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Transcription factors (TFs), which play crucial roles in the regulation of gene expression in the human genome, are highly regulated by a variety of mechanisms. A single extracellular stimulus can trigger multiple signaling pathways, and these in turn can activate multiple TFs to mediate the inducible expression of target genes. Alterations in the activities of TFs are often associated with human diseases, such as altered activating factor 1, estrogen receptor, and p53 function in cancer, nuclear factor kappaB in inflammatory diseases, and peroxisome proliferator-activated receptor gamma in obesity. A systematic assay for profiling the activation of TFs will aid in elucidating the mechanisms of TF activation, reveal altered TFs associated with human diseases, and aid in developing assays for drug discovery. Here, we developed a 24-plex fluorescent microsphere-based TF activation assay system with a 96-well plate format. The assay system enabled high-throughput profiling of the DNA binding activity of TFs in multiple samples with high sensitivity.

Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system.

Numerous studies have investigated the relationship between polymorphisms, in particular 677C-T and 1298A-C, of the methylene-tetrahydrofolate reductase (MTHFR) gene and coronary artery disease (CAD) with conflicting results. This study investigates the potential association of two point mutations inMTHFR, 677C-T and 1793G-A, along with other risk factors, with CAD. This is the first hospital-based study to investigate 1793G-A in this context. Genotype analysis was performed on 729 Caucasians and 66 African Americans undergoing coronary angiography using a novel PCR-based assay involving formation of Holliday junctions. Allelic frequencies for 677C-T were 66.2% C and 33.8% T for Caucasians and 90.9% C and 9.1% T for African Americans. With respect to the 1793G-A polymorphism, allelic frequencies were 94.7% G and 5.3% A for Caucasians and 99.2% G and 0.8% A for African Americans. Disease associations were examined in the Caucasian patients due to their greater genotype variability and larger number in the patient cohort. Results suggest that neither 677CT heterozygotes (OR-1.36; 95% CI 0.95 to 1.96) nor mutant homozygotes (OR-0.73; 95% CI 0.44 to 1.20) have either an increased or decreased risk for CAD compared to the 677CC genotype. Likewise, the 1793GA genotype did not demonstrate a statistically significant association with CAD compared to 1793GG patients (OR-0.79; 95% CI 0.47 to 1.33). Mean homocysteine levels (μmol/L) increased from normal to mutant for 677C-T (677CC: 10.2; 677CT: 11.0; 677TT: 11.6) and normal to heterozygous in 1793G-A (1793GG: 10.7; 1793GA: 11.5). TheseMTHFRpolymorphisms did not contribute to the prediction of clinically defined CAD in Caucasians.

Summary The hyperthermoacidophilic archaeon Sulfolobus shibatae contains group II chaperonins, known as rosettasomes, which are two nine-membered rings composed of three different 60 kDa subunits (TF55 alpha, beta and gamma). We sequenced the gene for the gamma subunit and studied the temperature-dependent changes in alpha, beta and gamma expression, their association into rosettasomes and their phylogenetic relationships. Alpha and beta gene expression was increased by heat shock (30 min, 86°C) and decreased by cold shock (30 min, 60°C). Gamma expression was undetectable at heat shock temperatures and low at normal temperatures (75–79°C), but induced by cold shock. Polyacrylamide gel electrophoresis indicated that in vitro alpha and beta subunits form homo-oligomeric rosettasomes, and mixtures of alpha, beta and gamma form hetero-oligomeric rosettasomes. Transmission electron microscopy revealed that beta homo-oligomeric rosettasomes and all hetero-oligomeric rosettasomes associate into filaments. In vivo rosettasomes were hetero-oligomeric with an average subunit ratio of 1α:1β:0.1γ in cultures grown at 75°C, a ratio of 1α:3β:1γ in cultures grown at 60°C and a ratio of 2α:3β:0γ after 86°C heat shock. Using differential scanning calorimetry, we determined denaturation temperatures (Tm) for alpha, beta and gamma subunits of 95.7°C, 96.7°C and 80.5°C, respectively, and observed that rosettasomes containing gamma were relatively less stable than those with alpha and/or beta only. We propose that, in vivo, the rosettasome structure is determined by the relative abundance of subunits and not by a fixed geometry. Furthermore, phylogenetic analyses indicate that archaeal chaperonin subunits underwent multiple duplication events within species (paralogy). The independent evolution of these paralogues raises the possibility that chaperonins have functionally diversified between species.

To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif.

