A common polymorphism in the oxygen-dependent degradation (ODD) domain of hypoxia inducible factor-1alpha (HIF-1alpha) does not impair Pro-564 hydroxylation

The hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the alpha subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1alpha in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).

Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera.

Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain.

A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis

The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the -subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel–Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

Ubiquitin-dependent proteolysis of trihydrophobin 1 (TH1) by the human papilloma virus E6-associated protein (E6-AP).

Human Papilloma virus E6-associated protein (E6-AP), which is known as an E3 ubiquitin ligase, mediates ubiquitination and subsequent degradation of a series of cellular proteins. In this paper, we identify here trihydrophobin 1 (TH1), an integral subunit of the human negative transcription elongation factor (NELF) complex, as a novel E6-AP interaction protein and a target of E6-AP-mediated degradation. Overexpression of E6-AP results in degradation of TH1 in a dose-dependent manner, whereas knock-down of endogenous E6-AP elevates the TH1 protein level. TH1 protein turnover is substantially faster, compared to controls, in cells that overexpressed E6-AP. Wild-type E6-AP promotes the ubiquitination of TH1, while a catalytically inactive point mutant of E6-AP abolishes its ubiquitination. Furthermore, in vitro ubiquitination assay also demonstrates that TH1 can be ubiquitinated by E6-AP. The degradation is blocked by treatment with proteasome inhibitor MG132. Herein, we provide strong evidence that TH1 is a specific substrate that is targeted for degradation through E6-AP-catalyzed polyubiquitination.

The role of a kinesin, KLP-20, on neural development and epidermal morphogenesis in Caenorhabditis elegans

The heterotrimeric kinesin-II motor in Caenorhabditis elegans consists of KLP-20, KLP-11, and KAP-1 subunits and broadly functions in cellular transport for the development of biological structures including cilia and axons. The results of this paper support the ubiquitous and necessary role kinesin-II motors have in development, particularly the KLP-20 microtubule-associating subunit. Mutations in klp-20 result in a variable abnormal (vab) phenotype characterized by observable epidermal defects, although the role of this gene in development and the mechanism by which the vab phenotype is produced is largely unknown. The vab phenotype is highly penetrant in the first larval stage (L1) of C. elegans, which supports that klp-20 functions in early development. Ciliated amphid sensory neurons can be stained with a fluorescent dye, DiI, to simultaneously test cilia structure and function, as well as the morphology of the amphid sensory organ. Reduced dye uptake in klp-20 mutant L1s suggests that the microtubule-based cilia are under-developed as a result of defective kinesin-II function. Consistent observations of the PLM mechanosensory neuron using the zdIs5 reporter suggest that klp-20 has an essential role in neuron development, as mutations to klp-20 result in under-developed PLM axons. Qualitative observations suggest there may be an interaction between the development of the overlying epidermis and the underlying nervous system, as a more severe vab phenotype is observed simultaneously with reduced dye uptake, and hence amphid sensory cilia under-development. Furthermore, a more severe vab phenotype manifested as large bumps on the posterior epidermis appears to be spatially correlated with PLM defects. The results presented and discussed in this paper suggest that KLP-20 has a necessary role in neurodevelopment and epidermal morphogenesis in C. elegans during embryogenesis.

Novel exon 12 mutations in the HIF2A gene associated with erythrocytosis.

Erythrocytosis can arise from deregulation of the erythropoietin (Epo) axis resulting from defects in the oxygen-sensing pathway. Epo synthesis is controlled by the hypoxia inducible factor (HIF) complex, composed of an a and a ß subunit. There are 2 main a subunits, HIF-1a and HIF-2a. Recently, a HIF-2a Gly537Trp mutation was identified in a family with erythrocytosis. This raises the possibility of HIF2A mutations being associated with other cases of erythrocytosis. We now report a subsequent analysis of HIF2A in a cohort of 75 erythrocytosis patients and identify 4 additional patients with novel heterozygous Met535Val and Gly537Arg mutations. All patients presented at a young age with elevated serum Epo. Mutations at Gly-537 account for 4 of 5 HIF2A mutations associated with erythrocytosis. These findings support the importance of HIF-2a in human Epo regulation and warrant investigation of HIF2A in patients with unexplained erythrocytosis.

