Inversed Effects of Nav1.2 Deficiency at Medial Prefrontal Cortex and Ventral Tegmental Area for Prepulse Inhibition in Acoustic Startle Response.

Numerous pathogenic variants of SCN2A gene, encoding voltage-gated sodium channel α2 subunit Nav1.2 protein, have been identified in a wide spectrum of neuropsychiatric disorders including schizophrenia. However, pathological mechanisms for the schizophrenia-relevant behavioral abnormalities caused by the variants remain poorly understood. Here in this study, we characterized mouse lines with selective Scn2a deletion at schizophrenia-related brain regions, medial prefrontal cortex (mPFC) or ventral tegmental area (VTA), obtained by injecting adeno-associated viruses (AAV) expressing Cre recombinase into homozygous Scn2a-floxed (Scn2afl/fl) mice, in which expression of the Scn2a was locally deleted in the presence of Cre recombinase. The mice lacking Scn2a in the mPFC exhibited a tendency for a reduction in prepulse inhibition (PPI) in acoustic startle response. Conversely, the mice lacking Scn2a in the VTA showed a significant increase in PPI. We also found that the mice lacking Scn2a in the mPFC displayed increased sociability, decreased locomotor activity, and increased anxiety-like behavior, while the mice lacking Scn2a in the VTA did not show any other abnormalities in these parameters except for vertical activity which is one of locomotor activities. These results suggest that Scn2a-deficiencies in mPFC and VTA are inversely relevant for the schizophrenic phenotypes in patients with SCN2A variants.

Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons.

Expressions of voltage-gated sodium channels Nav1.1 and Nav1.2, encoded by SCN1A and SCN2A genes, respectively, have been reported to be mutually exclusive in most brain regions. In juvenile and adult neocortex, Nav1.1 is predominantly expressed in inhibitory neurons while Nav1.2 is in excitatory neurons. Although a distinct subpopulation of layer V (L5) neocortical excitatory neurons were also reported to express Nav1.1, their nature has been uncharacterized. In hippocampus, Nav1.1 has been proposed to be expressed only in inhibitory neurons. By using newly generated transgenic mouse lines expressing Scn1a promoter-driven green fluorescent protein (GFP), here we confirm the mutually exclusive expressions of Nav1.1 and Nav1.2 and the absence of Nav1.1 in hippocampal excitatory neurons. We also show that Nav1.1 is expressed in inhibitory and a subpopulation of excitatory neurons not only in L5 but all layers of neocortex. By using neocortical excitatory projection neuron markers including FEZF2 for L5 pyramidal tract (PT) and TBR1 for layer VI (L6) cortico-thalamic (CT) projection neurons, we further show that most L5 PT neurons and a minor subpopulation of layer II/III (L2/3) cortico-cortical (CC) neurons express Nav1.1 while the majority of L6 CT, L5/6 cortico-striatal (CS), and L2/3 CC neurons express Nav1.2. These observations now contribute to the elucidation of pathological neural circuits for diseases such as epilepsies and neurodevelopmental disorders caused by SCN1A and SCN2A mutations.

In Vitro Antiviral Activity of Mentha cordifolia Plant Extract in HIV-1 Latently Infected Cells Using an Established Human Cell Line.

Emergence of drug resistance demands new therapeutic strategies against the human immunodeficiency virus (HIV). Currently, there is an increasing research focus on targeting gene expression-the crucial step wherein new viruses and new viral strains are amplified. Moreover, natural products are also being considered as potential candidates for new antivirals. We screened the extract obtained from a Philippine medicinal plant, Mentha cordifolia (Mc). In this study, we demonstrated that Mc ammonium sulfate extract has antiretroviral activity against HIV. HIV-1 latently infected cells (OM10.1) were pretreated with Mc extract and activated with TNFα. In treated cells, viral replication was inhibited in both cell culture supernatant and whole cell lysates. The level of viral production, as measured by the viral p24 protein concentration, was very much inhibited under noncytotoxic concentrations to the similar level without addition of TNFα. Luciferase assays, however, showed that Mc does not inhibit the HIV-1 long terminal repeat-driven gene expression. IκBα degradation and p65 nuclear translocation was also not affected as visualized through Western blot and immunofluorescence. These observations demonstrated that Mc possessed an antiviral component against HIV-1 and warrant further work to explore its target of action at a later step of gene expression. Our study introduces a potential source of a lead compound that targets steps in the HIV life cycle.

