Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events.

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

ADAM22 plays an important role in cell adhesion and spreading with the assistance of 14-3-3.

Cellular adhesion plays important roles in a variety of biological processes. The ADAM family contains disintegrin-like and metalloproteinase-like domains which potentially have cell adhesion and protease activities. Recent studies suggest that the interaction between 14-3-3zeta and ADAM22cyt can regulate cell adhesion and spreading, therefore it has a potential role in neural development and function. 14-3-3 family has seven highly conserved members that regulate various cellular functions. Using yeast two-hybrid method, we identified that ADAM22cyt bound some other 14-3-3 family members. The interaction was further confirmed by in vitro protein pull-down assay and co-immunoprecipitation. We also found that the overexpression of exogenous ADAM22 in HEK293 cells could significantly enhance cell adhesion and spreading, compared with the truncated ADAM22 lack of 14-3-3 binding motifs. These results strongly demonstrated a functional role for ADAM22/14-3-3 in cell adhesion and spreading.

bri3, a novel gene, participates in tumor necrosis factor-alpha-induced cell death.

bri3 was identified to be a novel gene up-regulated in TNF-treated cells with suppressed subtractive hybridization (SSH) in our laboratory. Previous studies showed that overexpression of BRI3 induced apoptosis in L929 cells. To further study the function of bri3, we disrupted its expression by expressing bri3 antisense RNA. The antisense RNA promoted resistance to TNF-induced cell death by more than 1000-fold in L929 cells, suggesting the involvement of BRI3 in TNF-induced cell death in this cell line. Analysis of cell death caused by other apoptotic inducers showed that the effect of BRI3 antisense RNA is highly specific to TNF-induced cell death. Taken together, bri3 appears to play an important role in TNF-induced cell death. Finally, we reported here that BRI3 may be localized to lysosome and function through lysosome.

A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding.

BMS-378806 is a recently discovered small molecule HIV-1 inhibitor that blocks viral entrance to cells. The compound exhibits potent inhibitory activity against a panel of R5-(virus using the CCR5 coreceptor), X4-(virus using the CXCR4 coreceptor), and R5/X4 HIV-1 laboratory and clinical isolates of the B subtype (median EC50 of 0.04 microM) in culture assays. BMS-378806 is selective for HIV-1 and inactive against HIV-2, SIV and a panel of other viruses, and exhibits no significant cytotoxicity in the 14 cell types tested (concentration for 50% reduction of cell growth, >225 microM). Mechanism of action studies demonstrated that BMS-378806 binds to gp120 and inhibits the interactions of the HIV-1 envelope protein to cellular CD4 receptors. Further confirmation that BMS-378806 targets the envelope in infected cells was obtained through the isolation of resistant variants and the mapping of resistance substitutions to the HIV-1 envelope. In particular, two substitutions, M426L and M475I, are situated in the CD4 binding pocket of gp120. Recombinant HIV-1 carrying these two substitutions demonstrated significantly reduced susceptibility to compound inhibition. BMS-378806 displays many favorable pharmacological traits, such as low protein binding, minimal human serum effect on anti-HIV-1 potency, good oral bioavailability in animal species, and a clean safety profile in initial animal toxicology studies. Together, the data show that BMS-378806 is a representative of a new class of HIV inhibitors that has the potential to become a valued addition to our current armamentarium of antiretroviral drugs.

Overexpression of human genes in Drosophila melanogaster by using GAL4 UAS system.

Many human genes determined by genomic sequencing have only few information about their functions. To fill this knowledge gap, the powerful Drosophila genetics was set as a model to elucidate human gene functions effectively. By using germline transformation together with GAL4-UAS system, we studied the possibility of expressing and functionally characterization of human genes in Drosophila. Fifty-four transgenic fly lines corresponding to 10 human genes have been established. When expressed individually by crossing to an array of 6 different GAL4 driver lines, one of these genes, the translation elongation factor 1 alpha 1 (EF1 alpha-1), resulted in abnormal notum and rough eye phenotypes. This study implies the feasibility of systematically screening human gene functions by overexpression in Drosophila.

The interaction between ADAM 22 and 14-3-3zeta: regulation of cell adhesion and spreading.

The ADAM family consists of a number of transmembrane proteins that contain disintegrin-like and metalloproteinase-like domains. Therefore, ADAMs potentially have cell adhesion and protease activities. 14-3-3 proteins are a highly conserved family of cytoplasmic proteins that associate with several intracellular signaling molecules in the regulation of various cellular functions. Here we report the identification of a novel interaction between the ADAM 22 cytoplasmic tail and the 14-3-3zeta isoform by a yeast two-hybrid screen. The interaction between the ADAM 22 cytoplasmic tail and 14-3-3zeta was confirmed by an in vitro protein pull-down assay as well as by co-immunoprecipitation, and the binding sites were mapped to the 28 amino acid residues of the C-terminus of the ADAM 22 cytoplasmic tail. Furthermore, we found that overexpression of the ADAM 22 cytoplasmic tail in human SGH44 cells inhibited cell adhesion and spreading and that deletion or mutation of the binding site for 14-3-3zeta within the ADAM 22 cytoplasmic tail abolished the ability of the overexpressed cytoplasmic tail to alter cell adhesion and spreading. Taken together, these results for the first time demonstrate an association between ADAM 22 and a 14-3-3 protein and suggest a potential role for the 14-3-3zeta/ADAM 22 association in the regulation of cell adhesion and related signaling events.

Screen and identification of proteins interacting with ADAM19 cytoplasmic tail.

