Use of Nonradioactive Detection Method for North- and South-Western Blot.

Many proteins bind to nucleic acids. For the first characterization of novel proteins, a fast and simple technique for testing their nucleic acid binding capabilities is desirable. Here we describe the use of a North-western and South-western blot protocol for the evaluation of the DNA and RNA binding abilities of a novel putative methyl transferase HSPC133 (METTL5).

Immunoblotting Using Radiolabeled Reagents for Detection.

Development of immunoblots is commonly performed using enzyme-labeled antibodies which convert soluble substrates into insoluble colored products. A simple, rapid, and sensitive alternative method which produces low background and allows a rapid quantitative evaluation is the use of radiolabeled antibodies or protein A conjugates. Here we describe the use of iodinated secondary antibodies for immunodetection of an autoantigen during HPLC purification.

Chlamydia psittaci inclusion membrane protein IncB associates with host protein Snapin.

Chlamydia (C.) psittaci, the causative agent of psittacosis in birds and humans, is the most important zoonotic pathogen of the family Chlamydiaceae. During a unique developmental cycle of this obligate intracellular pathogen, the infectious elementary body gains access to the susceptible host cell, where it transforms into the replicative reticulate body. C. psittaci uses dynein motor proteins for optimal early development. Chlamydial proteins that mediate this process are unknown. Two-hybrid screening with the C. psittaci inclusion protein IncB as bait against a HeLa Yeast Two-hybrid (YTH) library revealed that the host protein Snapin interacts with IncB. Snapin is a cytoplasmic protein that plays a multivalent role in intracellular trafficking. Confocal fluorescence microscopy using an IncB-specific antibody demonstrated that IncB, Snapin, and dynein were co-localized near the inclusion of C. psittaci-infected HEp-2 cells. This co-localization was lost when Snapin was depleted by RNAi. The interaction of Snapin with both IncB and dynein has been shown in vitro and in vivo. We propose that Snapin connects chlamydial inclusions with the microtubule network by interacting with both IncB and dynein.

Proteomic identification of PSF and p54(nrb) as TopBP1-interacting proteins.

TopBP1 is a BRCT domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and damage signalling. To further dissect its biological functions, we explored TopBP1-interacting proteins by co-immunoprecipitation assays and LC-ESI-MS-analyses. As TopBP1 binding partners we identified p54(nrb) and PSF, and confirmed the physical interactions by GST pull-down assays, co-immunoprecipitations and by yeast two-hybrid experiments. Recent evidence shows an involvement of p54(nrb) and PSF in DNA double-strand break repair (DSB) and radioresistance. To get a first picture of the physiological significance of the interaction of TopBP1 with p54(nrb) and PSF we investigated in real time the spatiotemporal behaviour of the three proteins after laser microirradiation of living cells. Localisation of TopBP1 at damage sites was noticed as early as 5 s following damage induction, whereas p54(nrb) and PSF localised there after 20 s. Both p54(nrb) and PSF disappeared after 20 s while TopBP1 was retained at damage sites significantly longer suggesting different functions of the proteins during DSB recognition and repair.

Functional interaction between type III-secreted protein IncA of Chlamydophila psittaci and human G3BP1.

Chlamydophila (Cp.) psittaci, the causative agent of psittacosis in birds and humans, is the most important zoonotic pathogen of the family Chlamydiaceae. These obligate intracellular bacteria are distinguished by a unique biphasic developmental cycle, which includes proliferation in a membrane-bound compartment termed inclusion. All Chlamydiaceae spp. possess a coding capacity for core components of a Type III secretion apparatus, which mediates specific delivery of anti-host effector proteins either into the chlamydial inclusion membrane or into the cytoplasm of target eukaryotic cells. Here we describe the interaction between Type III-secreted protein IncA of Cp. psittaci and host protein G3BP1 in a yeast two-hybrid system. In GST-pull down and co-immunoprecipitation experiments both in vitro and in vivo interaction between full-length IncA and G3BP1 were shown. Using fluorescence microscopy, the localization of G3BP1 near the inclusion membrane of Cp. psittaci-infected Hep-2 cells was demonstrated. Notably, infection of Hep-2 cells with Cp. psittaci and overexpression of IncA in HEK293 cells led to a decrease in c-Myc protein concentration. This effect could be ascribed to the interaction between IncA and G3BP1 since overexpression of an IncA mutant construct disabled to interact with G3BP1 failed to reduce c-Myc concentration. We hypothesize that lowering the host cell c-Myc protein concentration may be part of a strategy employed by Cp. psittaci to avoid apoptosis and scale down host cell proliferation.

Chlamydial protease CT441 interacts with SRAP1 co-activator of estrogen receptor alpha and partially alleviates its co-activation activity.