Feed-forward regulatory loop driven by IRF4 and NF-κB in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive hematological malignancy derived from mature CD4+ T-lymphocytes. Here, we demonstrate the transcriptional regulatory network driven by 2 oncogenic transcription factors, IRF4 and NF-κB, in ATL cells. Gene expression profiling of primary ATL samples demonstrated that the IRF4 gene was more highly expressed in ATL cells than in normal T cells. Chromatin immunoprecipitation sequencing analysis revealed that IRF4-bound regions were more frequently found in super-enhancers than in typical enhancers. NF-κB was found to co-occupy IRF4-bound regulatory elements and formed a coherent feed-forward loop to coordinately regulate genes involved in T-cell functions and development. Importantly, IRF4 and NF-κB regulated several cancer genes associated with super-enhancers in ATL cells, including MYC, CCR4, and BIRC3. Genetic inhibition of BIRC3 induced growth inhibition in ATL cells, implicating its role as a critical effector molecule downstream of the IRF4-NF-κB transcriptional network.

Quantification of the HIV transcriptional activator complex in live cells by image-based protein-protein interaction analysis.

The virus-encoded Tat protein is essential for HIV transcription in infected cells. The interaction of Tat with the cellular transcription elongation factor P-TEFb (positive transcriptional elongation factor b) containing cyclin T1 (CycT1) and cyclin-dependent kinase 9 (CDK9) is critical for its activity. In this study, we use the Fluoppi (fluorescent-based technology detecting protein-protein interaction) system, which enables the quantification of interactions between biomolecules, such as proteins, in live cells. Quantitative measurement of the molecular interactions among Tat, CycT1 and CDK9 has showed that any third molecule enhances the binding between the other two molecules. These findings suggest that each component of the Tat:P-TEFb complex stabilizes the overall complex, thereby supporting the efficient transcriptional elongation during viral RNA synthesis. These interactions may serve as appropriate targets for novel anti-HIV therapy.

Cooperation between the chloroplast psbA 5'-untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region.

Chloroplast mRNA translation is regulated by the 5'-untranslated region (5'-UTR). Chloroplast 5'-UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5'-UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5'-UTR with the E. coli phage T7 gene 10 5'-UTR, a highly active 5'-UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5'-UTR with a cognate 5'-coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5'-UTR and its coding region is important for translational initiation.

Molecular dynamics simulation and experimental verification of the interaction between cyclin T1 and HIV-1 Tat proteins.

The viral encoded Tat protein is essential for the transcriptional activation of HIV proviral DNA. Interaction of Tat with a cellular transcription elongation factor P-TEFb containing CycT1 is critically required for its action. In this study, we performed MD simulation using the 3D data for wild-type and 4CycT1mutants3D data. We found that the dynamic structural change of CycT1 H2' helix is indispensable for its activity for the Tat action. Moreover, we detected flexible structural changes of the Tat-recognition cavity in the WT CycT1 comprising of ten AAs that are in contact with Tat. These structural fluctuations in WT were lost in the CycT1 mutants. We also found the critical importance of the hydrogen bond network involving H1, H1' and H2 helices of CycT1. Since similar AA substitutions of the Tat-CycT1 chimera retained the Tat-supporting activity, these interactions are considered primarily involved in interaction with Tat. These findings described in this paper should provide vital information for the development of effective anti-Tat compound.

Functional characterization of human cyclin T1 N-terminal region for human immunodeficiency virus-1 Tat transcriptional activation.

