Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.11.2 (prolyl hydroxylase)
 1,814 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Skp1 is a subunit of SCF-E3 ubiquitin ligases and other protein complexes in the nucleus and cytoplasm of yeast and mammalian cells. In Dictyostelium, Skp1 is partially modified by an unusual pentasaccharide O-linked to hydroxyproline143. This modification was found to be susceptible to known prolyl hydroxylase inhibitors based on M(r)-shift analysis using SDS-polyacrylamide gel electrophoresis/Western blotting. In addition, Dictyostelium Skp1 consists of 2 genetic isoforms, Skp1A and Skp1B, which differ by a single amino acid and appear to be expressed throughout the life cycle based on reverse-transcription polymerase chain reactions. The significance of these structural variations was examined by expressing myc-tagged Skp1s and mutants that lacked the glycosylation site. Gel-based M(r)-shift studies showed that Skp1A and Skp1B are both nearly completely glycosylated during growth and early development, and mass spectrometry of glycopeptides showed that they were glycosylated similarly. Skp1 expressed later in prespore cells was not glycosylated, unlike bulk Skp1 persisting from earlier in development, but became glycosylated after return to growth medium. Skp1A and Skp1B were each concentrated in the nucleus and regions of the cytoplasm, based on immunofluorescence localization. However, when Skp1 glycosylation was blocked by mutation, prolyl hydroxylase inhibitors, or expression in prespore cells, nuclear concentration of Skp1 was not detected. Furthermore, nuclear concentration occurred in a mutant that attached only the core disaccharide to Skp1. Overall, there was no evidence for differential Skp1 isoform expression, glycosylation variants in the bulk Skp1 pool, or regulation of nuclear localization. However, these studies uncovered evidence that the glycosylation pathway is developmentally regulated and can function posttranslationally, and that core glycosylation is required for Skp1's nuclear concentration.
 ...
PMID:Analysis of Skp1 glycosylation and nuclear enrichment in Dictyostelium. 1135 77

Hypoxia-inducible factor (HIF) is central in coordinating many of the transcriptional adaptations to hypoxia. Composed of a heterodimer of alpha and beta subunits, the alpha subunit is rapidly degraded in normoxia, leading to inactivation of the hypoxic response. Many models for a molecular oxygen sensor regulating this system have been proposed, but an important finding has been the ability to mimic hypoxia by chelation or substitution of iron. A key insight has been the recognition that HIF-alpha is targeted for degradation by the ubiquitin-proteasome pathway through binding to the von Hippel-Lindau tumour suppressor protein (pVHL), which forms the recognition component of an E3 ubiquitin ligase complex leading to ubiquitylation of HIF-alpha. Importantly, the classical features of regulation by iron and oxygen availability are reflected in regulation of the HIF-alpha/pVHL interaction. It has recently been shown that HIF-alpha undergoes an iron- and oxygen-dependent modification before it can interact with pVHL, and that this results in hydroxylation of at least one prolyl residue (HIF-1alpha, Pro 564). This modification is catalysed by an enzyme termed HIF-prolyl hydroxylase (HIF-PH), and compatible with all previously described prolyl-4-hydroxylases HIF-PH also requires 2-oxoglutarate as a cosubstrate. The key position of this hydroxylation in the degradation pathway of HIF-alpha, together with its requirement for molecular dioxygen as a co-substrate, provides the potential for HIF-PH to function directly as a cellular oxygen sensor. However, the ability of these enzyme(s) to account for the full range of physiological regulation displayed by the HIF system remains to be defined.
 ...
PMID:Regulation of HIF by the von Hippel-Lindau tumour suppressor: implications for cellular oxygen sensing. 1179 92

