pGEX Vector Research Articles

CXXC5 variant in an immunodeficient patient with a progressive loss of hematopoietic cells

Partial loss of function variant in CXXC5 identified in an immunodeficient patient with progressive bone marrow failure, corrected by bone marrow transplant.

Prokaryotic expression, protein purification and functional verification of human homotypic fusion and vacuole protein sorting complex subunit

Homotypic fusion and vacuole protein sorting(HOPS) is a protein complex consisting of VPS11, VPS16, VPS18, VPS33, VPS39, VPS41 and regulates membrane transport in vivo through membrane fusion mechanisms. The evidence suggests that HOPS complex as a fusion factor, facilitates autophagosome-lysosome fusion. To determine whether the HOPS complex directly interacts with the autophagic SNARE protein STX17 in vitro, the coding sequence of the six genes were amplified from the existing plasmids by PCR, and then ligated to the prokaryotic expression vector pGEX 4T-1-GST or pET-His-NusA. After identification through colony PCR and DNA sequencing, 6 recombinant plasmids were constructed and transferred into Escherichia coli BL21 (DE3). The recombinant proteins were purified by glutathione sepharose 4B and nickel column. We used the tobacco etch virus protease to cut off the GST-tag or His-NusA-tag, to obtain HA-VPS11 protein of about 105 kDa, Flag-VPS16 protein of about 97 kDa, HA-VPS18 protein of about 108 kDa, Flag-VPS33 protein of about 70 kDa, HA-VPS39 protein of about 97 kDa, and Flag-VPS41 protein of about 98 kDa. The function of the purified proteins was verified by in vitro glutathione S-transferases pull-down assay, confirming that autophagic SNARE protein STX17 interacted directly with HOPS components. Our findings provide experimental basis to further study the function and mechanism of HOPS complex in the process of autophagosome-lysosome fusion.

Sortase A-aided Escherichia coli expression system for functional osteoprotegerin cysteine-rich domain

As a natural inhibitor of the receptor activator of nuclear factor-кB ligand (RANKL), osteprotegerin (OPG) is considered a promising treatment for metabolic bone diseases. Typical approaches for preparing recombinant OPG or its derivatives employ eukaryotic expression systems. Due to the advantages of a prokaryotic expression system, which include its convenience, low cost, and abundant production, in this study, we establish a strategy for preparing functional OPG using the Escherichia coli expression system. After initial failures in preparation of OPG and its truncation, OPG cysteine-rich domain (OPG-CRD/OPGT) by using pET and pGEX vectors, we constructed a sortase A (SrtA)-aided E. coli expression system, in which the expressed protein was a self-cleaving SrtA fusion protein. Using this system, we successfully prepared the recombinant OPGT protein. The BIAcore analyses indicated that the prepared OPGT had high affinities in binding with RANKL and TRAIL. Cell experiments confirmed the inhibitory effects of the prepared OPGT on RANKL-induced osteoclast differentiation and TRAIL-induced tumor cell apoptosis. The sortase A-aided E. coli expression system for OPGT established in this study may contribute to further studies and commercial applications of OPG.

Vaccination of rabbits with immunodominant antigens from Sarcoptes scabiei induced high levels of humoral responses and pro-inflammatory cytokines but confers limited protection.

BackgroundVaccination is an attractive ecological alternative to the use of acaricides for parasite control. However, effective anti-parasite vaccines against sarcoptic mange have not yet been developed. The purpose of this study was first to identify Sarcoptes scabiei immunodominant antigens and second to evaluate them as vaccine candidates in a rabbit/S. scabiei var. cuniculi model.MethodsThe S. scabiei Ssλ15 immunodominant antigen was selected by immunoscreening of a S. scabiei var. hominis cDNA. The full-length cDNA was sequenced and cloned into the pGEX vector and the recombinant protein expressed in BL21 (DE3) cells and purified. A vaccination trial was performed consisting of a test group (n = 8) immunised with recAgs (a mix of two recombinant antigens, Ssλ15 and the previously described Ssλ20∆B3) and a control group (n = 8) immunised with PBS. All analyses were performed with R Statistical Environment with α set at 0.050.ResultsThe full-length open reading frame of the 1,821 nt cloned cDNA encodes a 64 kDa polypeptide, the sequence of which had 96 % identity with a hypothetical protein of S. scabiei. Ssλ15 was localised by immunostaining of skin sections in the tegument surrounding the mouthparts and the coxa in the legs of mites. Rabbit immunisation with recAgs induced high levels of specific IgG (P < 0.010) and increased levels of total IgEs. However, no significant clinical protection against S. scabiei challenge was detected. Unexpectedly, the group immunised with the recAgs mix had significantly higher lesion scores (P = 0.050) although lower mean mite densities than those observed in the control group. These results might indicate that the lesions in the recAgs group were due not only to the mites density but also to an exacerbated immunological response after challenge, which is in agreement with the specific high levels of pro-inflammatory cytokines (IL-1 and TNFα) detected after challenge in this group.ConclusionsThe selected antigens delivered as recombinant proteins had no clinical protective efficacy against S. scabiei infestation although immunisation reduced mite density. However, these results pave the way for future studies on alternative production systems, adjuvants, delivery methods and combinations of antigens in order to manage stimulation of clinical protective immune responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1717-9) contains supplementary material, which is available to authorized users.