Transcription of the human immunodeficiency virus type 1 (HIV-1) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, positive transcription elongation factor b, with the viral Tat protein at the transactivation response (TAR) element of nascent viral transcripts. The involvement of the interaction between Tat and CycT1 is known to be through the Tat-TAR recognition motif (TRM) on CycT1. Here, we have further characterized this molecular interaction and clarified the role of the CycT1 N-terminal region in Tat action. We found crucial and distinctive roles of Q46, Q50 and F176 of human CycT1 protein in Tat-mediated transcription by creating various Ala substitution mutants of CycT1 based on its three-dimensional structure. We confirmed the involvement of these amino acid residues in binding to Tat with Q46 and Q50, and to a lesser extent with F176, by in vitro pull-down assay. Relative transactivation activities of wild-type CycT1 chimeras and mutant derivatives on the HIV-1 long terminal repeat were determined by luciferase reporter assays. Whereas CycT1 Q46A alone had impaired transcriptional activity, the CycT1(Q46A)-Tat chimeric protein retained almost full activity of the wild-type CycT1. However, CycT1 mutants (C261Y, Q50A or F176A) or their chimeric counterparts had lost the transactivation capacity. Moreover, a triple-mutant chimera containing Q46A, Q50A and F176A mutations completely abolished the transcriptional activity, indicating that these amino acid residues are involved through distinct mechanisms. These findings provide new insights for the development of anti-HIV drugs.

A-kinase-interacting protein 1 (AKIP1) acts as a molecular determinant of PKA in NF-kappaB signaling.

The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-kappaB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-kappaB activity. In this study, we have demonstrated the effect of PKA on NF-kappaB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-kappaB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-kappaB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-kappaB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-kappaB activation; PKA-activating agents enhanced NF-kappaB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-kappaB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-kappaB-induced breast cancer cell proliferation and multiple NF-kappaB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-kappaB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.

Impaired plant growth and development caused by human immunodeficiency virus type 1 Tat.

Previous attempts to express the human immunodeficiency virus 1 (HIV-1) Tat (trans-activator of transcription) protein in plants resulted in a number of physiological abnormalities, such as stunted growth and absence of seed formation, that could not be explained. In the study reported here, we expressed Tat in tomato and observed phenotypic abnormalities, including stunted growth, absence of root formation, chlorosis, and plant death, as a result of reduced cytokinin levels. These reduced levels were ascribed to a differentially expressed CKO35 in Tat-bombarded tomato. Of the two CKO isoforms that are naturally expressed in tomato, CKO43 and CKO37, only the expression of CKO37 was affected by Tat. Our analysis of the Tat confirmed that the Arg-rich and RGD motifs of Tat have functional relevance in tomato and that independent mutations at these motifs caused inhibition of the differentially expressed CKO isoform and the extracellular secretion of the Tat protein, respectively, in our Tat-bombarded tomato samples.

Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

Inhibition of human immunodeficiency virus type 1 replication by blocking IkappaB kinase with noraristeromycin.

Nuclear factor kappaB (NF-kappaB) is one of the critical transcription factors in inflammatory responses and replication of viruses such as human immunodeficiency virus (HIV). In fact, it has been demonstrated that various NF-kappaB inhibitors could block HIV replication. To explore more potent NF-kappaB inhibitors, we focused on carbocyclic adenine nucleosides that had been reported to have anti-inflammatory effects. We synthesized 15 carbocyclic adenine nucleoside compounds and examined their effects on the NF-kappaB-dependent gene expression using HEK293 cell line. Among these compounds, noraristeromycin (NAM) exhibited the most potent inhibitory effect on the NF-kappaB activity under the non-cytotoxic concentrations. NAM-inhibited IkappaBalpha phosphorylation and degradation upon stimulation of cells with tumour necrosis factor-alpha (TNF-alpha). In addition, NAM prevented p65 phoshorylation. These findings suggested that both IkappaB kinase-alpha (IKK-alpha) and -beta were targeted by NAM. Interestingly, in vitro kinase assay revealed that NAM inhibited the kinase activity of IKK-alpha more potently than that of IKK-beta. When we treated the cell lines, OM10.1 and Molt4/IIIB, in which HIV-1 is latently and chronically infected, we found a strong suppressive effect of NAM on HIV-1 viral replication upon stimulation with TNF-alpha.

Molecular docking analysis of the protein-protein interaction between RelA-associated inhibitor and tumor suppressor protein p53 and its inhibitory effect on p53 action.