It is becoming increasingly evident that the degradation of nuclear proteins requires nuclear-cytoplasmic trafficking of both the substrate proteins, as well as the E3 ubiquitin-ligases. Here, we show that nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein (VHL) is required for oxygen-dependent ubiquitination and degradation of the alpha subunits of hypoxia-inducible factor (HIF-alpha). VHL engages in a constitutive transcription-sensitive nuclear-cytoplasmic shuttle unaffected by oxygen tension or levels of nuclear substrate HIF-alpha. Ubiquitinated forms of HIF-alpha, as well as VHL/ubiquitinated HIF-alpha complexes, are found solely in the nuclear compartment of normoxic or reoxygenated VHL-competent cells. HIF-alpha localizes exclusively in the nucleus of hypoxic cells but is exported to the cytoplasm upon reoxygenation. Oxygen-dependent nuclear ubiquitination and nuclear export of HIF-alpha can be prevented by treatment with an HIF-specific prolyl hydroxylase inhibitor. Treatment with inhibitors of RNA polymerase II activity, which interfere with the ability of VHL to engage in nuclear export, also prevents cytoplasmic accumulation of HIF-alpha in reoxygenated cells. This caused a marked increase in the HIF-alpha half-life without affecting its nuclear ubiquitination. We present a model by which VHL-mediated ubiquitination of HIF-alpha and its subsequent degradation are dependent upon dynamic nuclear-cytoplasmic trafficking of both the E3 ubiquitin-ligase and the nuclear substrate protein.
 ...
PMID:Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein. 1210 Dec 28

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor induced by hypoxia. Under normoxic conditions, site-specific proline hydroxylation of the alpha subunits of HIF allows recognition by the von Hippel-Lindau tumor suppressor protein (VHL), a component of an E3 ubiquitin ligase complex that targets these subunits for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this hydroxylation is inhibited, allowing the alpha subunits of HIF to escape VHL-mediated degradation. Three enzymes, prolyl hydroxylase domain-containing proteins 1, 2, and 3 (PHD1, -2, and -3; also known as HIF prolyl hydroxylase 3, 2, and 1, respectively), have recently been identified that catalyze proline hydroxylation of HIF alpha subunits. These enzymes hydroxylate specific prolines in HIF alpha subunits in the context of a strongly conserved LXXLAP sequence motif (where X indicates any amino acid and P indicates the hydroxylacceptor proline). We report here that PHD2 has the highest specific activity toward the primary hydroxylation site of HIF-1alpha. Furthermore, and unexpectedly, mutations can be tolerated at the -5, -2, and -1 positions (relative to proline) of the LXXLAP motif. Thus, these results provide evidence that the only obligatory residue for proline hydroxylation in HIF-1alpha is the hydroxylacceptor proline itself.
 ...
PMID:Sequence determinants in hypoxia-inducible factor-1alpha for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and PHD3. 1218 24

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein is the cause of familial VHL disease and sporadic kidney cancer. The VHL gene product (pVHL) is a component of an E3 ubiquitin ligase complex that targets the hypoxia-inducible factor (HIF) 1 and 2 alpha subunits for polyubiquitylation. This process is dependent on the hydroxylation of conserved proline residues on the alpha subunits of HIF-1/2 in the presence of oxygen. In our effort to identify orphan HIF-like proteins in the data base that are potential targets of the pVHL complex, we report multiple splice variants of the human HIF-3 alpha locus as follows: hHIF-3 alpha 1, hHIF-3 alpha 2 (also referred to as hIPAS; human inhibitory PAS domain protein), hHIF-3 alpha 3, hHIF-3 alpha 4, hHIF-3 alpha 5, and hHIF-3 alpha 6. We demonstrate that the common oxygen-dependent degradation domain of hHIF-3 alpha 1-3 splice variants is targeted for ubiquitylation by the pVHL complex in vitro and in vivo. This activity is enhanced in the presence of prolyl hydroxylase and is dependent on a proline residue at position 490. Furthermore, the ubiquitin conjugation occurs on lysine residues at position 465 and 568 within the oxygen-dependent degradation domain. These results demonstrate additional targets of the pVHL complex and suggest a growing complexity in the regulation of hypoxia-inducible genes by the HIF family of transcription factors.
 ...
PMID:Multiple splice variants of the human HIF-3 alpha locus are targets of the von Hippel-Lindau E3 ubiquitin ligase complex. 1253 44

