Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1alpha

Stabilization of the hypoxia-inducible factor-1 (HIF-1) protein is essential for its role as a regulator of gene expression under low oxygen conditions. Here, employing a novel hydroxylation-specific antibody, we directly show that proline 564 of HIF-1alpha and proline 531 of HIF-2alpha are hydroxylated under normoxia. Importantly, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 hydroxylation is diminished with the treatment of hypoxia, cobalt chloride, desferrioxamine, or dimethyloxalyglycine, regardless of the E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) tumor suppressor gene. Furthermore, in VHL-deficient cells, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 had detectable amounts of hydroxylation following transition to hypoxia, indicating that the post-translational modification is not reversible. The introduction of v-Src or RasV12 oncogenes resulted in the stabilization of normoxic HIF-1alpha and the loss of hydroxylated Pro-564, demonstrating that oncogene-induced stabilization of HIF-1alpha is signaled through the inhibition of prolyl hydroxylation. Conversely, a constitutively active Akt oncogene stabilized HIF-1alpha under normoxia independently of prolyl hydroxylation, suggesting an alternative mechanism for HIF-1alpha stabilization. Thus, these results indicate distinct pathways for HIF-1alpha stabilization by different oncogenes. More importantly, these findings link oncogenesis with normoxic HIF-1alpha expression through prolyl hydroxylation.     

Localization of the yeast RNA polymerase I-specific subunits

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.     

Inhibition of antigen-specific T cell proliferation and cytokine production by protein kinase A type I

cAMP inhibits biochemical events leading to T cell activation by triggering of an inhibitory protein kinase A (PKA)-C-terminal Src kinase pathway assembled in lipid rafts. In this study, we demonstrate that activation of PKA type I by Sp-8-bromo-cAMPS (a cAMP agonist) has profound inhibitory effects on Ag-specific immune responses in peripheral effector T cells. Activation of PKA type I inhibits both cytokine production and proliferative responses in both CD4(+) and CD8(+) T cells in a concentration-dependent manner. The observed effects of cAMP appeared to occur endogenously in T cells and were not dependent on APC. The inhibition of responses was not due to apoptosis of specific T cells and was reversible by a PKA type I-selective cAMP antagonist. This supports the notion of PKA type I as a key enzyme in the negative regulation of immune responses and a potential target for inhibiting autoreactive T cells.     

Ammonia excretion and urea handling by fish gills: present understanding and future research challenges

In fresh water fishes, ammonia is excreted across the branchial epithelium via passive NH(3) diffusion. This NH(3) is subsequently trapped as NH(4)(+) in an acidic unstirred boundary layer lying next to the gill, which maintains the blood-to-gill water NH(3) partial pressure gradient. Whole animal, in situ, ultrastructural and molecular approaches suggest that boundary layer acidification results from the hydration of CO(2) in the expired gill water, and to a lesser extent H(+) excretion mediated by apical H(+)-ATPases. Boundary layer acidification is insignificant in highly buffered sea water, where ammonia excretion proceeds via NH(3) diffusion, as well as passive NH(4)(+) diffusion due to the greater ionic permeability of marine fish gills. Although Na(+)/H(+) exchangers (NHE) have been isolated in marine fish gills, possible Na(+)/NH(4)(+) exchange via these proteins awaits evaluation using modern electrophysiological and molecular techniques. Although urea excretion (J(Urea)) was thought to be via passive diffusion, it is now clear that branchial urea handling requires specialized urea transporters. Four urea transporters have been cloned in fishes, including the shark kidney urea transporter (shUT), which is a facilitated urea transporter similar to the mammalian renal UT-A2 transporter. Another urea transporter, characterized but not yet cloned, is the basolateral, Na(+) dependent urea antiporter of the dogfish gill, which is essential for urea retention in ureosmotic elasmobranchs. In ureotelic teleosts such as the Lake Magadi tilapia and the gulf toadfish, the cloned mtUT and tUT are facilitated urea transporters involved in J(Urea). A basolateral urea transporter recently cloned from the gill of the Japanese eel (eUT) may actually be important for urea retention during salt water acclimation. A multi-faceted approach, incorporating whole animal, histological, biochemical, pharmacological, and molecular techniques is required to learn more about the location, mechanism of action, and functional significance of urea transporters in fishes.     

Skin graft rejection elicited by beta 2-microglobulin as a minor transplantation antigen involves multiple effector pathways: role of Fas-Fas ligand interactions and Th2-dependent graft eosinophil infiltrates

Beta(2)-microglobulin (beta(2)m)-derived peptides are minor transplantation Ags in mice as beta(2)m-positive skin grafts (beta(2)m(+/+)) are rejected by genetically beta(2)m-deficient recipient mice (beta(2)m(-/-)). We studied the effector pathways responsible for the rejection induced by beta(2)-microglobulin-derived minor transplantation Ags. The rejection of beta(2)m(+/+) skin grafts by naive beta(2)m(-/-) mice was dependent on both CD4 and CD8 T cells as shown by administration of depleting mAbs. Experiments performed with beta(2)m(-/-)CD8(-/-) double knockout mice grafted with a beta(2)m(+/+) MHC class I-deficient skin showed that sensitized CD4 T cells directed at beta(2)m peptides-MHC class II complexes are sufficient to trigger rapid rejection. Rejection of beta(2)m(+/+) grafts was associated with the production of IL-5 in vitro, the expression of IL-4 and IL-5 mRNAs in the grafted tissue, and the presence within rejected grafts of a considerable eosinophil infiltrate. Blocking IL-4 and IL-5 in vivo and depleting eosinophils with an anti-CCR3 mAb prevented graft eosinophil infiltration and prolonged beta(2)m(+/+) skin graft survival. Lymphocytes from rejecting beta(2)m(-/-) mice also displayed an increased production of IFN-gamma after culture with beta(2)m(+/+) minor alloantigens. In vivo neutralization of IFN-gamma inhibited skin graft rejection. Finally, beta(2)m(+/+) skin grafts harvested from B6(lpr/lpr) donor mice, which lack a functional Fas molecule, survived longer than wild-type beta(2)m(+/+) skin grafts, showing that Fas-Fas ligand interactions are involved in the rejection process. We conclude that IL-4- and IL-5-dependent eosinophilic rejection, IFN-gamma-dependent mechanisms, and Fas-Fas ligand interactions are effector pathways in the acute rejection of minor transplantation Ags.     

Ribonucleotide reductases: divergent evolution of an ancient enzyme

Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron–sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their three-dimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family.