Prolyl Hydroxylase Paralogs in Nicotiana benthamiana Show High Similarity With Regard to Substrate Specificity.
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ABSTRACT:
SUBMITTER: Mocsai R
PROVIDER: S-EPMC7960765 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:L-Ascorbate (L-Asc), but not D-isoascorbate (D-Asc) and N-acetylcysteine (NAC) suppress HIF1 ODD-luc reporter activation induced by various inhibitors of HIF prolyl hydroxylase (PHD). The efficiency of suppression by L-Asc was sensitive to the nature of HIF PHD inhibitor chosen for reporter activation. In particular, the inhibitors developed to compete with alpha-ketoglutarate (αKG), were less sensitive to suppression by the physiological range of L-Asc (40-100 μM) than those having a strong iron chelation motif. Challenging those HIF activators in the reporter system with D-Asc demonstrated that the D-isomer, despite exhibiting the same reducing potency with respect to ferric iron, had almost no effect compared to L-Asc. Similarly, no effect on reporter activation was observed with cell-permeable reducing agent NAC up to 1 mM. Docking of L-Asc and D-Asc acid into the HIF PHD2 crystal structure showed interference of Tyr310 with respect to D-Asc. This suggests that L-Asc is not merely a reducing agent preventing enzyme inactivation. Rather, the overall results identify L-Asc as a co-substrate of HIF PHD that may compete for the binding site of αKG in the enzyme active center. This conclusion is in agreement with the results obtained recently in cell-based systems for TET enzymes and jumonji histone demethylases, where L-Asc has been proposed to act as a co-substrate and not as a reducing agent preventing enzyme inactivation.
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Project description:HIF prolyl-4-hydroxylase 2 (PHD2) is a non-heme Fe, 2-oxoglutarate (2OG) dependent dioxygenase that regulates the hypoxia inducible transcription factor (HIF) by hydroxylating two conserved prolyl residues in N-terminal oxygen degradation domain (NODD) and C-terminal oxygen degradation domain (CODD) of HIF-1?. Prior studies have suggested that the substrate preference of PHD2 arises from binding contacts with the ?2?3 loop of PHD2. In this study we tested the substrate selectivity of PHD2 by kinetic competition assays, varied ionic strength, and global protein flexibility using amide H/D exchange (HDX). Our results revealed that PHD2 preferred CODD by 20-fold over NODD and that electrostatics influenced this effect. Global HDX monitored by mass spectrometry indicated that binding of Fe(II) and 2OG stabilized the overall protein structure but the saturating concentrations of either NODD or CODD caused an identical change in protein flexibility. These observations imply that both substrates stabilize the ?2?3 loop to the same extent. Under unsaturated substrate conditions NODD led to a higher HDX rate than CODD due to its lower binding affinity to PHD2. Our results suggest that loop closure is the dominant contributor to substrate selectivity in PHD2.
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Project description:Protein hydroxylation extensively regulates cellular signaling by affecting protein stability, activity, and interactome, therefore contributing to the pathogenesis of various diseases including cancers. However, because of the transient nature of the hydroxylase-substrate interaction, identifying new prolyl hydroxylation substrates remains a daunting challenge. Here, by developing a novel enzyme-substrate trapping strategy coupled with TAP-TAG or orthogonal GST- purification followed by mass spectrometry, we identify Adenylosuccinate lyase (ADSL) as a bona fide EglN2 prolyl hydroxylase substrate in triple negative breast cancer (TNBC). ADSL expression is significantly higher in TNBC than other breast cancer subtypes or normal breast tissues. Functionally, ADSL knockout greatly impairs TNBC 2-D and 3-D cell proliferation and invasiveness in vitro, as well as TNBC tumorigenesis and lung colonization in xenograft models. Mechanistically, an integrated transcriptomics and metabolomics analysis revealed that ADSL promotes the activation of the oncogenic cMYC pathway by regulating cMYC protein level via a mechanism requiring ADSL hydroxylation on proline 24. Specifically, hydroxylation-proficient ADSL, by affecting adenosine levels, represses the expression of the long non-coding RNA MIR22HG, thus upregulating the oncogene cMYC protein level and its target gene expression. Our findings identify the critical role of ADSL hydroxylation in controlling cMYC and TNBC tumorigenesis.