The mechanism by which hypoxia induces gene transcription involves the inhibition of hypoxia-inducible factor (HIF)-1alpha prolyl hydroxylase activity, which prevents von Hippel-Lindau (vHL)-dependent targeting of HIF-1alpha to the ubiquitin-proteasome pathway. HIF-1alpha is stabilized, translocates to the nucleus, interacts with hypoxia-responsive elements, and promotes the activation of target genes. This report shows that cyclosporin A (CsA) interferes with the hypoxic signaling cascade in C6 glioma cells. CsA inhibits hypoxia-dependent gene transcription in a reporter gene assay and prevents the hypoxic accumulation of HIF-1alpha. Addition of the 530-603 C-terminal oxygen-dependent degradation (ODD) domain of HIF-1alpha to the green fluorescent protein (GFP) destabilized the protein in an oxygen-dependent manner. CsA prevented the hypoxic stabilization of an ODD.GFP fusion protein. An assay for 2-oxoglutarate-dependent dioxygenases was developed using a light mitochondrial kidney fraction as a source of enzyme. It uses the capacity of specific peptides to stimulate the degradation of [(14)C]2-oxoglutarate. CsA stimulated the enzymatic activity in the presence of a peptide that mimicked the 557-576 sequence of HIF-1alpha. The enzyme promoted [(35)S]vHL binding to glutathione S-transferase (GST).ODD fusion protein. This association increased in the presence of CsA. CsA effects were not observed when the proline residue corresponding to Pro-564 in the HIF-1alpha sequence was replaced by a hydroxyproline or an alanine residue. Finally, CsA increased vHL-ODD interaction during hypoxia. We conclude that CsA destabilizes HIF-1alpha by promoting hydroxylation of Pro-564 in the ODD domain. Such a mechanism may prevent hypoxic adaptation during CsA-induced nephrotoxicity and contribute to the adverse effects of this drug.
 ...
PMID:Cyclosporin A prevents the hypoxic adaptation by activating hypoxia-inducible factor-1alpha Pro-564 hydroxylation. 1258 29

Hypoxia-inducible factor (HIF)-1alpha, a master regulator of oxygen homeostasis, regulates genes crucial for cell growth and survival. In normoxia, HIF-1alpha is constantly degraded via the ubiquitin-proteasome pathway. The von Hippel-Lindau (VHL) E3 ubiquitin ligase binds HIF-1alpha through specific recognition of hydroxylated Pro-402 or Pro-564, both of which are modified by the oxygen-dependent HIF prolyl hydroxylases (PHDs/HPHs). Despite the identification of a conserved Leu-X-X-Leu-Ala-Pro motif, the molecular requirement of HIF-1alpha for PHDs/HPHs binding remains elusive. Recently, we demonstrated that Leu-574 of human HIF-1alpha--10 residues downstream of Pro-564--is essential for VHL recognition. We show here that the role of Leu-574 is to recruit PHD2/HPH2 for Pro-564 hydroxylation. An antibody specific for hydroxylated Pro-564 has been used to determine the hydroxylation status; mutation or deletion of Leu-574 results in a significant decrease in the ratio of the hydroxylated HIF-1alpha to the total amount. The nine-residue spacing between Pro-564 and Leu-574 is not obligatory for prolyl hydroxylation. Furthermore, mutation of Leu-574 disrupts the binding of PHD2/HPH2, a key prolyl hydroxylase for oxygen-dependent proteolysis of HIF-1alpha. Hence, our findings indicate that Leu-574 is essential for recruiting PHD2/HPH2, thereby providing a molecular basis for modulating HIF-1alpha activity.
 ...
PMID:Leu-574 of human HIF-1alpha is a molecular determinant of prolyl hydroxylation. 1508 14

