| Research Abstract |
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
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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
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