A Thermus thermophilus host strain of which the leuB gene was totally deleted was constructed from a delta pyrE strain by a two step method. First, the leuB gene was replaced with the pyrE gene. Second, the inserted pyrE gene was deleted by using 5-fluoroorotic acid. A plasmid vector with the leuB marker was constructed and the plasmid complemented the leuB deficiency of the host. When the leuB gene from Escherichia coli and its derivative encoding a stabilized enzyme were expressed with the host-vector system, their growth temperature reflected the stability of the enzyme. These results suggest that the gene replacement deletion method using the pyrE gene is useful for the construction of a reliable plasmid vector system and it can be applied to the selection of stabilized enzymes.

Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon Sulfolobus shibatae the two chaperonin proteins represent ≈4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations ≥ 0.5 mg/ml purified chaperonins form filaments in the presence of Mg 2+ and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist in vivo and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained S. shibatae cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins are associated with insoluble cellular structures and these structures appear to be filamentous in some areas, although they could not be uranyl-acetate-stained. The existence of chaperonin filaments in vivo suggests a mechanism whereby their protein-folding activities can be regulated. More generally, the filaments themselves may play a cytoskeletal role in Archaea.

Transcription factors are a group of proteins that modulate the expression of genes involved in many biological processes, such as cell growth and differentiation. Alterations in transcription factor function are associated with many human diseases, and therefore these proteins are attractive potential drug targets. A key issue in the development of such therapeutics is the generation of effective tools that can be used for high throughput discovery of the critical transcription factors involved in human diseases, and the measurement of their activities in a variety of disease or compound-treated samples. Here, a number of innovative arrays and 96-well format assays for profiling and measuring the activities of transcription factors will be discussed.

The role of electrostatic interaction between Lys96 and Glu47 of isocitrate dehydrogenase from Thermus thermophilus was investigated by site-directed mutagenesis. These two residues are located near the active site and involved in the interdomain interaction. Analyses of the catalytic properties and thermostability of the Glu147Gln mutant revealed that this interaction plays important roles in catalytic function and protein stability.

Hereditary hemochromatosis, one of the most common genetic diseases in Caucasians, is characterized by excessive iron deposition secondary to hyperabsorption of dietary iron and can potentially can lead to multiorgan failure if untreated. The C282Y and H63D mutations of the HFE gene are the most common mutations associated with symptomatic hemochromatosis. Recently, several genotyping methods have been used to identify hemochromatosis mutations and other single-nucleotide polymorphisms (SNPs) (1). There are advantages and limitations for each methodology in terms of cost and efficiency (2). We report here the application of a new SNP/point mutation genotyping platform developed by our group, the Holliday junction-based allele-specific genotyping (HAS) platform (3), to identify the C282Y and H63D mutations associated with hemochromatosis. For the HAS technology, we developed two detection modalities based on differences in the physical and biochemical properties between Holliday junctions (HJs) and duplex DNA or single-stranded DNA. The junctions that form in an allele-specific manner can be detected heterogeneously through gel electrophoresis (acrylamide or agarose; Fig. 1A⇓ ) or homogeneously through a fluorescence polarization (FP) competition assay (3). Using the HAS genotyping platform, we developed an assay for genotyping the C282Y and H63D mutations with both gel electrophoresis and FP for detection. Five primers (one forward, two reference, and two reverse primers) were designed for each of the two mutations (see Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue1/). PCR amplification was performed with a PTC-200 DNA Engine thermocycler (MJ Research, Inc.). For each point mutation locus, two PCR reactions in separate tubes were carried out in parallel for each DNA sample to amplify the target DNA with each of its two DNA references. Each PCR reaction contained four primers [one forward, one reference, reverse tail I, and reverse tail II (Table …

The hyperthermoacidophilic archaeon Sulfolobus shibatae contains group II chaperonins, known as rosettasomes, which are two nine-membered rings composed of three different 60 kDa subunits (TF55 alpha, beta and gamma). We sequenced the gene for the gamma subunit and studied the temperature-dependent changes in alpha, beta and gamma expression, their association into rosettasomes and their phylogenetic relationships. Alpha and beta gene expression was increased by heat shock (30 min, 86 degrees C) and decreased by cold shock (30 min, 60 degrees C). Gamma expression was undetectable at heat shock temperatures and low at normal temperatures (75-79 degrees C), but induced by cold shock. Polyacrylamide gel electrophoresis indicated that in vitro alpha and beta subunits form homo-oligomeric rosettasomes, and mixtures of alpha, beta and gamma form hetero-oligomeric rosettasomes. Transmission electron microscopy revealed that beta homo-oligomeric rosettasomes and all hetero-oligomeric rosettasomes associate into filaments. In vivo rosettasomes were hetero-oligomeric with an average subunit ratio of 1alpha:1beta:0.1gamma in cultures grown at 75 degrees C, a ratio of 1alpha:3beta:1gamma in cultures grown at 60 degrees C and a ratio of 2alpha:3beta:0gamma after 86 degrees C heat shock. Using differential scanning calorimetry, we determined denaturation temperatures (Tm) for alpha, beta and gamma subunits of 95.7 degrees C, 96.7 degrees C and 80.5 degrees C, respectively, and observed that rosettasomes containing gamma were relatively less stable than those with alpha and/or beta only. We propose that, in vivo, the rosettasome structure is determined by the relative abundance of subunits and not by a fixed geometry. Furthermore, phylogenetic analyses indicate that archaeal chaperonin subunits underwent multiple duplication events within species (paralogy). The independent evolution of these paralogues raises the possibility that chaperonins have functionally diversified between species.