The Skp1 protein, best known as a subunit of E3(SCF)-ubiquitin ligases, is subject to complex glycosylation in the cytoplasm of the cellular slime mold Dictyostelium. Pro143 of this protein is sequentially modified by a prolyl hydroxylase and five soluble glycosyltransferases (GT), to yield the structure Galalpha1,Galalpha1,3Fucalpha1,2Galbeta1,3GlcNAcalpha1-HyPro143. These enzymes are unusual in that they are expressed in the cytoplasmic compartment of the cell, rather than the secretory pathway where complex glycosylation of proteins usually occurs. The first enzyme in the pathway appears to be related to the soluble animal prolyl 4-hydroxylases (P4H), which modify the transcriptional factor subunit HIF-1alpha in the cytoplasm, and more distantly to the P4Hs that modify collagen and other proteins in the rER, based on biochemical and informatics analyses. The soluble alphaGlcNAc-transferase acting on Skp1 has been cloned and is distantly related to the mucin-type polypeptide N-acetyl-alpha-galactosaminyltransferase in the Golgi of animals. Its characterization has led to the discovery of a family of related polypeptide N-acetyl-alpha-glucosaminyltransferases in the Golgi of selected lower eukaryotes. The Skp1 GlcNAc is extended by a bifunctional diglycosyltransferase that sequentially and apparently processively adds beta1,3Gal and alpha1,2Fuc. Though this structure is also formed in the animal secretory pathway, the GTs involved are dissimilar. Conceptual translation of available genomes suggests the existence of this kind of complex cytoplasmic glycosylation in other eukaryotic microorganisms, including diatoms, oomycetes, and possibly Chlamydomonas and Toxoplasma, and an evolutionary precursor of this pathway may also occur in prokaryotes.
 ...
PMID:Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways. 1523 47

Skp1 is a cytoplasmic and nuclear protein of eukaryotes best known as an adaptor in SCF ubiquitin-protein isopeptide ligases. In Dictyostelium, Skp1 is subject to 4-hydroxylation at Pro(143) and subsequent O-glycosylation by alpha-linked GlcNAc and other sugars. Soluble cytosolic extracts have Skp1 prolyl 4-hydroxylase (P4H) activity, which can be measured based on hydroxylation-dependent transfer of [(3)H]GlcNAc to recombinant Skp1 by recombinant (Skp1-protein)-hydroxyproline alpha-N-acetyl-d-glucosaminyltransferase. The Dictyostelium Skp1 P4H gene (phyA) was predicted using a bioinformatics approach, and the expected enzyme activity was confirmed by expression of phyA cDNA in Escherichia coli. The purified recombinant enzyme (P4H1) was dependent on physiological concentrations of O(2), alpha-ketoglutarate, and ascorbate and was inhibited by CoCl(2), 3,4-dihydroxybenzoate, and 3,4-dihydroxyphenyl acetate, as observed for known animal cytoplasmic P4Hs of the hypoxia-inducible factor-alpha (HIFalpha) class. Overexpression of phyA cDNA in Dictyostelium yielded increased enzyme activity in a soluble cytosolic extract. Disruption of the phyA locus by homologous recombination resulted in loss of detectable activity in extracts and blocked hydroxylation-dependent glycosylation of Skp1 based on molecular weight analysis by SDS-PAGE, demonstrating a requirement for P4H1 in vivo. The sequence and functional similarities of P4H1 to animal HIFalpha-type P4Hs suggest that hydroxylation of Skp1 may, like that of animal HIFalpha, be regulated by availability of O(2), alpha-ketoglutarate, and ascorbate, which might exert novel control over Skp1 glycosylation.
 ...
PMID:The Skp1 prolyl hydroxylase from Dictyostelium is related to the hypoxia-inducible factor-alpha class of animal prolyl 4-hydroxylases. 1570 70

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.
 ...
PMID:Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene. 1592 19


1 2 3 4 Next >>