2019-10-09 | GSE136414 | GEO
Project description:Collagen prolyl 4-hydroxylases (CP4Hs) catalyze a prevalent posttranslational modification, the hydroxylation of (2S)-proline residues in protocollagen strands. The ensuing (2S,4R)-4-hydroxyproline residues are necessary for the conformational stability of the collagen triple helix. Prolyl peptide bonds isomerize between cis and trans isomers, and the preference of the enzyme is unknown. We synthesized alkene isosteres of the cis and trans isomers to probe the conformational preferences of human CP4H1. We discovered that the presence of a prolyl peptide bond is necessary for catalysis. The cis isostere is, however, an inhibitor with a potency greater than that of the trans isostere, suggesting that the cis conformation of a prolyl peptide bond is recognized preferentially. Comparative studies with a Chlamydomonas reinhardtii P4H, which has a similar catalytic domain but lacks an N-terminal substrate-binding domain, showed a similar preference for the cis isostere. These findings support the hypothesis that the catalytic domain of CP4Hs recognizes the cis conformation of the prolyl peptide bond and inform the use of alkenes as isosteres for peptide bonds.
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Project description:Posttranslational modifications can cause profound changes in protein function. Typically, these modifications are reversible, and thus provide a biochemical on-off switch. In contrast, proline residues are the substrates for an irreversible reaction that is the most common posttranslational modification in humans. This reaction, which is catalyzed by prolyl 4-hydroxylase (P4H), yields (2S,4R)-4-hydroxyproline (Hyp). The protein substrates for P4Hs are diverse. Likewise, the biological consequences of prolyl hydroxylation vary widely, and include altering protein conformation and protein-protein interactions, and enabling further modification. The best known role for Hyp is in stabilizing the collagen triple helix. Hyp is also found in proteins with collagen-like domains, as well as elastin, conotoxins, and argonaute 2. A prolyl hydroxylase domain protein acts on the hypoxia inducible factor alpha, which plays a key role in sensing molecular oxygen, and could act on inhibitory kappaB kinase and RNA polymerase II. P4Hs are not unique to animals, being found in plants and microbes as well. Here, we review the enzymic catalysts of prolyl hydroxylation, along with the chemical and biochemical consequences of this subtle but abundant posttranslational modification.
| S-EPMC2841224 | biostudies-literature
Project description:Protein hydroxylation affects protein stability, activity, and interactome, therefore contributing to various diseases including cancers. However, the transiency of the hydroxylation reaction hinders the identification of hydroxylase substrates. By developing an enzyme-substrate trapping strategy coupled with TAP-TAG or orthogonal GST- purification followed by mass spectrometry, we identify adenylosuccinate lyase (ADSL) as an EglN2 hydroxylase substrate in triple negative breast cancer (TNBC). ADSL expression is higher in TNBC than other breast cancer subtypes or normal breast tissues. ADSL knockout impairs TNBC cell proliferation and invasiveness in vitro and in vivo. An integrated transcriptomics and metabolomics analysis reveals that ADSL activates the oncogenic cMYC pathway by regulating cMYC protein level via a mechanism requiring ADSL proline 24 hydroxylation. Hydroxylation-proficient ADSL, by affecting adenosine levels, represses the expression of the long non-coding RNA MIR22HG, thus upregulating cMYC protein level. Our findings highlight the role of ADSL hydroxylation in controlling cMYC and TNBC tumorigenesis.
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Project description:Glucosinolates (GLSs) are amino acid-derived defense compounds characteristic of the Brassicales order. Cytochromes P450s of the CYP79 family are the entry point into the biosynthetic pathway of the GLS core structure and catalyze the conversion of amino acids to oximes. In Arabidopsis thaliana, CYP79A2, CYP79B2, CYP79B3, CYP79F1, and CYP79F2 have been functionally characterized and are responsible for the biosynthesis of phenylalanine-, tryptophan-, and methionine-derived GLSs, respectively. However, the substrate(s) for CYP79C1 and CYP79C2 were unknown. Here, we investigated the function of CYP79C1 and CYP79C2 by transiently co-expressing the genes together with three sets of remaining genes required for GLS biosynthesis in Nicotiana benthamiana. Co-expression of CYP79C2 with either the aliphatic or aromatic core structure pathways resulted in the production of primarily leucine-derived 2-methylpropyl GLS and phenylalanine-derived benzyl GLS, along with minor amounts of GLSs from isoleucine, tryptophan, and tyrosine. Co-expression of CYP79C1 displayed minor amounts of GLSs from valine, leucine, isoleucine, and phenylalanine with the aliphatic core structure pathway, and similar GLS profile (except the GLS from valine) with the aromatic core structure pathway. Additionally, we co-expressed CYP79C1 and CYP79C2 with the chain elongation and aliphatic core structure pathways. With the chain elongation pathway, CYP79C2 still mainly produced 2-methylpropyl GLS derived from leucine, accompanied by GLSs derived from isoleucine and from chain-elongated mono- and dihomoleucine, but not from phenylalanine. However, co-expression of CYP79C1 only resulted in GLSs derived from chain-elongated amino acid substrates, dihomoleucine and dihomomethionine, when the chain elongation pathway was present. This shows that CYP79 activity depends on the specific pathways co-expressed and availability of amino acid precursors, and that description of GLS core structure pathways as "aliphatic" and "aromatic" pathways is not suitable, especially in an engineering context. This is the first characterization of members of the CYP79C family. Co-expression of CYP79 enzymes with engineered GLS pathways in N. benthamiana is a valuable tool for simultaneous testing of substrate specificity against multiple amino acids.
