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

在本研究项目中，我一直试图实现以下目标：(1)阐明控制HIV基因表达的分子机制，包括下列子项目：(1-1)细胞转录激活因子的作用，即通过识别与核因子-kB主要亚基p65相互作用的蛋白质对(1-2)通过细胞内信号级联激活核因子-kB的机制(1-3)阐明细胞转录抑制物以了解在感染细胞中维持病毒潜伏的机制(1-4)通过阐明包括Tat-TAR(RNA靶标)-Cyclin T1(P-TEFb的一个主要亚基)的功能性三分子复合体的三维结构来分析病毒转录激活因子TAT的分子作用。(1-5)新型组蛋白脱乙酰酶化学抑制剂对潜伏感染细胞中艾滋病毒复制的影响。(2)开发新的st…以核因子-kB和Tat为靶分子阻断HEV的策略更多。在这两年的时间里，我们取得了以下成果：(1-1)-GT；我们鉴定了两种与NF-kB的p65亚基相互作用的新的细胞蛋白，即AKIP1(Gao，et.J Biol Chem 283：7834-7843,2008)和FKBP4(准备中)。通过酵母双杂交筛选初步鉴定了这些蛋白质，并在体外和体内(活细胞)证实了蛋白质之间的相互作用。特别是，AKIP1-p65的相互作用为解决cAMP-PKA信号对NF-kB激活信号的影响这一长期存在的问题提供了线索。我们发现AKIP1起着决定性的作用。当AKIP1缺失时，cAMP-PKA信号抑制了核因子-kB的激活。然而，当AKIP1在细胞中大量存在时，cAMP-PKA信号通过主动将NF-kB蛋白转移到细胞核并促进PKAc介导的P65的Ser276磷酸化而增强了NF-kB的激活信号。关于我们在1999年报道的RAI(J Biol Chem)，我们发现它在调节滋养层细胞分化方面具有新的作用(Minekawa等人)。(1-2)-我们发现cAMP-PKA信号在控制细胞阈值激活核因子-kB从而潜伏感染BIV前病毒方面具有双重作用，如(1-1)(1-3)-&gt；我们已经确定了一种细胞转录抑制蛋白AP-4，它涉及维持潜伏的HIV前病毒(Imai et at，J Biol Chem 281,12495-12505,2006年)。我们发现，AP-4通过阻断TBP与TATAbox的结合，使HDAC蛋白接近染色质结构，与HIV-1前病毒DNA中LTRTATAbox附近的靶点结合，从而抑制病毒转录。事实上，在HN-1潜伏感染的细胞中，AP-4被发现与HIV-1 LTR的TATAbox附近的靶部位结合，并在受到肿瘤坏死因子信号和NF-kB激活等刺激后立即从HIV LTR中释放出来。(1-4)-&gt；我们已经使用已发表的TAT、TAR和Cyclin T1的坐标以及一个新的分子对接软件(Tomoda等人)在电子计算机中阐明了TAT-TAR-Cyclin T1的三分子结构。《癌症科学》，2008，出版中)。通过诱变使接触氨基酸发生突变，以确定其复杂结构。不过，为了进一步巩固数据，我们还在分析接触面的细节。我们还启动了可能的TAT抑制剂的分子设计，并确定了几个这样的候选药物。这些化合物的实际抑制作用仍在进行中。(1-5)-gt；我们发现一些新的HDAC抑制剂可以有效地激活潜伏的HIV-1，而不依赖于NF-kB。这篇论文已经提交给了一本杂志。(2)-我们发表了一些额外的论文，描述了抑制核因子-kB的新化合物的作用和鉴定(Victoriano等人，Antimicr。化学试剂50：547-555,2006；田中等人，《欧洲药学杂志》565：212-219,2007)。较少

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