RelA-associated inhibitor (RAI) was initially identified as a protein that interacts with the p65 subunit (RelA) of nuclear factor-kappaB. It was recently found to interact with the p53 tumor suppressor protein. RAI is a structural homolog of the p53-binding protein 2 and I kappaB family proteins, and is known to inhibit the DNA-binding activities of p65 and p53. In the present study, we have attempted to predict the 3-dimensional structure of RAI in complex with p53 using computational chemistry. In order to evaluate the predicted structure model, we created a series of RAI mutants in which the amino acid residues involved in the interaction with p53 were mutated, and examined their activities in blocking p53-mediated bax gene expression. Our observations support the validity of the predicted 3-dimensional model of the p53-RAI protein complex. Based on the p53-RAI complex model, we have demonstrated the biological importance of the R248 and R273 residues of p53, and the D775 and E795 residues of RAI, in the protein-protein interaction between p53 and RAI and the biological actions of these proteins. These findings will further clarify the biological actions of RAI in carcinogenesis and can be used for the development of a novel strategy in blocking the actions of RAI. The possible biological implications of RAI are also discussed.

AKIP1 enhances NF-kappaB-dependent gene expression by promoting the nuclear retention and phosphorylation of p65.

In this study, we have identified protein kinase A-interacting protein 1 (AKIP1) as a binding partner of NF-kappaB p65 subunit, and AKIP1 enhances the NF-kappaB-mediated gene expression. AKIP1 is a nuclear protein and known to interact with the catalytic subunit of PKA (PKAc). We identified AKIP1 by a yeast two-hybrid screen using the N terminus region of p65 as bait. The interaction between AKIP1 and p65 was confirmed by glutathione S-transferase pull-down assay in vitro and immunoprecipitation-Western blotting assay in vivo. We found that the PKAc was present in the AKIP1.p65 complex and enhanced the transcriptional activity of NF-kappaB by phosphorylating p65. In a transient luciferase assay, AKIP1 cotransfection efficiently increased the transcriptional activity of NF-kappaB induced by phorbol 12-myristate 13-acetate (PMA). When AKIP1 was knocked down by RNA interference, the PMA-mediated NF-kappaB-dependent gene expression was abolished, indicating a physiological role of AKIP1. We found that PKAc, which is maintained in an inactive form by binding to IkappaBalpha and NF-kappaB in resting cells, was activated by PMA-induced signaling and could phosphorylate p65. Overexpression of AKIP1 increased the PKAc binding to p65 and enhanced the PKAc-mediated phosphorylation of p65 at Ser-276. Interestingly, this p65 phosphorylation promoted nuclear translocation of p65 and enhanced NF-kappaB transcription. In fact, we observed that AKIP1 colocalized with p65 within the cells and appeared to retain p65 in nucleus. These findings indicate a positive role of AKIP1 in NF-kappaB signaling and suggest a novel mechanism by which AKIP1 augments the transcriptional competence of NF-kappaB.

A single-nucleotide synonymous mutation in the gag gene controlling human immunodeficiency virus type 1 virion production.

Viral factors as well as host ones play major roles in the disease progression of human immunodeficiency virus type 1 (HIV-1) infection. We have examined cytotoxic T-lymphocyte activity and HIV-1 DNA PCR results of 312 high-risk seronegative drug users in northern Thailand and identified four seronegative cases positive for both assays. Furthermore, we have identified a synonymous mutation in nucleotide position 75 of the gag p17 gene (A426G) of HIV-1 that belongs to the CRF01_AE virus circulating in Thailand. The replication-competent HIV-1 clone containing the A426G mutation demonstrated a dramatic reduction of virion production and perturbation of viral morphogenesis without affecting viral protein synthesis in cells.

Inhibition of human immunodeficiency virus type 1 replication in latently infected cells by a novel IkappaB kinase inhibitor.

