TR Domain Research Articles

Identification and expression analysis of Oxfibrillin gene involved in the regeneration process of Ophryotrocha xiamen (Annelida, Dorcilleidae)

Regeneration of lost body parts is a widespread phenomenon across annelids. However, the molecular inducers of the cell sources for this reparative morphogenesis have not been identified. We have identified a regeneration-related gene Oxfibrillin from the transcriptome analysis of a polychaeta, Ophryotrocha xiamen, which is found to be a well-suited model to study the mechanisms of regeneration. Fibrillins are large glycoproteins that assemble to form the microfibrils and regulate growth factors or other transfer processes. Here, we obtained the 31,274 bp genomic DNA sequences of Oxfibrillin. The coding sequence length was 5784 bp encoding 1927 amino acids with a VWD domain, EGF/cb-EGF domains, a TR domain, and a transmembrane domain. Oxfibrillin was positioned within the subgroup of invertebrates and showed low scores for homology to mammalian fibrillin. In gene expression analysis, Oxfibrillin genes were constantly upregulated during the early regeneration process and then remained stable until the formation of the complete tail which indicated that it might be a vital factor to affect posterior regeneration process. Therefore, the Oxfibrillin of O. xiamen might play important roles in the regeneration process.

The Singer transfer for infinite real projective space

Abstract The article is devoted to studying the Singer transfer. The image of the Singer transfer Tr * ℝ ⁢ ℙ ∞ {\operatorname{Tr}_{*}^{\mathbb{R}\mathbb{P}^{\infty}}} for the infinite real projective space is proved to be a module over the image of the transfer Tr * {\operatorname{Tr}_{*}} for the sphere. Further, the algebraic Kahn–Priddy homomorphism is shown to be an epimorphism from Im ⁡ Tr * ℝ ⁢ ℙ ∞ {\operatorname{Im}\operatorname{Tr}_{*}^{\mathbb{R}\mathbb{P}^{\infty}}} onto Im ⁡ Tr * {\operatorname{Im}\operatorname{Tr}_{*}} in positive stems. The indecomposable elements h ^ i {\widehat{h}_{i}} for i ≥ 1 {i\geq 1} and c ^ i , d ^ i , e ^ i , f ^ i , p ^ i {\widehat{c}_{i},\widehat{d}_{i},\widehat{e}_{i},\widehat{f}_{i},\widehat{p}_{% i}} for i ≥ 0 {i\geq 0} are in the image of the Singer transfer Tr * ℝ ⁢ ℙ ∞ {\operatorname{Tr}_{*}^{\mathbb{R}\mathbb{P}^{\infty}}} , whereas the ones g ^ i {\widehat{g}_{i}} for i ≥ 1 {i\geq 1} and D ^ 3 ⁢ ( i ) , p ^ i ′ {\widehat{D}_{3}(i),\widehat{p}^{\prime}_{i}} for i ≥ 0 {i\geq 0} are not in its image. This transfer is shown to be not monomorphic in every positive homological degree. The transfer behavior is also investigated near “critical elements”. The squaring operation on the domain of Tr * ℝ ⁢ ℙ ∞ {\operatorname{Tr}_{*}^{\mathbb{R}\mathbb{P}^{\infty}}} is proved to be eventually isomorphic. This phenomenon leads to the so-called “stability” of the Singer transfer for the infinite real projective space under the iterated squaring operation.

Quality of life and burden in family caregivers of patients with advanced cancer receiving specialized palliative care.