Chaperonins are multisubunit protein complexes that can be isolated from cells as high-molecular-weight structures that appear as double rings in the electron microscope. We recently discovered that chaperonin double rings isolated from the hyperthermophilic archaeon Sulfolobus shibatae, when incubated at physiological temperatures in the presence of ATP and Mg2+, stacked into filaments; we hypothesized that these filaments are related to filaments seen inside S. shibatae cells and that chaperonins exist as filaments in vivo (J. D. Trent et al., 1997, Proc. Natl. Acad. Sci. USA 94, 5383-5388). This paper elucidates the conditions under which we have observed S. shibatae chaperonins to form filaments and evaluates native polyacrylamide gel electrophoresis (PAGE), TEM, spectrophotometry, and centrifugation as methods for studying these filaments. We observed that in the presence of Mg2+ combined with ATP, ADP, ATPgammaS, or GTP, native PAGE indicated that chaperonin subunits assembled into double rings and that the conformation of these double rings was effected by nucleotide binding, but we saw no indication of chaperonin filament formation. Under these same conditions, however, TEM, spectroscopy, and centrifugation methods indicated that chaperonin subunits and double rings had assembled into filaments. We determined that this discrepancy in the representation of the chaperonin structure was due to the native PAGE method itself. When we exposed chaperonin filaments to the electrophoretic field used in native PAGE, the filaments dissociated into double rings. This suggests that TEM, spectrophotometry, and centrifugation are the preferred methods for studying the higher-order structures of chaperonins, which are likely to be of biological significance.

Chaperonins are multi-subunit double-ring complexed composed of 60-kDa proteins that are believed to mediate protein folding in vivo. The chaperonins in the hyperthermophilic archaeon Sulfolobus shibatae are composed of the organism`s two most abundant proteins, which represent 4% of its total protein and have an intracellular concentration of {ge} 3.0 mg/ml. At concentrations of 1.0 mg/ml, purified chaperonin proteins aggregate to form ordered filaments. Filament formation, which requires Mg{sup ++} and nucleotide binding (not hydrolysis), occurs at physiological temperatures under conditions suggesting filaments may exist in vivo. If the estimated 4,600 chaperonins per cell, formed filaments in vivo, they could create a matrix of filaments that would span the diameter of an average S. shibatae cell 100 times. Direct observations of unfixed, minimally treated cells by intermediate voltage electron microscopy (300 kV) revealed an intracellular network of filaments that resembles chaperonin filaments produced in vitro. The hypothesis that the intracellular network contains chaperonins is supported by immunogold analyses. The authors propose that chaperonin activity may be regulated in vivo by filament formation and that chaperonin filaments may serve a cytoskeleton-like function in archaea and perhaps in other prokaryotes.

The substrate-binding sites of NADP-dependent isocitrate dehydrogenase and NAD-dependent 3-isopropylmalate dehydrogenase from Thermus thermophilus were analyzed by site-directed mutagenesis. Ser97 and Asn99 of isocitrate dehydrogenase were identified to be involved in the isocitrate recognition. In 3-isopropylmalate dehydrogenase, the corresponding residues, Leu90 and Leu91, appear to recognize the substrate by forming a hydrophobic pocket. Double mutation of Asp78 and Glu87 revealed that negative charge of these residues plays a crucial role in discriminating isopropylmalate from isocitrate.