Selection with melphalan or paclitaxel (Taxol) yields variants with different patterns of multidrug resistance, integrin expression and in vitro invasiveness

A melphalan-resistant variant (Roswell Park Memorial Institute (RPMI)-2650M1) and a paclitaxel-resistant variant (RPMI-1650Tx) of the drug-sensitive human nasal carcinoma cell line, RPMI-2650. were established. The multidrug resistance (MDR) phenotype in the RPMI-2650Tx appeared to be P-glycoprotein (PgP)-mediated. Overexpression of multidrug resistant protein (MRP) family members was observed in the RPMI-2650M1 cells, which were also much more invasive in vitro than the parental cell line or the paclitaxel-resistant variant. Increased expression of alpha (2), alpha (5), alpha (6), beta (1) and beta (4) integrin subunits, decreased expression of alpha (4) integrin subunit, stronger adhesion to collagen type IV, laminin, fibronectin and matrigel, increased expression of MMP-2 and MMP-9 and significant motility compared with the parental cells were observed, along with a high invasiveness in the RPMI-7650M1 cells. Decreased expression of the alpha (2) integrin subunit, decreased attachment to collagen type IV, absence of cytokeratin 18 expression, no detectable expression of gelatin-degrading proteases and poor motility may be associated with the non-invasiveness of the RPMI-2650Tx variant. These results suggest that melphalan exposure can result in not only a MDR phenotype. but could also make cancer cells more invasive, whereas paclitaxel exposure resulted in MDR without increasing the in vitro invasiveness in the RPMI-2650 cells. (C) 2001 Elsevier Science Ltd. All rights reserved.

Molecular characterization of Nosema bombi (Microsporidia : Nosematidae) and a note on its sites of infection in Bombus terrestris (Hymenoptera : Apoidea)

Investigations of queen, worker and male bumble bees (Bombus terrestris) showed that all individuals became infected with Nosema bombi. Infections were found in Malpighian tubules, thorax muscles, fat body tissue and nerve tissue, including the brain. Ultrastructural studies revealed thin walled emptied spores in host cell cytoplasm interpreted as autoinfective spores, besides normal spores (environmental spores) intended for parasite transmission between hosts. The nucleotide sequence of the gene coding for the small subunit rRNA (SSU-rRNA) from Microsporidia isolated from B. terrestris, B. lucorum, and B. hortorum were identical, providing evidence that N. bombi infects multiple hosts. The sequence presented here (GenBank Accession no AY008373) is different from an earlier submission to GenBank (Accession no U26158) of a partial sequence of the same gene based on material collected from B. terrestris. It still remains to be investigated if there is species diversity among Microsporidia found in bumble bees.

The invasive genus Asparagopsis (Bonnemaisoniaceae, Rhodophyta): Molecular systematics, morphology, and ecophysiology of Falkenbergia isolates

The genus Asparagopsis was studied using 25 Falkenbergia tetrasporophyte strains collected worldwide. Plastid (cp) DNA RFLP revealed three groups of isolates, which differed in their small subunit rRNA gene sequences, temperature responses, and tetrasporophytic morphology (cell sizes). Strains from Australia, Chile, San Diego, and Atlantic and Mediterranean Europe were identifiable as A. armata Harvey, the gametophyte of which has distinctive barbed spines. This species is believed to be endemic to cold-temperate waters of Australia and New Zealand and was introduced into Europe in the 1920s. All isolates showed identical cpDNA RFLPs, consistent with a recent introduction from Australia. Asparagopsis taxiformis (Delile) Trevisan, the type and only other recognized species, which lacks spines, is cosmopolitan in warm-temperate to tropical waters. Two clades differed morphologically and ecophysiologically and in the future could be recognized as sibling species or subspecies. A Pacific/Italian clade had 4-8degrees C lower survival minima and included a genetically distinct apomictic isolate from Western Australia that corresponded to the form of A. taxiformis originally described as A. sanfordiana Harvey. The second clade, from the Caribbean and the Canaries, is stenothermal (subtropical to tropical) with some ecotypic variation. The genus Asparagopsis consists of two or possibly three species, but a definitive taxonomic treatment of the two A. taxiformis clades requires study of field-collected gametophytes.