The caregivers of advanced cancer patients face many physical, psychological, social, and economic problems. In this study, the quality of life and burden in the primary family caregiver of patients with advanced cancer receiving inpatient palliative care were investigated. A total of 200 patients with advanced cancer hospitalized at palliative care center and primary caregivers were included. Functional capacities of patients were evaluated with Karnofsky Performance Scale and need of care with Katz index. The Turkish version of World Health Organization Quality of Life Instrument, Short Form (WHOQOL-BREF TR) was used to assess the quality of life of caregivers. The median Karnofsky Performance score of patients was found to be 30% and Katz Index score to be 2. The mean WHOQOL-BREF TR domain scores of family caregivers were 48.96 (Standard deviation (SD) =12.67) for physical health, 59.21 (SD=14.09) for psychological status, 56.83 (SD=20.91) for social relations, and 55.67 (SD=14.13) for environmental domain. Scores of psychological and environmental subscales were lower in women caregivers. The environmental subscale showed a significant difference in terms of education. The score of social relations subscale of the care giving spouse was lower than caregiving children and siblings. The score of environmental subscale of caregivers with insufficient income was found to be lower than caregivers with sufficient income. Karnofsky Performance and Katz Index scores and subscales of WHOQOL-BREF TR did not reveal any significant relation. This study showed that all subscales of quality of life are impaired in caregivers of advanced cancer patients, physical health being the most prominent.

The role of calcium in the interaction between calmodulin and a minimal functional construct of eukaryotic elongation factor 2 kinase.

Eukaryotic elongation factor 2 kinase (eEF-2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF-2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM-regulated kinases. We had previously identified a minimal construct of eEF-2K (TR) that is activated similarly to the wild-type enzyme by CaM in vitro and retains its ability to phosphorylate eEF-2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1-methyls of TR and Ile δ1- and Met ε-methyls of CaM, as probes of their mutual interaction and the influence of Ca2+ thereon. We find that in the absence of Ca2+ , CaM exclusively utilizes its C-terminal lobe (CaMC ) to engage the N-terminal CaM-binding domain (CBD) of TR in a high-affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca2+ -loaded N-terminal lobe of CaM (CaMN ) at increased Ca2+ levels serves to enhance the affinity further. These latter interactions under Ca2+ saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca2+ -free state. Our data are consistent with a scenario in which Ca2+ enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM-bound species without significantly modifying the conformation of TR within the final, active complex.

Molecular cloning, characterization and expression analysis of glucose transporters fromRachycentron canadum

It is very necessary to clarify the mechanism of carbohydrate metabolism in fish due to their poor ability to utilize carbohydrates. Glucose transporters (GLUTs) are important carriers involved in glucose transport across the plasma membrane, which is the first rate-limiting step of carbohydrate metabolism. In this study, five glucose transporters (RcGLUT1, RcGLUT2, RcGLUT3, RcGLUT5 and RcGLUT9) were obtained from Rachycentron canadum. The complete cDNAs open reading frames (ORF) of RcGLUT1, RcGLUT2, RcGLUT3, RcGLUT5 and RcGLUT9 were 1,476 bp, 1,530 bp, 1,551 bp, 1,539 bp and 1,578 bp (GenBank accession no. MK560257, MK560258, MH184460, MH184461 and MH184462), encoding 491, 509, 516, 512 and 525 amino acid (aa) residues respectively. All five RcGLUTs contained a Sugar_tr domain, which is an important feature of the GLUT gene family. The multiple sequence alignment and phylogenetic relationship analyses showed that RcGLUTs were highly conserved and homologous from fish to mammals. Examination of tissue distribution showed that RcGLUTs were expressed constitutively in R. canadum. RcGLUT1-5 and RcGLUT9 had the highest expression in the foregut, kidneys, haemocytes, muscles, liver and heart respectively. All six RcGLUTs first increased to peak levels of expression and then reduced both in the liver and muscle after fasting. The exception is that the expression levels of RcGLUT4 and RcGLUT5 in muscle were significantly lower than the control. In response to hypoxia, only RcGLUT2 in the liver and muscle and RcGLUT9 in muscle were expressed at a significantly lower level relative to the control. In sum, all RcGLUTs in the liver and muscles showed significant changes in response to fasting and hypoxia, suggesting that GLUT genes may play a role in the response to common physiological stresses.

In Vitro Preparation and Crystallization of Vertebrate Telomerase Subunits.