Early cancer diagnosis and risk assessment are powerful drivers in discovery of oncology biomarkers for diagnostic tests and simultaneous improvement of already identified biomarkers. Molecular tools such as RNA Colorimetric in situ hybridization (CISH) and immunohistochemistry (IHC) combined with conventional histopathology promise to increase the sensitivity and specificity of cancer staging and overall survival prediction. Changes in oncogene and proto-oncogene expression are a hallmark of cancer. For this reason, simultaneous quantification of mRNA and protein expression using RNA CISH and IHC combined with image-analysis is of interest. One such proto-oncogene is HER2, which is encoded by ERBB2 gene located at the long arm of human chromosome 17 (17q12). Here, we combine RNA CISH and IHC with image-analysis to develop a reliable quantitative HER2 assay that enables quantification of HER2 mRNA and protein expression in the context of human breast cancer. Since HER2 is only over-expressed in a subset of human breast cancers, we initially focused our analysis by screening tissue microarrays (TMAs) using RNA CISH and IHC. TMAs containing 20 normal and 20 breast tumor tissues were tested and whole-slide imaging was used to obtain CISH and IHC signals. HER2 mRNA and protein expression were analyzed, quantified, and compared both at tissue- and individual cell-levels using proprietary image-analysis based tools. Moreover, we compared heterogeneity of RNA and protein expression and further extended our studies to several other cancer-related genes. Finally, since oncogene expression plays a role in cancer pathologies, successful quantification of oncogene in situ expression in human cancer tissues may prove to be beneficial to further understanding of cancer pathology, for drug screening and development of biomarkers and ultimately diagnostic tests. Citation Format: Mirza Peljto, Joseph Krueger, Erik Hagendorn, David Young, Steve Potts, Quan Nguyen, Botoul Maqsodi, Takuro Yaoi, Gary McMaster. Tissue image analysis tool to quantify HER2 RNA and protein expression by colorimetric in situ hybridization and immunohistochemistry for breast cancer diagnosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 44. doi:10.1158/1538-7445.AM2013-44

The coenzyme specificity of isocitrate dehydrogenase from an extreme thermophilic bacterium was converted from NADP-dependent to NAD-dependent by replacing a "module" involved in the coenzyme binding site. The conversion was not possible with the replacement of a few residues that interact with the coenzyme. In addition, the module-replaced mutant dehydrogenase was as stable as the original, wild type enzyme. The results support a previous hypothesis that a module is a structural and functional unit of a protein.

Isocitrate dehydrogenase from the thermophilic bacterium, Thermus thermophilus HB8, was crystallized by the vapor diffusion hanging drop method with polyethylene glycol 6000 as the precipitant. Pillar-like crystals of about 0.6 x 0.6 x 0.3 mm3 were obtained. Analysis of a series of oscillation photographs indicated that the orthorhombic crystals belonged to the I222 or I2(1)2(1)2(1) space group with unit cell dimensions of a = 100.1 A, b = 150.4 A, and c = 87.4 A. Intensity data were collected up to 2.5 A resolution.

The coenzyme binding site of NAD(+)-dependent 3-isopropylmalate dehydrogenase from Thermus thermophilus was analyzed by chemical modification and site-directed mutagenesis, and His273 of the enzyme was identified to be involved in the coenzyme binding.

Isocitrate dehydrogenase (ICDH) from an extreme thermophile, Thermus thermophilus HB8, was overexpressed in Escherichia coli. The enzyme was easily purified to homogeneity by a combination of heat treatment (70 degrees C, 20 min) and column chromatography. The N-terminal sequence of the protein thus purified coincided with that of the protein extracted from the thermophile. The substrate specificity of the enzyme was mutationally analyzed and engineered to recognize 3-alkyl-malate as a substrate. Based on the three-dimensional structure of E. coli isocitrate dehydrogenase, Ser97 qnd Asn99 of the thermophile enzyme were speculated to participate in the substrate recognition, and these residues were replaced with threonine and leucine, respectively. Molecular recognition of the mutant enzymes, [S97T]ICDH, [N99L]ICDH, and [S97T, N99L]ICDH, were studied using isocitrate, 3-isopropylmalate, and 3-ethylmalate. The affinity toward isocitrate was reduced in the cases of [S97T]ICDH and [N99L]ICDH, confirming the importance of the residues for the reaction. Though none of the mutants acted on 3-isopropylmalate, [N99L]ICDH was competitively inhibited by 3-isopropylmalate with a higher affinity than that of the wild-type enzyme. [N99L]ICDH showed an approximately 10(3)-fold higher value of (kcat/Km)3-ethylmalate/(kcat/Km)isocitrate than the wild-type enzyme, indicating that the single mutation of Asn99 to leucine switched the substrate specificity of the enzyme away from isocitrate and toward 3-ethylmalate.

The coenzyme binding site of isocitrate dehydrogenase from Thermus thermophilus was analyzed by site-directed mutagenesis. The mutation analysis revealed that Arg231 and Tyr284 are involved in the discrimination between NAD and NADP, suggesting that these two residues interact with 2′-phosphate group of NADP.