Abstract 1587: GEP100-Arf6 pathway enhanced by Grb2 expression plays important roles for node-metastasis of lung cancer

Abstract BACKGROUND Invasive and metastatic activities are the most challenging hallmark of cancer in clinical settings. Our previous reports have shown that GEP100 activates Arf6 by its binding to activated ErbB family receptors, such as EGFR. They also have revealed the phosphorylation at specific tyrosines in the C-terminal EGFR is necessary for the binding to the plekstrin homology (PH) domain of GEP100, which tyrosines are well known to be Grb2-binding sites as well. Additionally, this GEP100-Arf6 signal pathway is pivotal for epithelio-mesenchymal transition (EMT) leading to invasion and metastasis in various types of tumors. Here we have examined the augmentation effect of Grb2 on the binding of GEP100 with EGFR and the Arf6 activation. Furthermore, the significance of co-expression of Grb2 and GEP100 in clinical settings was analyzed MATERIALS AND METHODS GST-tagged proteins including PH domain of GEP100 or SH2/SH3 domain of Grb2 inserted into pGEX vector were expressed in bacteria with glutathione-beads purification. They were used for the mutual binding assays. A549 cells which HA-Grb2 or HA alone vector was transfected, were starved for 18hours and stimulated with EGF. And, their lysates were applied for the immunoprecipitation assay against GEP100. Then, Arf6 activities of these cells were examined using the pulldown assay with GST-tagged GGA protein. Furthermore the in vitro invasive activities of those cells were measured by Matrigel invasion assays. The expression levels of Grb2 and GEP100 of the tumor cells by immunohistochemical staining methods were examined in resected human lung adenocarcinoma specimens. The expression data of the two molecules integrated with their clinicopathological factors and EMT status information previously published were analyzed with regard to their invasive and metastatic activities. RESULTS Our results revealed that endogenous Grb2 was immunoprecipitaed with GEP100. These two molecules were physically associated through the PH domain of GEP100 and both the SH2 and N-terminal SH3 domain of Grb2, not its C-terminal SH3 domain. Exogenously aberrant expression of Grb2 in A549 lung cancer cells enhanced the association between activated EGFR and GEP100, consequently, Arf6 activation, and in vitro invasive activity, according to the expression level of Grb2. Among 239 lung adenocarcinoma specimens on tissue microarrays, 131 (55%) and 65 (27%) cases of patients were positive for Grb2 and GEP100, respectively. Tumors with double-positive for Grb2 and GEP100 (45 cases) showed significantly more aggressive EMT status (p = 0.0116) and higher node-metastatic potential (p = 0.0082, node-positive/negative; 12/33 to 7/77) than the double-negative one (84 cases). CONCLUSION Grb2 augments the binding of GEP100 to EGFR leading to Arf6 activation, and promotes lung cancer invasion and metastasis via GEP100-Arf6 pathway. Citation Format: Toshi Menju, Shigeto Nishikawa, Koji Takahashi, Shinya Neri, Takao Nakanishi, Hiroyuki Cho, Kei Shikuma, Terumasa Sowa, Makoto Sonobe, Stephan M. Feller, Hisataka Sabe, Hiroshi Date. GEP100-Arf6 pathway enhanced by Grb2 expression plays important roles for node-metastasis of lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1587.