Telomerase is a unique reverse transcriptase that replicates the telomeric DNA at most eukaryotic chromosomal ends. The telomerase consists of the catalytic protein subunit TERT and the RNA component TR that provides the template for telomeric DNA synthesis. In vitro reconstitution of telomerase core components in large quantity is the prerequisite to studying the catalytic mechanisms of telomerase at the structural level; however, large-scale preparation of recombinant telomerase, especially that of higher eukaryotes, has been a big challenge for a long time. It has been known that the CR4/5 domain of the vertebrate TR binds to the TRBD domain of TERT and the interaction is essential to the assembly and enzymatic activity of telomerase. We assembled the TRBD-CR4/5 ribonucleoprotein complex of the medaka fish telomerase in vitro and determined its atomic structure through X-ray crystallography. Our study provides the structural insight into the RNA-protein recognition mechanism that is common to most eukaryotic telomerase. The methods of our study are also applicable to large-scale preparations of other ribonucleoprotein complexes for structural studies.

A novel mutation of the THRB gene in a Japanese family with resistance to thyroid hormone

Resistance to thyroid hormone (RTH; OMIM 190160) is an inherited syndrome of reduced sensitivity to thyroid hormone. RTH, in majority, is caused by monoallelic inactivating mutation of THRB, which encodes the thyroid hormone receptor β (TRβ) (1). RTH is endocrinologically characterized by high serum thyroid hormones together with inappropriately normal TSH. The severity of hormonal resistance varies among different tissues in an affected individual, due to differences in the relative expression of TRβ and thyroid hormone receptor alpha (TRα) in different tissues (2). To date, more than 100 THRB mutations have been reported among RTH patients. Most mutations are located in the ligand-binding domain of TR. They interfere with the function of the normal TR because of their dominant negative effect (1). We report a novel mutation of the THRB gene in a family with RTH.

P.1.g.031 The neuroprotective and anti-amnestic effects of carbamylated erythropoietin derivates in ischemic brain injury

Purpose: To compare the neuroprotection of carbamylated erythropoietin (CEPO) and EPO fusion protein containing TR domain from glycoprotein MUC1 (CEPO-TR), and EPO fusion protein with modified Fc fragments of IgG1 (CEPO-Fc) against ischemic brain injury, including behavioral disturbances in a bilateral focal ischemic infarction of the rat prefrontal cortex. Methods: The carbamylated erythropoietin and erythropoietin derivates were produced by treatment of purified proteins with potassium cyanate in borate buffer. The resulting carbamylated erythropoietin and derivates exhibit no erythropoietic activity in UT-7/EPOR cell viability assay. Bilateral focal ischemic infarction of the prefrontal cortex (areas Frl and Fr2) was induced by the method of photochemical thrombosis in rats. After passive avoidance training and testing rats were treated, respectively, with following regimens: saline, 50mg/kg CEPO, 50mg/kg CEPO-TR, 50mg/kg CEPO-Fc. The substance was injected intraperitoneally in 1 h after operation. Neurological deficit scores and infarct volume were assessed at 4 and 7 days after operation. Functional state of CNS was determined by conditioned passive avoidance response (PA), i.e. by the latency of transition from light compartment to dark compartment (in sec). Morphometric measurements of the areas and volumes of the ischemic focus on serial slices were carried out on animal brain fixed by plunging into formalinethanol-acetic acid mixture (2:7:1). The data were statistically processed using Statistica 6.0 software. Summary of results: Additional oligosaccharides, linked to the erythropoietin, prolong its half-life and increase bioactivity in vivo. For this purpose we, for the first time, used TR domain from glycoprotein MUC1, bearing 5 additional sites for O-glycosylation. Our result indicated that ligation of TR-domain to the coding sequence of EPO did not affect secretion of the chimeric protein into the medium, receptor binding affinity in vitro bioactivity, compared with EPO wild type. However, both the in vitro potency and half-life in circulation of EPO bearing TR or Fc fragments were significantly enhanced. After photothrombosis and injection of saline, the latency of entry into the dark compartment decreased from 300 s to 76 s. Treatment of rats with CEPO, CEPOTR, and CEPO-Fc after photothrombosis restored latency of PA to 243, 258, 227 s, respectively, on day 4. Only injection of CEPOTR retains passive avoidance latency 207 s on the day 7. Ischemic damage volume in animals treated with CEPO-TR on 7th day after operation was 15.8±5.4 against 24.22±4.7 with control injection of saline (P< 0.05). This phenomenon on 7th day was observed only with CEPO-TR. Protection efficiency coefficient, calculated from these experiments represented 34%. Another erythropoietin derivate did not give statistically significant results. Conclusion: Treatment of rats with CEPO and CEPO fusion proteins after photothrombosis of cortex resulted in restoration of passive avoidance response and diminishing of volume of ischemic damage. Those, our study indicates that CEPO-Fc and CEPO-TR display neuroprotectionand anti-amnestic activity, while CEPO-TR demonstrates prolongation ability.