Chlamydiae are obligate intracellular pathogens which secrete host-interactive proteins capable of directly modulating eukaryotic pathways. Using the PDZ domain of the protease CT441 of Chlamydia trachomatis as a bait in a yeast two-hybrid screen, we identified the SRAP1 co-activator of estrogen receptor alpha (ERalpha) as an interacting protein. SRAP1 is a unique modulator of steroid receptor activity, as it is able to mediate its co-regulatory effects both as a RNA and a protein. GST pull-down experiments confirmed the interaction of CT441 and SRAP1 in vitro. Furthermore, it was shown that the CT441-PDZ domain fused to a nuclear localization signal was able to bind and to target SRAP1 to the nucleus in mammalian cells. CT441 did not cleave SRAP1, but retained the protein in the cytoplasm and thereby partially alleviated its co-activation of ERalpha in a heterologous yeast system and in mammalian cells. Possible implications of chlamydial regulation of host metabolism by targeting ERalpha activity are discussed. Moreover, the property of CT441-PDZ domain to specifically sequester SRA1 protein but not SRA1 RNA may be used to distinguish between the cellular functions of the SRA1 RNA and protein. This has clinical relevance as it has been proposed that disturbance of the balance between SRAP1-coding and non-coding SRA1 RNAs in breast tumor tissues might be involved in breast tumorigenesis.

GD3-7-aldehyde is an apoptosis inducer and interacts with adenine nucleotide translocase.

We prepared GD3-7-aldehyde (GD3-7) and determined its apoptotic potential. GD3-7 proved to be more efficient to induce pro-apoptotic mitochondrial alterations than GD3 when tested on mouse liver mitochondria. GD3-7-induced mitochondrial swelling and depolarization was blocked by cyclosporin A (CsA) supporting a critical role of the permeability transition pore complex (PTPC) during GD3-7-mediated apoptosis. In contrast to GD3, GD3-7 was able to induce channel formation in proteoliposomes containing adenine nucleotide translocase (ANT). This suggests that ANT is the molecular target of GD3-7. Using a specific antiserum, GD3-7 was detected in the lipid extract of the myeloid tumor cell line HL-60 after apoptosis induction, but not in living cells. Therefore, GD3-7 might be a novel mediator of PTPC-dependent apoptosis in cancer cells.

Use of non-radioactive detection method for north- and southwestern blot.

Many proteins bind to nucleic acids. For the first characterization of novel proteins, a fast and simple technique for testing their nucleic acid binding capabilities is desirable. Here, we describe the use of a northwestern and southwestern blot protocol for the evaluation of the DNA and RNA binding abilities of a novel putative methyl transferase HSPC133 (METTL5).

Immunoblotting using radiolabeled reagents for detection.

Development of immunoblots is commonly performed using enzyme-labeled antibodies, which convert soluble substrates into insoluble colored products. A simple, rapid, and sensitive alternative method that produces low background and allows a rapid quantitative evaluation is the use of radiolabeled antibodies or protein A conjugates. Here, we describe the use of iodinated secondary antibodies for immunodetection of an autoantigen during HPLC purification.

Characterization of the interaction between the human DNA topoisomerase IIbeta-binding protein 1 (TopBP1) and the cell division cycle 45 (Cdc45) protein.

TopBP1 (topoisomerase IIbeta-binding protein 1) is a BRCT [BRCA1 (breast-cancer susceptibility gene 1) C-terminal]-domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and DNA damage signalling. Experiments with fission yeast and Xenopus revealed that the TopBP1 homologues of these organisms are required for chromatin loading of the replication protein Cdc45 (cell division cycle 45). To improve our understanding of the physiological functions of human TopBP1, we investigated the interplay between human TopBP1 and Cdc45 proteins in synchronized HeLa-S3 cells. Using GST (glutathione transferase) pull-down and co-immunoprecipitation techniques, we showed a direct interaction between TopBP1 and Cdc45 in vitro and in vivo. The use of deletion mutants in GST pull-down assays identified the first and second as well as the sixth BRCT domains of TopBP1 to be responsible for the functional interaction with Cdc45. Moreover, the interaction between Cdc45 and the first and second BRCT domains of TopBP1 inhibited their transcriptional activation both in yeast and mammalian one-hybrid systems. Both proteins interacted exclusively at the G(1)/S boundary of cell cycle; only weak interaction could be found at the G(2)/M boundary. The overexpression of the sixth BRCT domain led to diminished loading of Cdc45 on to chromatin. These results suggest that human TopBP1 is involved in the formation of the initiation complex of replication in human cells and is required for the recruitment of Cdc45 to origins of DNA replication.

