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Detection of human immunodeficiency virus type 1 Nef and CD4 physical interaction in living human cells by using bioluminescence resonance energy transfer.

Author(s) : Cluet D, Bertsch C, Beyer C, Gloeckler L, Erhardt M, Gut J, Galzi J, Aubertin A,
Journal : J Virol
CD4 down-regulation by human immunodeficiency virus type 1 (HIV-1) Nef protein is a key function for virus virulence. This activity may be mediated by a direct Nef-CD4 interaction. We investigated the formation, in situ, of such a complex between proteins using bioluminescence resonance energy transfer technology and co-immunoprecipitations. Our data clearly demonstrate that Nef and CD4 interact in intact human cells. Moreover, our results clearly indicate that the dileucinemotif of the CD4 cytoplasmic domain, critical for the Nef-induced CD4 down-regulation, is not implicated in the Nef/CD4 complex formation in the cellular context.

Human papillomavirus type 18 E6 protein binds the cellular PDZ protein TIP-2/GIPC, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome.

Author(s) : Favre-Bonvin A, Reynaud C, Kretz-Remy C, Jalinot P,
Journal : J Virol
Several viral proteins expressed by DNA or RNA transforming viruses have the particular property of binding via their C-terminal end to various cellular proteins with PDZ domains. This study is focused on the PDZ protein TIP-2/GIPC, which was originally identified in two-hybrid screens performed with two different baits: the human T-cell leukemia virus type 1 Tax oncoprotein and the regulator of G signaling RGS-GAIP. Further studies have shown that TIP-2/GIPC isalso able to associate with the cytoplasmic domains of various transmembrane proteins. In this report we show that TIP-2/GIPC interacts with the E6 protein of human papillomavirus type 18 (HPV-18). This event triggers polyubiquitination and proteasome-mediated degradation of the cellular protein. In agreement with this observation, silencing of E6 by RNA interference in HeLa cells causes an increase in the intracellular TIP-2/GIPC level. This PDZ protein has been previously found to be involved in transforming growth factor beta (TGF-beta) signaling by favoring expression of the TGF-beta type III receptor at the cell membrane. In line with this activity of TIP-2/GIPC, we observed that depletion of this protein in HeLa cells hampers induction of the Id3 gene by TGF-beta treatment and also diminishes the antiproliferative effect of this cytokine. Conversely, silencing of E6 increases the expression of Id3 and blocks proliferation of HeLa cells. These results support the notion that HPV-18 E6 renders cells less sensitive to the cytostatic effect of TGF-beta by lowering the intracellular amount of TIP-2/GIPC.

Modeling the emergence of multi-protein dynamic structures by principles of self-organization through the use of 3DSpi, a multi-agent-based software.

Author(s) : Soula H, Robardet C, Perrin F, Gripon S, Beslon G, Gandrillon O,
Journal : BMC Bioinformatics
BACKGROUND: There is an increasing need for computer-generated models that can be used for explaining the emergence and predicting the behavior of multi-protein dynamic structures in cells. Multi-agent systems (MAS) have been proposed as good candidates to achieve this goal. RESULTS: We have created 3DSpi, a multi-agent based software that we used to explore the generation of multi-protein dynamic structures. Being based on a very restricted set of parameters, it is perfectly suited for exploring the minimal set of rules needed to generate large multi-protein structures. It can therefore be used to test the hypothesis that such structures are formed and maintained by principles of self-organization. Weobserved that multi-protein structures emerge and that the system behavior is very robust, in terms of the number and size of the structures generated. Furthermore, the generated structures very closely mimic spatial organization ofreal life multi-protein structures. CONCLUSION: The behavior of 3DSpi confirms the considerable potential of MAS for modeling subcellular structures. It demonstrates that robust multi-protein structures can emerge using a restricted set of parameters and allows the exploration of the dynamics of such structures.A number of easy-to-implement modifications should make 3DSpi the virtual simulator of choice for scientists wishing to explore how topology interacts with time, to regulate the function of interacting proteins in living cells.

Photoreceptor differentiation in Drosophila: from immature neurons to functional photoreceptors.

Author(s) : Mollereau B, Domingos P,
Journal : Dev Dyn
How a pool of equipotent cells acquires a multitude of distinct fates is a majorquestion in developmental biology. The study of photoreceptor (PR) cell differentiation in Drosophila has been used to address this question. PR differentiation is a process that extends over a period of 5 days: It begins in the larval eye imaginal disc when PRs are recruited and commit to particular PR fates, and it culminates in the pupal eye disc with the morphogenesis of the rhabdomeres and the initiation of rhodopsin expression. Several models for PR specification agree that the Ras and Notch signaling pathways are important for the specification of different PR subtypes (Freeman [1997] Development 124:261-270; Cooper and Bray [2000] Curr. Biol. 10:1507-1510; Tomlinson and Struhl [2001] Mol. Cell. 7:487-495). In the first part of this review, we briefly describe the different signaling pathways and transcription factors required forthe specification and differentiation of the different PR subtypes in the larvaleye disc. In the second part, we review the roles of several transcription factors, which are required for the terminal photoreceptor differentiation and rhodopsin expression.

Role of conformational heterogeneity in domain swapping and adapter function of the Cks proteins.