Apical MUC1 expression revealed on the foveolar epithelium in H. pylori gastritis

Background:The membrane mucin MUC1 is altered in its pattern of expression in cancer, and also in other pathological situations, including Helicobacter pylori gastritis. Here we investigate the basis for the loss of apical staining of the gastric foveolar epithelium in H. pylori gastritis.Methods:MUC1 was examined in the gastric antrum from cases of H. pylori gastritis and normal controls. We used tissue sections that were either treated or not treated with periodate to effect deglycosylation, and the monoclonal antibodies LICRLonM8, MUSE-11, CT2 and BC2.Results:We show that the epitopes on the TR domain of MUC1 are partially cryptic due to glycosylation and that MUC1 is present on the apical surface of the gastric foveolar epithelium of gastritis patients.Conclusion:This observation suggests that there is no substantial loss of the mucin domain of MUC1 from the apical surface in gastritis, as suggested by others, but rather the H. pylori influences the glycosylation of MUC1. This paper highlights the issue of epitope specificity of monoclonal antibodies directed against disease-associated markers, specifically when they are glycoproteins, as is the case for many cancer markers.

Functional consequences of mutating conserved SF2 helicase motifs in the Type III restriction endonuclease EcoP15I translocase domain

For efficient DNA hydrolysis, Type III restriction endonuclease EcoP15I interacts with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of two methylation (Mod) subunits and a single restriction (Res) subunit yielding a multifunctional enzyme complex able to methylate or to hydrolyse DNA. Comprehensive sequence alignments, limited proteolysis and mass spectroscopy suggested that the Res subunit is a fusion of a motor or translocase (Tr) domain of superfamily II helicases and an endonuclease domain with a catalytic PD…EXK motif. In the Tr domain, seven predicted helicase motifs (I, Ia, II-VI), a recently discovered Q-tip motif and three additional regions (IIIa, IVa, Va) conserved among Type III restriction enzymes have been identified that are predicted to be involved in DNA binding and ATP hydrolysis. Because DNA unwinding activity for EcoP15I (as for bona fide helicases) has never been found and EcoP15I ATPase rates are only low, the functional importance of the helicase motifs and regions was questionable and has never been probed systematically. Therefore, we mutated all helicase motifs and conserved regions predicted in Type III restriction enzyme EcoP15I and examined the functional consequences on EcoP15I enzyme activity and the structural integrity of the variants by CD spectroscopy. The resulting eleven enzyme variants all, except variant IVa, are properly folded showing the same secondary structure distribution as the wild-type enzyme. Classical helicase motifs I-VI are important for ATP and DNA cleavage by EcoP15I and mutations therein led to complete loss of ATPase and cleavage activity. Among the catalytically inactive enzyme variants three preserved the ability to bind ATP. In contrast, newly assigned motifs Q-tip, Ia and Va are not essential for EcoP15I activity and the corresponding enzyme variants were still catalytically active. DNA binding was only marginally reduced (2-7 fold) in all enzyme variants tested.

Analysis of sequence polymorphism and population structure of tomato chlorotic dwarf viroid and potato spindle tuber viroid in viroid-infected tomato plants.