ADAM family plays important roles in neurogenesis. The cytoplasmic tail of ADAM19 (ADAM19-CT) contains 193 residues. The presence of two putative SH3 ligand-binding sites suggests potential interactions with cytosolic proteins, which could be possibly linked to the functions of ADAM19. To address these issues, a yeast two-hybrid screen was performed in human fetal brain cDNA library to isolate proteins that interact with the cytoplasmic tail of ADAM19. Four proteins were obtained, ArgBP1, beta-cop, ubiquitin and a novel protein. GST-Pulldown assay has confirmed the interaction between AdAM19 and ArgBP1. By constructing series of deletion mutants of ADAM19-CT and ArgBP1 respectively, the interaction regions have been identified. They are the SH3 binding sites in ADAM19-CT and the P4 region in ArgBP1. And the interaction is specific. ArgBP1 does not bind to ADAM22, ADAM29 or ADAM9 (mouse). ArgBP1 may be the key protein, which accounts for the physiological function of ADAM19.

The interaction between ADAM22 and 14-3-3beta.

ADAM family consists of a number of transmembrane proteins that contain a disintegrin and metalloprotease domain. ADAMs are involved in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis and inflammatory response. The ADAM proteins have both cell adhesion and protease activities. Adam22 is highly expressed in human brain. The adam22-/- mice presented severe ataxia and died before weaning, but the function of ADAM22 is still unknown. 14-3-3 beta interacting with ADAM22 was detected by using yeast two-hybrid assay. The specificity of interaction between ADAM22 and 14-3-3beta was proved by in vitro binding assay and immunoprecipitation. The major 14-3-3beta binding site was located in the last 28 amino acid residues of ADAM22 cytoplasmic tail. Protein 14-3-3beta is abundant and plays an important role in mediating cell diffusion, migration and cell cycle control. The interaction of ADAM22 and 14-3-3beta suggests that the ADAM22 may play a crucial role in neural function and development.

Cloning and characterization of a gene encoding glutathione-regulated potassium-efflux system protein KefKL from the endosymbiont Wolbachia.

The maternally inherited intracellular symbiont Wolbachia is well known for inducing a variety of reproductive and developmental abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, feminization of genetic males and male killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these abnormalities have not yet been determined. In our study, representational difference analysis (RDA) was used to analyze the genomic difference between different Wolbachia strains. A gene encoding glutathione-regulated potassium-efflux system protein KefKL from Wolbachia in Drosophila simulans Riverside (w Ri) was isolated. The homologous genes from Wolbachia in Drosophila melanogaster yw67c23 (wMel) and Wolbachia in Drosophila melanogaster CantonS (wMelCS) were also cloned and sequenced. Sequence analysis showed that these deduced amino acid sequences contained two important motifs: Na+/H+ antiportor and NAD binding domain, which shared conserved sequences among different strains. Considering the crucial function of KefKL for ionic homeostasis, this gene might play an important role in Wolbachia physiology. Further study indicated that there was no homologue detected from Wolbachia in Drosophila simulans DSW/Mau (wMa) and Wolbachia in Drosophila simulans Noumea (wNo). Whether Wolbachia contained KefKL (or the homologous gene) was consistent with the phylogenetic studies using wsp sequences, which showed that wMa and wNo were grouped into one branch, while w Ri, wMel and wMelCS were more closely related.

Aggregation of the Intracellular Domain of the Human Thrombopoietin Receptor c-MPL.

Thrombopoietin (TPO) is the major cytokine which is involved in platelet production and exerts its effects via the receptor c-MPL. The yeast two-hybrid system has been used to study the aggregation of the intracellular domain of c-MPL in TPO signal transduction. First, the cDNA fragment of MPLP intracellular domain was amplified and cloned by using RT-PCR method from the total RNA of HEL cells. Then the cDNA fragment was sequenced and subcloned into two-hybrid vectors pAS2 and pGAD424, respectively, and the recombinants are named as pASMM and pGADMM. Co-transformation of these plasmids into yeast activated his3 and lacZ reporter genes, demonstrating in vivo interaction of the MPLP receptor intracellular domain itself. The aggregation may be important in TPO signal transduction.

Cloning and Expression of a Fused Gene Encoding TNFRI Death Domain and Chloramphenicol Acetyltransferase.

By designing four primers for an overlapping PCR, we created a fusion gene Ddlcat encoding human TNF receptor I death domain and chloramphenicol acetyltransferase (CAT). By DNA sequencing, the whole sequence of the fusion gene is confirmed to be correct. Two hours after induction with IPTG, we could see a 39 kD extra protein band on SDS-PAGE pattern. We proved that this 39 kD protein band is DdLcat protein by Western blotting. Then we purified this protein to the purity of 95% through Q-Sepharose chromatography.

High Expression of a cDNA Coding for Human Lymphotoxin alpha Derivative in E. coli and Purification of Its Protein Product.

Using PCR, a cDNA coding for human lymphotoxin alpha derivative (hLTalphaDa) lacking 27 amino acids at N-terminal of natural hLT was constructed. The expression construct was expressed in E. coli BL21 (DE3). The product of expression was in the form of inclusion bodies, and accounted for 60%-80% of total bacterial proteins. The product protein was purified to over 95% by treatments of inclusion bodies. The specific activity of hLTalphaDa was above 2x10(8) IU(per mg protein). Its cytotoxic activity can be neutralized by monoclone antibody against hLT. The anti-tumor effects of hLTalphaDa were also tested in vitro and in vivo.