Changben Li

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-bianding 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, β-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). ArgBP1may be the key protein, which accounts for the physiological function of ADAM19.

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 yw 67c23 ( w Mel) and Wolbachia in Drosophila melanogaster CantonS ( w MelCS) 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 ( w Ma) and Wolbachia in Drosophila simulans Noumea ( w No). Whether Wolbachia contained KefKL (or the homologous gene) was consistent with the phylogenetic studies using wsp sequences, which showed that w Ma and w No were grouped into one branch, while w Ri, w Mel and w MelCS were more closely related.

Phytogeny of genusGlossina (Diptera:Glossinidae) according to ITS2 sequences

The flies of genusGlossina (Diptera: Glossinidae) are an important vector of African trypanosomiases which cause diseases in humans and animals. The ribosomal DNA Internal Transcribed Spacer-2 (ITS-2) region sequences from differentGlossina species were PCR-amplified and analyzed in order to construct a molecular phylogeny for genusGlossina. Trees generated by parsimony confirmed the monophyletic taxonomic placement of genusGlossina wherefusca group species formed the deepest branch followed bymorsitans andpalpalis groups, respectively. The placement ofGlossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on ITS-2 locus sequence analysis, suggest thatGlossina austeni can be placed into a separate subgenerus which forms a sister-group relationship with themorsitans group species.

SGK and 14-3-3 protein are involved in the serine/ threonine phosphorylation mechanism for TPO/MPL signal transduction

Thrombopioetin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Yeast two-hybrid screening was performed to isolate the proteins interacting with the cytoplasmic domain of c-Mpl. 48 positive clones were isolated from 5 × 106 independent transformants. The results of sequence analysis demonstrate that they represent 13 different protein encoding sequences. Among them there are a partial coding sequence of serine/threonine protein kinase SGK (serum and glucocorticoid-inducible kinase) and 14-3-3 theta protein partial coding sequence. GST-pull-down assay and co-immunoprecipitation in mammal cells have confirmed the interaction between these two proteins and c-Mpl. By constructing a series of deleted c-Mpl cytoplasmic domain, the interaction region in c-Mpl cytoplasmic tail was localized in amino acids 523–554. At the same time, the directed interaction between SGK and 14-3-3 proteins also has been verified by yeast two-hybrid assay. The present note is the first time to report that two proteins act with c-Mpl at the same time and put forward that SGK and 14-3-3 protein may be involved in the serine/threonine phosphorylation mechanism for signal transduction.

Identification of the Binding Domain of Signal-Transduction-Related Proteins on c-Mpl

cMpl is the receptor of thrombopoietin, the critical regulator of platelet production. Proteins SGK, 1433, Siva, Tom1, Cop9S3 and cMplBP have recently been proved to interact with cMpl. With the intent to identify the binding domains of the 6 proteins on cMpl, serial deletions and point mutations of the cytotail of cMpl have been generated and their interaction with the proteins mentioned above have been examined by yeast two hybrid system. The results indicate that Box1 (a highly conserved region within the cytokine receptor superfamily) is the critical domain for binding. Considering the similar role Box1 plays in other cytokine receptors, the results suggest that Box1 is probably a widely used proteinbinding domain in cytokine receptors. Amino acids adjacent to Box1 and the Cterminal region also contribute to the interaction. Point mutations of Ser531 have negative effects on the interaction, which indicates the importance of the Box1 region in the protein interaction.

Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

Thrombopoietin (TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl. The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl. First, the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2, and the resulting plasmid is designated as pASMM. Then a human placenta cDNA library was screened using the pASMM as a target plasmid. Seven positive clones were isolated from 150 000 independent transformants. Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3′ end and flanking non-translation region was obtained. Therefore, there is an interaction between vimentin and TPO receptor. The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.

Study of thrombopoietin for gene therapy of thrombocytopenia

Thrombopoietin (TPO) is likely to he a potent, specific and reliable medication in the treatment of thrombocytopenia. A TPO-highly-expressed plasmid pcDNA3-TPO was constructed and a primary study was made on the expression of TPO cDNAin vitro and gene transfer study for thrombocytopeniain vivo. rhTPO showed complete and stable bioactivity by a series of indicators. High expression of TPO was detected in plasma from healthy mice or thrombocytopenia mice model receiving direct intramuscular injection of pcDNA3-TPO. And the platelet level of healthy mice peaked to 1.9-fold of baseline. Mice with CTX-induced thrombocytopenia achieved profound nadirs, acceleration of recovery, even 1.8–2.0-fold supranormal levels of peripheral platelet counts. The results offered experimental support for clinical application of gene therapy for thrombocytopenia via direct intramuscular injection of TPO cDNA.

Murine neurofibroma reversion by antisense RNA for HTLV-I tax*

Neurofibroma cell lines derived from mice transgenic for HTLV-I LTR tax express high levels of HTLV-I tax mRNA and protein and exhibit a transformed phenotype. A retrovirus vector carrying HTLV-I tax cDNA in reversed transcriptional orientation was stably transfected into the neurofibroma cells. Antisense RNA inhibited expression of the tax gene with a decrease of more than 40% in both tax mRNA and protein. Tax antisense RNA reversed the transformed phenotype as exhibited by dramatic changes in cell morphology and growth characteristics. Expression of several cellular genes which are activated by Tax protein including GM-CSF, IL-6, LT/TNF, c-myc and LIF was down-regulated, while M-CSF and c-src proto-oncogene expressions were up-regulated. Accumulation of β-actin mRNA was not affected. The changes that occurred in the tax antisense expressing neurofibroma cells could be the consequence of the decreased concentration of Tax protein. These results also indicate that HTLV-I Tax protein is crucial for maintaining the transformed cell morphology, growth and proliferation of murine neurofibroma cells and suggest that deregulation of endogenous cellular genes by Tax protein is the mechanism through which neurofibromas occur in tax mice.