The sequence polymorphism and population structure of Tomato chlorotic dwarf viroid (TCDVd) (isolate Trust) and Potato tuber spindle viroid (PSTVd) (isolate FN) in tomato plants were investigated. Of the 9 and 35 TCDVd clones sequenced from 2 different TCDVd-infected plants, 2 and 4 sequence variants were identified, respectively, leading to a total of 4 sequence variants of 360 nucleotides in length. Variant I was identical to AF162131, the first TCDVd sequence to be reported, and the rest exhibited 1 to 3 nucleotide differences, all in the TR domain, from AF162131/variant I. Of the 33 and 29 PSTVd clones sequenced from 2 different PSTVd-infected plants, 8 and 9 sequence variants were found, respectively, leading to a total of 15 variants ranging in length from 356 to 359 nucleotides. The variant I was identical to EF044303, a PSTVd reported in Russia. The rest exhibited 1 to 11 nucleotide differences scattering in all five domains from EF044303/variant I. The results demonstrated for the first time that TCDVd, like many other viroids including PSTVd, exists in host plants as a collective group comprised of various sequence variants. However, in comparison to PSTVd, TCDVd is less polymorphic in tomato plants as fewer variants and lower haplotype/nucleotide diversities were observed.

Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity

For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod2Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.

Monoclonal Antibodies Recognizing the Non-Tandem Repeat Regions of the Human Mucin MUC4 in Pancreatic Cancer

The MUC4 mucin is a high molecular weight, membrane-bound, and highly glycosylated protein. It is a multi-domain protein that is putatively cleaved into a large mucin-like subunit (MUC4α) and a C-terminal growth-factor like subunit (MUC4β). MUC4 plays critical roles in physiological and pathological conditions and is aberrantly overexpressed in several cancers, including those of the pancreas, cervix, breast and lung. It is also a potential biomarker for the diagnosis, prognosis and progression of several malignancies. Further, MUC4 plays diverse functional roles in cancer initiation and progression as evident from its involvement in oncogenic transformation, proliferation, inhibition of apoptosis, motility and invasion, and resistance to chemotherapy in human cancer cells. We have previously generated a monoclonal antibody 8G7, which is directed against the TR region of MUC4, and has been extensively used to study the expression of MUC4 in several malignancies. Here, we describe the generation of anti-MUC4 antibodies directed against the non-TR regions of MUC4. Recombinant glutathione-S-transferase (GST)-fused MUC4α fragments, both upstream (MUC4α-N-Ter) and downstream (MUC4α-C-Ter) of the TR domain, were used as immunogens to immunize BALB/c mice. Following cell fusion, hybridomas were screened using the aforementioned recombinant proteins ad lysates from human pancreatic cell lines. Three anti MUC4α-N-Ter and one anti-MUC4α-C-Ter antibodies were characterized by several inmmunoassays including enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunofluorescene, flow cytometry and immunoprecipitation using MUC4 expressing human pancreatic cancer cell lines. The antibodies also reacted with the MUC4 in human pancreatic tumor sections in immunohistochemical analysis. The new domain-specific anti-MUC4 antibodies will serve as important reagents to study the structure-function relationship of MUC4 domains and for the development of MUC4-based diagnostics and therapeutics.

The evolutionary trajectory of mitochondrial carrier family during metazoan evolution

BackgroundExploring metabolic evolution is a way to understand metabolic complexity. The substrate transport of mitochondrial carrier family (MCF) influences direct metabolic activities, making it possible to understand indirectly metabolic evolution from the evolution of substrate transport of MCF. However, the evolutionary study of substrate transport of MCF does not mean that all the concrete structures of mitochondrial carriers (MCs) must first be gained.ResultsHere we studied the alternation of MCF structure and potential correlated functions of MCF during metazoan evolution. The data analysis indicates that the types of substrates transported by MCF as a whole were maintained during metazoan evolution. However, the size of the substrates transported by members of MCs continuously diminished during the evolutionary process. We have found that the ratio of hydrophobic amino acids at specific helix-helix interfaces increases significantly during vertebrate evolution. Amino acid's spatial positioning and the calculating of packing values both indicate the increase in the number of hydrophobic amino acids would lead to a more "tight" structure of the TR domain, which is in agreement with the trend of diminishing size of substrates transported by MCs. In addition, there was a significant increase in the number of carriers of MCF during vertebrate evolution.ConclusionsWe propose that the more "tight" TR structure generated by the increase of the hydrophobic amino acids at specific helix-helix interfaces during vertebrate evolution enhances the substrate selectivity of MCF, reflecting the evolutionary trajectory of MCF during metazoan evolution.

