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

在这个研究项目中，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，其中包括以下子项目：（1-1）细胞转录激活因子的作用；通过鉴定与NF-kB主要亚基p65相互作用的蛋白伙伴，即NF-kB（1-2）通过细胞内信号级联激活NF-kB的机制（1-3）阐明细胞转录抑制因子以了解维持病毒在感染细胞中的潜伏期的机制（1-4）通过阐明由Tat- tar （RNA靶点）-Cyclin T1（主要的细胞周期蛋白）组成的功能性三分子复合物的3d结构分析病毒转录激活因子Tat的分子作用P-TEFb亚基，正转录延伸因子。（1-5）新型组蛋白去乙酰化酶（HDAC）化学抑制剂对潜伏感染细胞中HIV病毒复制的影响。(2)开发以NF-kB和Tat为靶分子阻断HEV的新策略。在这两年中，我们已经取得了以下成果：(1-1)->我们已经确定了两种新的细胞蛋白与NF-kB的p65亚基相互作用，即AKIP1（Gao等人，J Biol Chem 283:7834-7843, 2008）和FKBP4（正在制备中）。这些蛋白最初通过酵母双杂交筛选鉴定，并在体外和体内（活细胞）证实了蛋白-蛋白相互作用。特别是，AKIP1-p65相互作用为解决camp - pka信号传导对NF-kB激活信号传导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NF-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，促进pac介导的p65的Ser 276磷酸化，从而增强NF-kB激活信号。关于我们在1999年报道的RAI (J Biol Chem)，我们发现了它在调节滋养细胞分化中的新作用（Minekawa et al.）。中华内分泌学杂志（英文版），2007)。我们发现cAMP-PKA信号在控制NF-kB激活的细胞阈值中具有双重作用，因此（1-1）(1-3)->中描述的(1-3)->中描述的(1-3)->。我们已经确定了一种细胞转录抑制蛋白AP-4，参与维持潜伏的HIV前病毒（Imai et at, J Biol Chem 281,12495-12505, 2006）。我们发现AP-4组成性地结合在HIV 1原病毒DNA LTR的TATAbox附近的靶点上，并通过阻断TBP与TATAbox的结合并使HDAC蛋白关闭染色质结构来抑制病毒转录。事实上，在HN-1潜伏感染的细胞中，AP-4被发现与HIV-1 LTR TATAbox附近的靶点结合，并在TNF信号和NF-kB激活等刺激下立即从HIV LTR中释放出来。我们利用已发表的Tat、TAR和Cyclin T1的坐标和一种新的分子对接软件（Tomoda et al.）在计算机上阐明了Tat-TAR-Cyclin T1的三分子结构。癌症科学，2008，已出版)。通过诱变对接触氨基酸进行突变，以确定其复杂结构。然而，为了进一步巩固数据，我们仍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。我们发现一些新的HDAC抑制剂可以独立于NF-kB有效地激活潜伏的HIV-1。这篇论文已提交给一家杂志。(2) ->我们发表了一些额外的论文，描述了抑制NF-kB的新化合物的作用和鉴定（Victoriano et al, Antimicr）。中华医学杂志，2006；Tanaka等人，欧洲医学杂志565:212-219,2007)。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