Molecular cloning, prokaryotic expression and promoter analysis of squalene synthase gene from Schizochytrium Limacinum

Squalene synthase (SQS) is an important enzyme in the steroid biosynthetic pathways which condenses two molecules of farnesyl pyrophosphate into a squalene. In this study, the gene encoding SQS was isolated from Schizochytrium limacinum and characterized. The full-length cDNA of S. limacinum SQS gene (SlSQS) is 1605 bp in length, it contains a 1293 bp ORF encoding a polypeptide of 430 amino acids. Multiple amino acid sequence alignment showed that the SlSQS protein sequence shared 5 conserved signature domains and a hydrophobic carboxy-terminal part with other known SQS protein sequences. C-terminal-truncated SlSQS was constructed into expression vector pGEX and successfully expressed in Escherichia coli cells. The expressed fusion protein was confirmed to have SQS activity. In addition, a 724 bp promoter region of SlSQS was also cloned and several cis-acting elements were predicted. These results might be helpful to understand the structure and expression regulation of SQS in S. limacinum.

Global Impact of Salmonella Pathogenicity Island 2-secreted Effectors on the Host Phosphoproteome

During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated.

Chemotaxis in Epulopiscium

The purpose of this study was to further understand the protein‐protein interactions that occur during chemotaxis of Epulopiscium. Epulopiscium is a very large, unculturable, gram positive bacterium that lives symbiotically in the gut of the surgeonfish. Chemotaxis is the process in which cells move in response to different chemical stimuli. The specific proteins being studied were methyl accepting chemotaxis sensory transducers (MCP3 and 6), CheA, a sensory kinase, and CheW, a coupling protein. These proteins were selected to study because of DNA sequence homology with E. coli. We hypothesized that the Epulo MCP proteins should interact with CheW and/or CheA based on the behavior of the homologous proteins in E. coli. The Epulopiscium proteins were produced recombinantly in E. coli through the use of pGEX and pET‐24b vectors. CheA and CheW were cloned into E. coli using the pET‐24b vector and MCP‐3, MCP‐6, and CheW were cloned and expressed in E. coli using the pGEX vector. These proteins were purified via affinity chromatography and interactions were analyzed using a GST pull down assay.

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.

A Rab8 Guanine Nucleotide Exchange Factor-Effector Interaction Network Regulates Primary Ciliogenesis

Primary cilia are microtubule-based solitary membrane projections on the cell surface that play important roles in signaling and development. Recent studies have demonstrated that polarized vesicular trafficking involving the small GTPase Rab8 and its guanine nucleotide exchange factor Rabin8 is essential for primary ciliogenesis. In this study, we show that a highly conserved region of Rabin8 is pivotal for its activation as a guanine nucleotide exchange factor for Rab8. In addition, in its activated conformation, Rabin8 interacts with Sec15, a subunit of the exocyst and downstream effector of Rab8. Expression of constitutively activated Rab8 promotes the association of Sec15 with Rabin8. Using immunofluorescence microscopy, we found that Sec15 co-localized with Rab8 along the primary cilium. Inhibition of Sec15 function in cells led to defects in primary ciliogenesis. The Rabin8-Rab8-Sec15 interaction may couple the activation of Rab8 to the recruitment of the Rab8 effector and is involved in the regulation of vesicular trafficking for primary cilium formation.

Assembly of a Filamin Four-domain Fragment and the Influence of Splicing Variant-1 on the Structure

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed.

Telomerase Inhibitor PinX1 Provides a Link between TRF1 and Telomerase to Prevent Telomere Elongation

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.

Purification of enzymatically inactive peptidylarginine deiminase type 6 from mouse ovary that reveals hexameric structure different from other dimeric isoforms

The murine peptidylarginine deiminase (PAD) has five isoforms encoded by different genes and participates in a variety of cellular functions through the citrullination of target proteins. The crystal structure of human PAD4 with a dimeric form was previously solved because of the enzyme’s relevance to rheumatoid arthritis. PAD6, abundant in mouse oocytes and eggs, is believed to take part in early events of embryogenesis, but its biochemical properties are little understood. Here we have purified and characterized a recombinant PAD6. A PAD6 cDNA was cloned from mouse ovary RNA and expressed in Escherichia coli through pET29 and pGEX vectors. When benzoyl-L-arginine ethyl ester was used as a substrate, no appreciable activity was detected with a cell homogenate under conditions where a human PAD4 cDNA caused significant activity. Both proteins were affinity-purified to near homogeneity. The circular dichroism spectra of PAD6 and human PAD4 were similar in the far ultraviolet region. On molecular sieving, PAD6 was eluted faster than human PAD4. The cross-linking of PAD6 with dimethyl suberimidate clearly showed six bands on an sodium dodecyl sulfate-polyacrylamide gel. These results indicate that PAD6 can constitute a hexameric structure. The purified PAD6 still showed no enzymatic activity. This unique structure and loss in enzymatic activity is strongly suggested to favor the formation of egg cytoplasmic sheets as the architectural protein.