First Report of Citrus viroid V in Moro Blood Sweet Orange in Iran

Viroids are nonencapsidated, small, circular, single-stranded RNAs that replicate autonomously when inoculated in their host plants in which they may elicit diseases (sensitive hosts) or replicate as latent infections (tolerant hosts). Citrus viroid V (CVd-V) was initially identified in Spain (1) and later found to be present in the United States, Nepal, and the Sultanate of Oman (2). CVd-V is a member of the Apscaviroid genus within the Pospiviroidae family. Like other members of this genus, CVd-V has a restricted host range but it is able to infect a wide range of citrus and citrus related species (1,2). Within the framework of a comprehensive survey of the sanitary status of the citrus industry in Iran, a sample from a private orchard of symptomless Moro blood sweet orange (Citrus sinensis) trees grafted on Mexican lime (C. aurantifolia) located at Javanan in the southern inland region was found to be infected with CVd-V. Briefly, RNAs of nucleic acid preparations from bark tissues were separated by 5% polyacrylamide gel electrophoresis (PAGE), electrotransferred to positively charged nylon membranes, immobilized by UV cross-linking, and hybridized with a full length CVd-V specific digoxigenin (DIG)-labeled DNA probe (2). A positive identification of CVd-V was made in these extracts. This positive detection of CVd-V was confirmed by reverse transcription-PCR using CVd-V specific primers of opposite polarity (5'-GACGAAGGCCGGTGAGCAGTAAGCC-3') and (5'-GACGACGACAGGTGAGTACTTTC-3') corresponding to CVd-V positions 90 to 114 and 69 to 89, respectively. Analysis of the sequence of the 293-bp amplicon (Genbank Accession No. GQ466068) revealed 99% identity with the reference sequence (Genbank Accession No. NC010165) of CVd-V. The rod-like predicted minimum free energy secondary structure of this new variant has 68.3% paired nucleotides. The changes with respect to the reference CVd-V variant are: (i) a deletion (48→-U) located in a loop of the V domain; (ii) a substitution (155A→C) located in a loop of the TR domain of the viroid secondary structure; and (iii) two compensatory substitutions located in the upper (46A→G) and lower (244U→C) strands of the viroid secondary structure. As shown earlier, the genome of CVd-V allows little variation with a large loop located in the segment I of the secondary structure (2) being the most amenable for mutations/changes. Among the viroids that have been found naturally infecting citrus, the members of the genus Apscaviroid are not associated with specific diseases but they cause a reduction of tree size and fruit harvest (3), an effect that is enhanced when several viroids coinfect the same plant (4). Therefore, the presence of CVd-V should be considered in further indexing tests aimed at the production and distribution of pathogen-free plants in Iran.

Gelsolin: A Novel Thyroid Hormone Receptor-β Interacting Protein that Modulates Tumor Progression in a Mouse Model of Follicular Thyroid Cancer