In human immunodeficiency virus type 1 (HIV-1) latently infected cells, NF-kappaB plays a major role in the transcriptional induction of HIV-1 replication. Hence, downregulation of NF-kappaB activation has long been sought for effective anti-HIV therapy. Tumor necrosis factor alpha (TNF-alpha) stimulates IkappaB kinase (IKK) complex, a critical regulator in the NF-kappaB signaling pathway. A novel IKK inhibitor, ACHP {2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl-nicotinonitrile}, was developed and evaluated as a potent and specific inhibitor for IKK-alpha and IKK-beta. In this study, we examined the ability of this compound to inhibit HIV-1 replication in OM10.1 cells latently infected with HIV. When these cells were pretreated with ACHP, TNF-alpha-induced HIV-1 replication was dramatically inhibited, as measured by the HIV p24 antigen levels in the culture supernatants. Its 50% effective concentration was approximately 0.56 microM, whereas its 50% cytotoxic concentration was about 15 microM. Western blot analysis revealed inhibition of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 nuclear translocation, and p65 phosphorylation. ACHP was also found to suppress HIV-1 long terminal repeat (LTR)-driven gene expression through the inhibition of NF-kappaB activation. Furthermore, ACHP inhibited TNF-alpha-induced NF-kappaB (p65) recruitment to the HIV-1 LTR, as assessed by chromatin immunoprecipitation assay. These findings suggest that ACHP acts as a potent suppressor of TNF-alpha-induced HIV replication in latently infected cells and that this inhibition is mediated through suppression of IKK activity.

53BP2 induces apoptosis through the mitochondrial death pathway.

The p53 binding protein 2 (53BP2) has been identified as the interacting protein to p53, Bcl-2, and p65 subunit of nuclear factor kappaB (NF-kappaB). The TP53BP2 gene encodes two splicing variants, 53BP2S and 53BP2L, previously known as apoptosis stimulating protein 2 of p53 (ASPP2). We found that these 53BP2 proteins are located predominantly in the cytoplasm and induce apoptosis as demonstrated by cleavage of poly ADP ribose polymerase (PARP) and annexin V staining. Furthermore, we demonstrate that 53BP2 is located in the mitochondria and induces apoptosis associated with depression of the mitochondrial trans-membrane potential (DeltaPsim) and activation of caspase-9. From these findings we conclude that 53BP2 induces apoptosis through the mitochondrial death pathway.

Replication-defective HIV isolated from seronegative individuals at high risk for HIV infection.

We have identified a mutant human immunodeficiency virus type 1 (HIV-1) CRF01_AE that contains a single nucleotide mutation in gag gene from 4 HIV-1 seronegative drug users in Thailand. We found A to G mutation at the nucleotide position 75 of gag p17 gene (A75G) not changing the amino acid sequence. The mutant HIV-1 molecular clones were examined for their replication capability. Although the mutation dramatically reduced the level of virion production, it did not affect the amounts of viral protein synthesis within the transfected cells. In addition, this mutation did not affect the levels of Gag polyproteins. Furthermore, electron microscopic examinations have revealed a dramatic reduction of the virion production and perturbation of viral morphogenesis at the cytoplasmic membrane. These results indicate that the A75G mutation is attributable to the long-term sero-negativity of individuals at high risk of HIV-1 infection and suggest a novel mechanism that regulates HIV production.

Expression of 53BP2 and ASPP2 proteins from TP53BP2 gene by alternative splicing.

The p53 binding protein 2 (53BP2) has been initially identified as an interacting protein to p53 and subsequent studies have shown that it also interacts with Bcl-2 and NF-kappaB p65 subunit. We have previously found that the TP53BP2 gene encoding 53BP2 protein is a single copy gene and has been mapped to the long arm of chromosome 1 at q42.1. The subsequent studies revealed that TP53BP2 encodes two proteins, 53BP2 and ASPP2, of 1005 and 1128 amino acids, respectively. ASPP2 contains additional 123 amino acids to the N-terminus of 53BP2. In this study, we have examined the genomic organization of TP53BP2 transcripts and found that it encodes two mRNA species, either with (53BP2) or without exon 3 (ASPP2), by alternative splicing in various cell lines and tissues. Thus, we propose to call these proteins as 53BP2S (short) and 53BP2L (long), respectively.