Negative Regulation of TGFβ Signaling by the Kinase LKB1 and the Scaffolding Protein LIP1

Signal transduction by the Smad pathway elicits critical biological responses to many extracellular polypeptide factors, including TGFβ and bone morphogenetic protein. Regulation of Smad signaling imparts several cytoplasmic and nuclear mechanisms, some of which entail protein phosphorylation. Previous work established a protein complex between Smad4 and the scaffolding protein LKB1-interacting protein 1 (LIP1). LKB1 is a well studied tumor suppressor kinase that regulates cell growth and polarity. Here, we analyzed the LKB1-LIP1 and the Smad4-LIP1 protein complexes and found that LIP1 can self-oligomerize. We further demonstrate that LKB1 is capable of phosphorylating Smad4 on Thr77 of its DNA-binding domain. LKB1 inhibits Smad4 from binding to either TGFβ- or bone morphogenetic protein-specific promoter sequences, which correlates with the negative regulatory effect LKB1 exerts on Smad4-dependent transcription. Accordingly, LKB1 negatively regulates TGFβ gene responses and epithelial-mesenchymal transition. Thus, LKB1 and LIP1 provide negative control of TGFβ signaling.

The Protein Stability of Axin, a Negative Regulator of Wnt Signaling, Is Regulated by Smad Ubiquitination Regulatory Factor 2 (Smurf2)

Axin is a negative regulator of Wnt/β-catenin signaling via regulating the level of β-catenin, which is a key effector molecule. Therefore, controlling the level of Axin is a critical step for the regulation of Wnt/β-catenin signaling. It has been shown that ubiquitination-mediated proteasomal degradation may play a critical role in the regulation of Axin; however, the E3 ubiquitin ligase(s), which attaches ubiquitin to a target protein in combination with an E2 ubiquitin-conjugating enzyme, for Axin has not yet been identified. Here, we show that Smurf2 is an E3 ubiquitin ligase for Axin. Transient expression of Smurf2 down-regulated the level of Axin and increased the ubiquitination of Axin. Conversely, shRNA specific to Smurf2 blocked Axin ubiquitination. Essential domains of Axin responsible for Smurf2 interaction as well as Smurf2-mediated down-regulation and ubiquitination were identified. In vitro ubiquitination assays followed by analysis using mass spectroscopy revealed that Smurf2 specifically ubiquitinylated Lys(505) of Axin and that the Axin(K505R) mutant resisted degradation. Knockdown of endogenous Smurf2 increased the level of endogenous Axin and resulted in reduced β-catenin/Tcf reporter activity. Overall, our data strongly suggest that Smurf2 is a genuine E3 ligase for Axin.

P38 Regulates Expression of Osteoblast-specific Genes by Phosphorylation of Osterix

Osterix, a zinc finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osx-null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that, in several mesenchymal and osteoblastic cell types, BMP-2 induces an increase in expression of the two isoforms of Osterix arising from two alternative promoters. We identified a consensus Sp1 sequence (GGGCGG) as Osterix binding regions in the fibromodulin and the bone sialoprotein promoters in vitro and in vivo. Furthermore, we show that Osterix is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-73 and Ser-77 are the regulatory sites phosphorylated by p38. Our data also demonstrate that Osterix is able to increase recruitment of p300 and Brg1 to the promoters of its target genes fibromodulin and bone sialoprotein in vivo and that it directly associates with these cofactors through protein-protein interactions. Phosphorylation of Osterix at Ser-73/77 increased its ability to recruit p300 and SWI/SNF to either fibromodulin or bone sialoprotein promoters. We therefore propose that Osterix binds to Sp1 sequences on target gene promoters and that its phosphorylation by p38 enhances recruitment of coactivators to form transcriptionally active complexes.

Regulation of Sealing Ring Formation by L-plastin and Cortactin in Osteoclasts

The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be involved in the initial and maturation phases of sealing ring formation, respectively. siRNA to cortactin inhibits this maturation process but not the formation of actin aggregates. Osteoclasts treated as above but with TNF-α demonstrated very similar effects as observed with RANKL. Osteoclasts treated with a neutralizing antibody to TNF-α displayed podosome-like structures in the entire subsurface and at the periphery of osteoclast. It is possible that TNF-α and RANKL-mediated signaling may play a role in the early phase of sealing ring configuration (i.e. either in the disassembly of podosomes or formation of actin aggregates). Furthermore, osteoclasts treated with alendronate or αv reduced the formation of the sealing ring but not actin aggregates. The present study demonstrates a novel mechanistic link between L-plastin and cortactin in sealing ring formation. These results suggest that actin aggregates formed by L-plastin independent of integrin signaling function as a core in assembling signaling molecules (integrin αvβ3, Src, cortactin, etc.) involved in the maturation process.