rbcL analysis supports the tribe Gymnothamnieae Kajimura (Ceramiaceae, Rhodophyta)

The tribe Gymnothamnieae Kajimura was proposed for the monotypic ceramiacean genus Gymnothamnion J. Agardh, previously placed either in the Ptiloteae Cramer or the Antithamnieae Hommersand. A bisporangial isolate of G. elegans (Schousboe ex C. Agardh) J. Agardh from Morocco formed only bisporangia in culture. Its smaller uninucleate cells and sporangia than those of tetrasporophytes suggested that bisporophytes may be haploid as in another member of the Ceramiaceae, Aglaothamnion diaphanum L'Hardy-Halos et Maggs. Phylogenetic analyses of the gene for the large subunit of rubisco (rbcL) from Gymnothamnion and representatives of eight other tribes of the Ceramiaceae confirmed that the removal of Gymnothamnion from the Ptiloteae and the Antithamnieae was warranted. Whereas all tribes with two or more representatives in our analyses were moderately or robustly resolved, Gymnothamnion did not form a strong clade with any other taxa. Analysis of rbcL sequences failed to resolve relationships between tribes, probably due to saturation at the high levels of sequence divergence found. In addition to reproductive features previously reported and interpreted as primitive, G. elegans shows a primitive vegetative feature and it is suggested that Gymnothamnion may be one of the most basal of the taxa presently included in the Ceramiaceae.

Rapid annulation of tropolone units via a pyrylium ylide 1,3-dipolar cycloaddition reaction

The tropolone subunit of the naturally occurring alkaloid rubrolone aglycon is synthesized via a short reaction sequence starting with a 1,3-dipolar cycloaddition of a pyrylium ylide and indenone, followed by enone oxidation, oxygen bridge elimination and finally hydroxy group oxidation.

Erythrocytosis-associated HIF-2alpha mutations demonstrate a critical role for residues C-terminal to the hydroxylacceptor proline.

Abstract A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an alpha subunit (HIF-alpha) and a beta subunit (HIF-beta). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-alpha in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-alpha for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-alpha and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosis-associated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2alpha. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2alpha in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-alpha.

FACT facilitates chromatin transcription by RNA polymerases I and III

Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III-transcribed genes.

IL-12R2 promotes the development of CD4+CD25+ regulatory T cells

We have previously shown that mice lacking the IL-12-specific receptor subunit ß2 (IL-12Rß2) develop more severe experimental autoimmune encephalomyelitis than wild-type (WT) mice. The mechanism underlying this phenomenon is not known; nor is it known whether deficiency of IL-12Rß2 impacts other autoimmune disorders similarly. In the present study we demonstrate that IL-12Rß2-/- mice develop earlier onset and more severe disease in the streptozotocin-induced model of diabetes, indicating predisposition of IL-12Rß2-deficient mice to autoimmune diseases. T cells from IL-12Rß2-/- mice exhibited significantly higher proliferative responses upon TCR stimulation. The numbers of naturally occurring CD25+CD4+ regulatory T cells (Tregs) in the thymus and spleen of IL-12Rß2-/- mice were comparable to those of WT mice. However, IL-12Rß2-/- mice exhibited a significantly reduced capacity to develop Tregs upon stimulation with TGF-ß, as shown by significantly lower numbers of CD25+CD4+ T cells that expressed Foxp3. Functionally, CD25+CD4+ Tregs derived from IL-12Rß2-/- mice were less efficient than those from WT mice in suppressing effector T cells. The role of IL-12Rß2 in the induction of Tregs was confirmed using small interfering RNA. These findings suggest that signaling via IL-12Rß2 regulates both the number and functional maturity of Treg cells, which indicates a novel mechanism underlying the regulation of autoimmune diseases by the IL-12 pathway. Copyright © 2008 by The American Association of Immunologists, Inc.

Ribosomal 18S RNA processing by the IGF-I-responsive WDR3 protein is integrated with p53 function in cancer cell proliferation.

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G1 phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.