LINC, a human complex that is related to pRB-containing complexes in invertebrates regulates the expression of G2/M genes.

Here we report the identification of the LIN complex (LINC), a human multiprotein complex that is required for transcriptional activation of G2/M genes. LINC is related to the recently identified dREAM and DRM complexes of Drosophila and C. elegans that contain homologs of the mammalian retinoblastoma tumor suppressor protein. The LINC core complex consists of at least five subunits including the chromatin-associated LIN-9 and RbAp48 proteins. LINC dynamically associates with pocket proteins, E2F and B-MYB during the cell cycle. In quiescent cells, LINC binds to p130 and E2F4. During cell cycle entry, E2F4 and p130 dissociate and LINC switches to B-MYB and p107. Chromatin Immunoprecipitation experiments demonstrate that LINC associates with a large number of E2F-regulated promoters in quiescent cells. However, RNAi experiments reveal that LINC is not required for repression. In S-phase, LINC selectively binds to the promoters of G2/M genes whose products are required for mitosis and plays an important role in their cell cycle dependent activation.

Enzymatic treatment of duck hepatitis B virus: topology of the surface proteins for virions and noninfectious subviral particles.

The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles' interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: 1--preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was accessible, 2--phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions, 3--iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions.

Autoimmunity as a result of escape from RNA surveillance.

In previous studies, we detected a frame shift mutation in the gene encoding the autoantigen La of a patient with systemic lupus erythematosus. The mutant La mRNA contains a premature termination codon. mRNAs that prematurely terminate translation should be eliminated by RNA quality control mechanisms. As we find Abs specific for the mutant La form in approximately 30% of sera from anti-La-positive patients, we expected that mutant La mRNAs circumvent RNA control and the expression of mutant La protein could become harmful. Indeed, real-time PCR, immunostaining, and immunoblotting data of mice transgenic for the mutant La form show that mutant La mRNAs are not repressed in these animals and are translated to mutant La protein. In addition to the mutant La protein, we detected a minor portion of native human La in the mutant La-transgenic mice. Therefore, ribosomal frame shifting may allow the mutant La mRNA to escape from RNA control. Interestingly, expression of the mutant La mRNA results in a lupus-like disease in the experimental mice. Consequently, escape of mutant La mRNA from RNA control can have two effects: it 1) results in the expression of an immunogenic (neo)epitope, and 2) predisposes to autoimmunity.

Global analysis of host tissue gene expression in the invasive front of colorectal liver metastases.

Host cell reactions are a crucial determinant for tumor invasion. We analyzed on a genomewide scale gene expression differences between microdissected tissues taken from unaffected liver tissue of a human colorectal tumor (LS174) growing in the livers of nude mice and tissue from the host part of the invasive front. Due to the low degree of interspecies cross-hybridization of 15% as determined on Affymetrix microarrays, our xenograft model allowed for the distinction of genes of murine versus human origin even if the respective tissues could not be isolated separately. Using the gene ontology (GO) classification, we were able to determine patterns of up- and downregulated genes in the liver part of the invasive front. We observed a pronounced overrepresentation, e.g., of the GO terms "extracellular matrix," "cell communication," "response to biotic stimulus," "structural molecule activity" and "cell growth," indicating a very pronounced host cell response to tumor invasion. On the single gene level, hepatic stellate cell (HSC) activation markers were overrepresented in the liver part of the invasion front. Immunohistochemistry and qPCR confirmed an activation of HSC as well as an increased number of HSC in the invasive front as compared to the noninvaded liver tissue. In summary, our data demonstrate the feasibility of an interspecies differential gene expression approach on a genomewide scale.

Reversible membrane association of dinitrogenase reductase activating glycohydrolase in the regulation of nitrogenase activity in Rhodospirillum rubrum; dependence on GlnJ and AmtB1.

In the photosynthetic bacterium Rhodospirillum rubrum nitrogenase activity is regulated by reversible ADP-ribosylation of dinitrogenase reductase in response to external so called "switch-off" effectors. Activation of the modified, inactive form is catalyzed by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the ADP-ribose moiety. This study addresses the signal transduction between external effectors and DRAG. R. rubrum, wild-type and P(II) mutant strains, were studied with respect to DRAG localization. We conclude that GlnJ clearly has an effect on the association of DRAG to the membrane in agreement with the effect on regulation of nitrogenase activity. Furthermore, we have generated a R. rubrum mutant lacking the putative ammonium transporter AmtB1 which was shown not to respond to "switch-off" effectors; no loss of nitrogenase activity and no ADP-ribosylation. Interestingly, DRAG was mainly localized to the cytosol in this mutant. Overall the results support our model in which association to the membrane is part of the mechanism regulating DRAG activity.