Author(s) : Seeliger M, Spichty M, Kelly S, Bycroft M, Freund S, Karplus M, Itzhaki L,
Journal : J Biol Chem
Cks proteins are adapter molecules that coordinate the assembly of multiprotein complexes. They share the ability to domain swap by exchanging a beta-strand, beta4. Here we use NMR spectroscopy and molecular dynamics simulations to investigate the dynamic properties of human Cks1 and its response on assembly with components of the SCF(Skp2) ubiquitin ligation machinery. In the NMR experiment with the free form of Cks1, a subset of residues displayed elevated R2 values and the cross-peaks of neighboring residues were missing from the spectrum, indicating a substantial conformational exchange contribution on the microsecond to millisecond time scale. Strikingly the region of greatest conformational variability was the beta4-strand that domain swaps to form the dimer. Binding of the ligand common to all Cks proteins, Cdk2, suppressed the conformational heterogeneity. This response was specific to Cdk2 binding; in contrast, binding of Skp2, a ligand unique to human Cks1, did not alter the dynamic behavior. Short time (<5 ns) molecular dynamics simulations indicate that residues of Cks1 that form the binding site for phosphorylated ligands are considerably more flexible in the free form of Cks1 than they are in the Cdk2-Cks1 complex. A cooperative interaction between Cdk2 and Cks1 is suggested,which reduces the configurational entropy of Cks1 and therefore facilitates phosphoprotein binding. Indications of an unusual dynamic behavior of strand beta4 in the free form of Cks1 were obtained from longer time scale (50 ns) dynamics simulations. A spontaneous reversible unzipping of hydrogen bonds between beta4 and beta2 was observed, suggesting an early intermediate structurefor unfolding and/or domain swapping. We propose that the dynamic properties of the beta-sheet and its modification upon ligand binding underlie the domain swapping ability and the adapter function of Cks proteins.

Serial analysis of gene expression in the silkworm, Bombyx mori.

Author(s) : Huang J, Miao X, Jin W, Couble P, Mita K, Zhang Y, Liu W, Zhuang L, Shen Y, Keime C, Gandrillon O, Brouilly P, Briolay J, Zhao G, Huang Y,
Journal : Genomics
The silkworm Bombyx mori is one of the most economically important insects and serves as a model for Lepidoptera insects. We used serial analysis of gene expression (SAGE) to derive profiles of expressed genes during the developmentallife cycle of the silkworm and to create a reference for understanding silkworm metamorphosis. We generated four SAGE libraries, one from each of the four developmental stages of the silkworm. In total we obtained 257,964 SAGE tags, ofwhich 39,485 were unique tags. Sorted by copy number, 14.1% of the unique tags were detected at a median to high level (five or more copies), 24.2% at lower levels (two to four copies), and 61.7% as single copies. Using a basic local alignment search tool on the EST database, 35% of the tags matched known silkworm expressed sequence tags. SAGE demonstrated that a number of the genes were up- or down-regulated during the four developmental phases of the egg, larva, pupa, andadult. Furthermore, we found that the generation of longer cDNA fragments from SAGE tags constituted the most efficient method of gene identification, which facilitated the analysis of a large number of unknown genes.

Silencing of human Int-6 impairs mitosis progression and inhibits cyclin B-Cdk1 activation.

Author(s) : Morris C, Jalinot P,
Journal : Oncogene
The Int-6 protein has been originally identified as the product of a mouse gene being a frequent integration site of the mouse mammary tumour virus. Here, we show that reducing Int-6 expression by RNA interference in HeLa cells markedly alters mitosis progression. Defects in spindle formation, chromosome segregationand cytokinesis were observed. These abnormalities of mitosis completion are correlated with an inhibition of cyclin B-Cdk1 kinase activity, due to a prolonged inhibitory phosphorylated state of Cdk1. In line with this observation, the Wee1 tyrosine kinase that negatively controls Cdk1 was less efficiently inactivated during G2 in Int-6-depleted cells. These findings support the notionthat the oncogenic properties associated with alteration of Int-6 originate fromchromosomal instability.

Up-regulation of the ubiquitous alternative splicing factor Tra2beta causes inclusion of a germ cell-specific exon.

Author(s) : Venables J, Bourgeois C, Dalgliesh C, Kister L, Stevenin J, Elliott D,
Journal : Hum Mol Genet
We have discovered a new exon of the homeodomain-interacting kinase HipK3 that incorporates a premature stop codon and is included only in the human testis. Toinvestigate this, we tested the effects of transfecting cells with green fluorescent protein fusions of RNA-binding proteins implicated in spermatogenesis using a novel assay based on multi-fraction fluorescence-activated cell sorting (MF-FACS). This allows the effect of a controlled titration of any splicing factor on the splicing of endogenous genes to be studied in vivo. We found that Tra2beta recapitulates testis-specific splicing of endogenous HipK3 in a concentration-dependent manner and binds specifically to a long purine-rich sequence in the novel exon. This sequence was also specifically bound by hnRNP A1, hnRNP H, ASF/SF2 and SRp40, but not by 9G8. Consistent with these observations, in vitro studies showed that this sequence shifts splicing to a downstream 5' splice site within a heterologous pre-mRNA substrate in the presence of Tra2beta, ASF/SF2 and SRp40, whereas hnRNP A1 specifically inhibits this choice. By mutating the purine-rich sequence in the context of the HipK3 gene, we also show that it is the major determinant of Tra2beta- and hnRNP A1-mediated regulation. Tra2 is essential for sex determination and spermatogenesis in flies, and Tra2beta protein was most highly expressed in testis out of six mouse tissues, whereas hnRNP A1 is down-regulated during germ cell development. Therefore, our data imply an evolutionarily conserved role forTra2 proteins in spermatogenesis and suggest that an elevated concentration of Tra2beta may convert it into a tissue-specific splicing factor.