Follicular thyroid cancer (FTC) is known to metastasize to distant sites via hematogenous spread; however, the underlying pathways that contribute to metastasis remain unknown. Recent creation of a knockin mutant mouse that expresses a mutant thyroid hormone receptor-beta (TRbeta(PV/PV) mouse) that spontaneously develops thyroid cancer with metastasis similar to humans has provided new opportunities to study contributors to FTC metastasis. This study evaluates the role of gelsolin, an actin-regulatory protein, in modulating the metastatic potential of FTC. Gelsolin was previously found by cDNA microarray analysis to be down-regulated in TRbeta(PV/PV) mice as compared with wild-type mice. This study found an age-dependent reduction of gelsolin protein abundance in TRbeta(PV/PV) mice as tumorigenesis progressed. Knockdown of gelsolin by small interfering RNA resulted in increased tumor cell motility and increased gelsolin expression by histone deacetylase inhibitor (trichostatin A) led to decreased cell motility. Additional biochemical analyses demonstrated that gelsolin physically interacted with TRbeta1 or PV in vivo and in vitro. The interaction regions were mapped to the C terminus of gelsolin and the DNA binding domain of TR. The physical interaction of gelsolin with PV reduced its binding to actin, leading to disarrayed cytoskeletal architectures. These results suggest that PV-induced alteration of the actin/gelsolin cytoskeleton contributes to increased cell motility. Thus, the present study uncovered a novel PV-mediated oncogenic pathway that could contribute to the local tumor progression and metastatic potential of thyroid carcinogenesis.

Activation of phosphatidylinositol 3-kinase signaling by a mutant thyroid hormone β receptor

Activation of the phosphatidylinositol 3-kinase (PI3K)-AKT/protein kinase B signaling pathway has been associated with multiple human cancers. Recently we showed that AKT is activated in both the thyroid and metastatic lesions of a mouse model of follicular thyroid carcinoma [thyroid hormone beta receptor (TRbeta)(PV/PV) mice]. This TRbeta(PV/PV) mouse harbors a knock-in mutant TRbeta gene (TRbetaPV mutant) that spontaneously develops thyroid cancer and distant metastasis similar to human follicular thyroid cancer. Here we show that in thyroid tumors, PV mutant bound significantly more to the PI3K-regulatory subunit p85alpha, resulting in a greater increase in the kinase activity than did TRbeta1 in wild-type mice. By GST pull-down assays, the ligand-binding domain of TR was identified as the interaction site with p85alpha. By confocal fluorescence microscopy, p85alpha was shown to colocalize with TRbeta1 or PV mainly in the nuclear compartment of cultured tumor cells from TRbeta(PV/PV) mice, but cytoplasmic p85alpha/PV or p85alpha/TRbeta1 complexes were also detectable. Further biochemical analysis revealed that the activation of the PI3K-AKT-mammalian target of the rapamycin-p70(S6K) pathway was observed in both the cytoplasmic and nuclear compartments, whereas the activation of the PI3K-integrin-linked kinase-matrix metalloproteinase 2 pathway was detected mainly in the extranuclear compartments. These results suggest that PV, via the activation of p85alpha, could act to affect PI3K downstream signaling in both the nuclear and extranuclear compartments, thereby contributing to thyroid carcinogenesis. Importantly, the present study unveils a mechanism by which a mutant TR acts to activate PI3K activity via protein-protein interactions.

Human immunodeficiency virus type 1 Tat binding protein-1 is a transcriptional coactivator specific for TR.

The DNA-binding domain of nuclear hormone receptors functions as an interaction interface for other transcription factors. Using the DNA-binding domain of TRbeta1 as bait in the yeast two-hybrid system, we cloned the Tat binding protein-1 that was originally isolated as a protein binding to the human immunodeficiency virus type 1 Tat transactivator. Tat binding protein-1 has subsequently been identified as a member of the ATPase family and a component of the 26S proteasome. Tat binding protein-1 interacted with the DNA-binding domain but not with the ligand binding domain of TR in vivo and in vitro. TR bound to the amino-terminal portion of Tat binding protein-1 that contains a leucine zipper-like structure. In mammalian cells, Tat binding protein-1 potentiated the ligand-dependent transactivation by TRbeta1 and TRalpha1 via thyroid hormone response elements. Both the intact DNA-binding domain and activation function-2 of the TR were required for the transcriptional enhancement in the presence of Tat binding protein-1. Tat binding protein-1 did not augment the transactivation function of the RAR, RXR, PPARgamma, or ER. The intrinsic activation domain in Tat binding protein-1 resided within the carboxyl-terminal conserved ATPase domain, and a mutation of a putative ATP binding motif but not a helicase motif in the carboxyl-terminal conserved ATPase domain abolished the activation function. Tat binding protein-1 synergistically activated the TR-mediated transcription with the steroid receptor coactivator 1, p120, and cAMP response element-binding protein, although Tat binding protein-1 did not directly interact with these coactivators in vitro. In contrast, the N-terminal portion of Tat binding protein-1 directly interacted in vitro and in vivo with the TR-interacting protein 1 possessing an ATPase activity that interacts with the activation function-2 of liganded TR. Collectively, Tat binding protein-1 might function as a novel DNA-binding domain-binding transcriptional coactivator specific for the TR probably in cooperation with other activation function-2-interacting cofactors such as TR-interacting protein 1.