Localization and Characterization of GTP-Binding Protein CT703 in the Chlamydia trachomatis-Infected Cells

To localize and characterize the GTP-binding protein encoded by the chlamydial ORF CT703 in the Chlamydia trachomatis-infected cells, the gene coding for CT703 in the Chlamydia trachomatis serovar L2 genome was cloned into the prokaryotic expression vector pGEX and expressed as GST fusion protein in the E. coli BL21 strain. The GST-CT703 fusion protein was purified and used to raise antigen-specific antibodies. Using the anti-fusion protein antibodies, we localized the endogenous CT703 protein inside the chlamydial inclusion using an indirect immunofluorescence assay (IFA). We also detected a significantly decreased level of CT703 in cultures that were induced to undergo persistent infection. These observations suggest that CT703 may be an important regulator for promoting chlamydial productive infection.

Direct Ubiquitination of β-Catenin by Siah-1 and Regulation by the Exchange Factor TBL1

Beta-catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, beta-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin beta-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated beta-catenin substrate, we have investigated Siah-1-mediated ubiquitination of beta-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of beta-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate beta-catenin. In addition, TBL1 was shown to play a role in protecting beta-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of beta-catenin, suggesting that polyubiquitination of beta-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.

383 CHARACTERIZATION OF β4-INTEGRIN-MEDIATED CYTOTOXIC EFFECT OF AEXU, A TYPE THREE SECRETION SYSTEM EFFECTOR, ON HUMAN PROSTATE CANCER PC3 CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2010383 CHARACTERIZATION OF β4-INTEGRIN-MEDIATED CYTOTOXIC EFFECT OF AEXU, A TYPE THREE SECRETION SYSTEM EFFECTOR, ON HUMAN PROSTATE CANCER PC3 CELLS Masafumi Kumano, Hideaki Miyake, Takeshi Honda, and Masato Fujisawa Masafumi KumanoMasafumi Kumano Kobe, Japan More articles by this author , Hideaki MiyakeHideaki Miyake Kobe, Japan More articles by this author , Takeshi HondaTakeshi Honda Osaka, Japan More articles by this author , and Masato FujisawaMasato Fujisawa Kobe, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.451AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Type three secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the host cells. AexU, one of TTSS effectors, has been shown to play an important role during A. hydrophila SSU strain pathogenesis through the ribosylation of ADP. Recently, several investigators have suggested the cytotoxic activities of AexU against a wide variety of tumor cells through the modulation of β4-integrin function. The objectives of this study were to evaluate the cytotoxic effect of AexU on human androgen-independent prostate cancer PC3 cells, and to investigate the mechanism of cytotoxicity induced by AexU focusing on the significance of β4-integrin expression in PC3 cells. METHODS We established pGEX vector encoding GST-AexU-HA fusion protein, and purified AexU protein by affinity chromatography. The cytotoxic effects of AexU either alone or in combination with chemotherapeutic agents on PC3 cells were evaluated. We then established PC3 cells in which the expression vector containing short hairpin RNA (shRNA) targeting β4-integrin was introduced (PC3/sh-In). The sensitivity to AexU in PC3/sh-In cells was compared with that in PC3 cells transfected with control vector alone (PC3/Co). RESULTS Expression level of β4-integrin in PC3 cells was significantly greater than those in other human prostate cancer LNCaP and DU145 cells. The cytotoxic effect of AexU on PC3 cells was more significant than those in the remaining two cell lines. Treatment of PC3 cells with AexU resulted in the upregulation of Bax as well as the downregulation of phosphorylated Akt. The IC50 of docetaxel and cisplatin in PC3 cells were decreased by approximately 90% and 80%, respectively, by the combined treatment with sublethal dose of AexU. The cytotoxic effect of AexU on PC3/Co cells was significantly greater than that on PC3/sh-In cells, accompanied by the upregulation of phosphorylated Akt in PC3/sh-In cells compared with that in PC3/Co cells. CONCLUSIONS These findings suggest that treatment with AexU could be a useful therapeutic option against androgen-independent prostate cancer, particularly that overexpressing β4-integrin, through the growth inhibition and the enhanced chemosensitivity. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e151-e152 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Masafumi Kumano Kobe, Japan More articles by this author Hideaki Miyake Kobe, Japan More articles by this author Takeshi Honda Osaka, Japan More articles by this author Masato Fujisawa Kobe, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...