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Project description:Bacteria are the principal consumers of dissolved organic carbon (DOC) in the ocean and predation of bacteria makes organic carbon available to higher trophic levels. The efficiency with which bacteria convert the consumed carbon (C) into biomass (i.e., carbon growth efficiency, Y) determines their ecological as well as biogeochemical role in marine ecosystems. Yet, it is still unclear how changes in temperature will affect Y and, hence, the transfer of consumed C to higher trophic levels. Here, we experimentally investigated the effect of temperature on metabolic functions of coastal microbial communities inoculated in both nutrient-limited chemostats and nutrient-unlimited turbidostats. We inoculated chemostats and turbidostats with coastal microbial communities into seawater culture medium augmented with 20 and 100 ?mol L-1 of glucose respectively and measured CO2 production, carbon biomass and cell abundance. Chemostats were cultured between 14 and 26°C and specific growth rates (?) between 0.05 and 6.0 day-1, turbidostats were cultured between 10 and 26°C with specific growth rates ranging from 28 to 62 day-1. In chemostats under substrate limitation, which is common in the ocean, the specific respiration rate (r, day-1) showed no trend with temperature and was roughly proportional to ?, implying that carbon growth efficiency (Y) displayed no tendency with temperature. The response was very different in turbidostats under temperature-limited, nutrient-repleted growth, here ? increased with temperature but r decreased resulting in an increase of Y with temperature (Q10: 2.6). Comparison of our results with data from the literature on the respiration rate and cell weight of monospecific bacteria indicates that in general the literature data behaved similar to chemostat data, showing no trend in specific respiration with temperature. We conclude that respiration rates of nutrient-limited bacteria measured at a certain temperature cannot be adjusted to different temperatures with a temperature response function similar to Q10 or Arrhenius. However, the cellular respiration rate and carbon demand rate (both: mol C cell-1 day-1) show statistically significant relations with cellular carbon content (mol C cell-1) in chemostats, turbidostats, and the literature data.
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Project description:We describe covalently binding modulators of the activity of human prolyl hydroxylase domain 2 (PHD2) and studies towards a strategy for photocapture of PHD2 substrates. Reversible active site binding of electrophile bearing compounds enables susbsequent covalent reaction with a lysine residue (K408) in the flexible C-terminal region of PHD2 to give a modified protein that retains catalytic activity.
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Project description:Hypoxia-inducible factor (HIF) has a pivotal role in oxygen homeostasis and cardioprotection mediated by ischemic preconditioning. Its stability is regulated by HIF prolyl 4-hydroxylases (HIF-P4Hs), the inhibition of which is regarded as a promising strategy for treating diseases such as anemia and ischemia. We generated a viable Hif-p4h-2 hypomorph mouse line (Hif-p4h-2(gt/gt)) that expresses decreased amounts of wild-type Hif-p4h-2 mRNA: 8% in the heart; 15% in the skeletal muscle; 34-47% in the kidney, spleen, lung, and bladder; 60% in the brain; and 85% in the liver. These mice have no polycythemia and show no signs of the dilated cardiomyopathy or hyperactive angiogenesis observed in mice with broad spectrum conditional Hif-p4h-2 inactivation. We focused here on the effects of chronic Hif-p4h-2 deficiency in the heart. Hif-1 and Hif-2 were stabilized, and the mRNA levels of glucose transporter-1, several enzymes of glycolysis, pyruvate dehydrogenase kinase 1, angiopoietin-2, and adrenomedullin were increased in the Hif-p4h-2(gt/gt) hearts. When isolated Hif-p4h-2(gt/gt) hearts were subjected to ischemia-reperfusion, the recovery of mechanical function and coronary flow rate was significantly better than in wild type, while cumulative release of lactate dehydrogenase reflecting the infarct size was reduced. The preischemic amount of lactate was increased, and the ischemic versus preischemic [CrP]/[Cr] and [ATP] remained at higher levels in Hif-p4h-2(gt/gt) hearts, indicating enhanced glycolysis and an improved cellular energy state. Our data suggest that chronic stabilization of Hif-1alpha and Hif-2alpha by genetic knockdown of Hif-p4h-2 promotes cardioprotection by induction of many genes involved in glucose metabolism, cardiac function, and blood pressure.
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