The role of hinge domain in heterodimerization and specific DNA recognition by nuclear receptors

Four structural domains are characteristic of the members of the nuclear receptor superfamily. The hinge (D) domain which is located between the DNA binding (C) domain and the ligand binding (EF) domain, is less conserved among the nuclear receptors. In this study, we investigated the effects of the D domain on receptor function with regard to ligand binding, protein–protein interaction and DNA recognition. We found that EF domain of TR lacked T3 binding activity and additional D domain was required for its ligand binding. Using pull down assays and two-hybrid assays, we also demonstrated that the EF domain of TR did not dimerize with TR or RXR in solution, while the DEF domain was able to homo-and heterodimerize with RXR. In contrast, the RXR EF domain alone was able to heterodimerize with TR. The D domain of TR is required but that of RXR is not necessary for the interaction. We further demonstrated that the D domain was required for receptor specific DNA recognition. The ABC domain of vitamin D receptor (VDR) and TR(DEF) chimeric receptor could not bind to VDR response element (VDRE). Addition of own D domain of VDR to the ABC domain enables the chimeric receptor to bind VDRE and transactivate. The D domain of TR cannot substitute for that of VDR in context of specific DNA recognition. These data suggest that the D domain is important to maintain the integrity of the functional structure of the nuclear receptors.

Nitroreductase reactions of Arabidopsis thaliana thioredoxin reductase

Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) catalyzed redox cycling of aromatic nitrocompounds, including the explosives 2,4,6-trinitrotoluene and tetryl, and the herbicide 3,5-dinitro- o-cresol. The yield of nitro anion radicals was equal to 70–90%. Redox cycling of tetryl was accompanied by formation of N-methylpicramide. Bimolecular rate constants of nitroaromatic reduction ( k cat/ K m) and reaction catalytic constants ( k cat) increased upon an increase in oxidant single-electron reduction potential ( E 1 7). Using compounds with an unknown E 1 7 value, the reactivity of TR increased parallelly to the increase in reactivity of ferredoxin:NADP + reductase of Anabaena PCC 7119 (EC 1.18.1.2). This indicated that the main factor determining reactivity of nitroaromatics towards TR was their energetics of single-electron reduction. Incubation of reduced TR in the presence of tetryl or 2,4-dinitrochlorobenzene resulted in a loss of thioredoxin reductase activity, most probably due to modification of reduced catalytic disulfide, whereas nitroreductase reaction rates were unchanged. This means that on the analogy of quinone reduction by TR (D. Bironait≐, Ž. Anusevičius, J.-P. Jacquot, N. Č≐nas, Biochim. Biophys. Acta 1383 (1998) 82–92), FAD and not catalytic disulfide of TR was responsible for the reduction of nitroaromatics. Tetryl, 2,4,6-trinitrotoluene and thioredoxin increased the FAD fluorescence intensity of TR. This finding suggests that nitroaromatics may bind close to the thioredoxin-binding site at the catalytic disulfide domain of TR, and induce a conformational change of enzymes (S.B. Mulrooney, C.H. Williams Jr., Protein Sci. 6 (1997) 2188–2195). Our data indicate that certain nitroaromatic herbicides, explosives and other classes of xenobiotics may interfere with the reduction of thioredoxin by plant TR, and confer prooxidant properties to this antioxidant enzyme.