Tomoda等人Cancer Sci，2008年，印刷中）。通过诱变突变接触氨基酸以确认复杂结构。但是，为了进一步巩固数据，我们仍在分析接触表面的细节。我们还启动了可能的TAT抑制剂的分子设计，并确定了几个此类候选者。这些化合物的实际抑制作用仍在进行中。（1-5） - >我们发现一些新型的HDAC抑制剂可以有效地从NF-KB独立地激活潜在的HIV-1。该论文已提交给期刊。（2） - >我们发表了一些其他论文，描述了抑制NF-KB的新型化合物的作用和鉴定（Victoriano等，Antimicr。Antimicr。ChemothersChemother Chemother 50：547-555，2006; Tanaka等人Tanaka等人，Tanaka等人Eur J Pharm 565：212-219，2007）。较少的

项目成果

Sensitive and specific enzyme-linked immunosorbent assay using chemiluminescence for detection of severe acute respiratory syndrome viral infection

• DOI: 10.1128/jcm.01006-07
• 发表时间: 2008-01-01
• 期刊: JOURNAL OF CLINICAL MICROBIOLOGY
• 影响因子: 9.4
• 作者: Fujimoto, Kotaro;Chan, Kwok-Hung;Okamoto, Takashi
• 通讯作者: Okamoto, Takashi

Production of Immunogenic Tat Proteinsin Tomato Plants

番茄植物中免疫原性 Tat 蛋白的生产

• 发表时间: 2007
• 作者: M Cueno;Antonio C.Laurena;Yasutomi Y;Barzaga N.and Okamoto T.
• 通讯作者: Barzaga N.and Okamoto T.

Tat-TAR-PTEFb(Cyclint1)を標的としたin sillico薬剤スクリーニング

针对 Tat-TAR-PTEFb (Cyclint1) 的计算机药物筛选

• 发表时间: 2007
• 作者: 朝光 かおり;日比 悠里名;岡本 尚
• 通讯作者: 岡本 尚

Sawanpanyalert, P., Yanai H., Hara T., Yamazaki S., Yamamoto N., Okamoto T.: A single nucleotide synonymous mutation in gag gene controlling human immuno-deficiency virus type 1 virion production

Sawanpanyalert，P.，Yanai H.，Hara T.，Yamazaki S.，Yamamoto N.，Okamoto T.：控制人类免疫缺陷病毒1型病毒粒子产生的gag基因中的单核苷酸同义突变

• 发表时间: 2007
• 期刊: J. Virol 81
• 作者: Hamano;T.;Matsuo;K.;Hibi;Y.;Victoriano;A-F;B.;Takahashi;N.;Mabuchi;Y.;Soji;T.;Irie;S
• 通讯作者: S

Magnolia ovoata extract and its active component magnolol prevnt skin photoaging via inhibition of nuclear fator κB

卵形厚朴提取物及其活性成分厚朴酚通过抑制核因子κB预防皮肤光老化

• 发表时间: 2007
• 期刊: Eur.Journal of Pharmacology 565
• 作者: Itoh;Y.;Tanaka.K.
• 通讯作者: Tanaka.K.