The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.
The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1–3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel–Lindau protein (VHL)–elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.
We describe here a classical molecular modeling exercise that was carried out to provide a basis for the design of novel antagonist ligands of the CCR2 receptor. Using a theoretical model of the CCR2 receptor, docking studies were carried out to define plausible binding modes for the various known antagonist ligands, including our own series of indole piperidine compounds. On the basis of these results, a number of site-directed mutations (SDM) were designed that were intended to verify the proposed docking models. From these it was clear that further refinements would be necessary in the model. This was aided by the publication of a crystal structure of bovine rhodopsin, and a new receptor model was built by homology to this structure. This latest model enabled us to define ligand-docking hypotheses that were in complete agreement with the results of the SDM experiments.
Animals respond to the challenge of limited oxygen availability by a coordinated response that works to increase oxygen supply and minimize tissue damage. The chronic hypoxic response is mediated by the alpha,beta-hypoxia inducible transcription factor (HIF) that enables the expression of a gene array. Because this array includes genes encoding for proteins that regulate processes including red blood cell and blood vessel formation, manipulation of the HIF system has potential for the treatment of ischemic diseases, anaemia and tumours. Hydroxylase enzymes act as oxygen sensors by regulating both the lifetime of HIF-alpha and its transcriptional activity. This tutorial review aims to provide a non-expert introduction to the HIF field by providing a background to current work, summarising molecular knowledge on the HIF system, and outlining opportunities for therapeutic intervention.
Background: The hypoxia-inducible factor (HIF) hydroxylases (FIH and PHDs) regulate hypoxia sensing in animals.Results: FIH·HIF-α reacts faster with O2 than PHD2·HIF-α but slower than FIH·ankyrin complexes.Conclusion: The kinetics of catalysis by isolated FIH and PHD2 reflect their cellular activities.Significance: We provide a kinetic rationale for cellular observations that FIH activity is more hypoxia-tolerant than that of the PHDs.
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