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Joona Tapio Riikka Halmetoja Elitsa Y. Dimova Joni M. Mki Anu Laitala Gail Walkinshaw Johanna Myllyharju Raisa Serpi Peppi Koivunen 《The Journal of biological chemistry》2022,298(8)
Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs 1–3) are druggable targets in renal anemia, where pan-HIF-P4H inhibitors induce an erythropoietic response. Preclinical data suggest that HIF-P4Hs could also be therapeutic targets for treating metabolic dysfunction, although the contributions of HIF-P4H isoenzymes in various tissues to the metabolic phenotype are inadequately understood. Here, we used mouse lines that were gene-deficient for HIF-P4Hs 1 to 3 and two preclinical pan-HIF-P4H inhibitors to study the contributions of these isoenzymes to the anthropometric and metabolic outcome and HIF response. We show both inhibitors induced a HIF response in wildtype white adipose tissue (WAT), liver, and skeletal muscle and alleviated metabolic dysfunction during a 6-week treatment period, but they did not alter healthy metabolism. Our data indicate that HIF-P4H-1 contributed especially to skeletal muscle and WAT metabolism and that its loss lowered body weight and serum cholesterol levels upon aging. In addition, we found HIF-P4H-3 had effects on the liver and WAT and its loss increased body weight, adiposity, liver weight and triglyceride levels, WAT inflammation, and cholesterol levels and resulted in hyperglycemia and insulin resistance, especially during aging. Finally, we demonstrate HIF-P4H-2 affected all tissues studied; its inhibition lowered body and liver weight and serum cholesterol levels and improved glucose tolerance. We found very few HIF target metabolic mRNAs were regulated by the inhibition of three isoenzymes, thus suggesting a potential for selective therapeutic tractability. Altogether, these data provide specifications for the future development of HIF-P4H inhibitors for the treatment of metabolic diseases. 相似文献
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Koivunen P Hirsilä M Kivirikko KI Myllyharju J 《The Journal of biological chemistry》2006,281(39):28712-28720
Three hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the HIFs by hydroxylating prolines at two separate sites in the oxygen-dependent degradation domain (ODDD) of their alpha subunits. We compared in vitro hydroxylation by purified recombinant human HIF-P4Hs of 19-20- and 35-residue peptides corresponding to the two sites in HIF-alphas and purified recombinant HIF-1alpha and HIF-2alpha ODDDs of 248 and 215 residues. The increase in the length of peptides representing the C-terminal site from 19 to 20 to 35 residues reduced the K(m) values to 90-800 nm, i.e. to 0.7-11% of those for the shorter peptides, whereas those representing the N-terminal site were 10-470 microm, i.e. 10-135%. The K(m) values of HIF-P4H-1 for the recombinant HIF-alpha ODDDs were 10-20 nm, whereas those of HIF-P4H-2 and -3 were 60-140 nm, identical values being found for the wild-type HIF-1alpha ODDD and its N site mutant. The K(m) values for the C site mutant were about 5-10 times higher but only 0.2-3% of those for the 35-residue N site peptides, and this marked difference suggested that the HIF-P4Hs may become bound first to the C-terminal site of an ODDD and that this binding may enhance subsequent binding to the N-terminal site. The K(m) values of HIF-P4H-2 for oxygen determined with the HIF-1alpha ODDD and both its mutants as substrates were all about 100 microm, being 40% of those reported for the three HIF-P4Hs with a 19-residue peptide. Even this value is high compared with tissue O(2) levels, indicating that HIF-P4Hs are effective oxygen sensors. 相似文献
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Li D Hirsilä M Koivunen P Brenner MC Xu L Yang C Kivirikko KI Myllyharju J 《The Journal of biological chemistry》2004,279(53):55051-55059
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Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family 总被引:14,自引:0,他引:14
Hewitson KS McNeill LA Riordan MV Tian YM Bullock AN Welford RW Elkins JM Oldham NJ Bhattacharya S Gleadle JM Ratcliffe PJ Pugh CW Schofield CJ 《The Journal of biological chemistry》2002,277(29):26351-26355
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Cell-permeating alpha-ketoglutarate derivatives alleviate pseudohypoxia in succinate dehydrogenase-deficient cells
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MacKenzie ED Selak MA Tennant DA Payne LJ Crosby S Frederiksen CM Watson DG Gottlieb E 《Molecular and cellular biology》2007,27(9):3282-3289
Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are components of the tricarboxylic acid (TCA) cycle and tumor suppressors. Loss of SDH or FH induces pseudohypoxia, a major tumor-supporting event, which is the activation of hypoxia-inducible factor (HIF) under normoxia. In SDH- or FH-deficient cells, HIF activation is due to HIF1alpha stabilization by succinate or fumarate, respectively, either of which, when in excess, inhibits HIFalpha prolyl hydroxylase (PHD). To reactivate PHD, we focused on its substrate, alpha-ketoglutarate. We designed and synthesized cell-permeating alpha-ketoglutarate derivatives, which build up rapidly and preferentially in cells with a dysfunctional TCA cycle. This study shows that succinate- or fumarate-mediated inhibition of PHD is competitive and is reversed by pharmacologically elevating intracellular alpha-ketoglutarate. Introduction of alpha-ketoglutarate derivatives restores normal PHD activity and HIF1alpha levels to SDH-suppressed cells, indicating new therapy possibilities for the cancers associated with TCA cycle dysfunction. 相似文献
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Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor 总被引:28,自引:0,他引:28
Hirsilä M Koivunen P Günzler V Kivirikko KI Myllyharju J 《The Journal of biological chemistry》2003,278(33):30772-30780
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Stolze IP Tian YM Appelhoff RJ Turley H Wykoff CC Gleadle JM Ratcliffe PJ 《The Journal of biological chemistry》2004,279(41):42719-42725
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An endoplasmic reticulum transmembrane prolyl 4-hydroxylase is induced by hypoxia and acts on hypoxia-inducible factor alpha 总被引:3,自引:0,他引:3
Koivunen P Tiainen P Hyvärinen J Williams KE Sormunen R Klaus SJ Kivirikko KI Myllyharju J 《The Journal of biological chemistry》2007,282(42):30544-30552
Prolyl 4-hydroxylases (P4Hs) act on collagens (C-P4Hs) and the oxygen-dependent degradation domains (ODDDs) of hypoxia-inducible factor alpha subunits (HIF-P4Hs) leading to degradation of the latter. We report data on a human P4H possessing a transmembrane domain (P4H-TM). Its gene is also found in zebrafish but not in flies and nematodes. Its sequence more closely resembles those of the C-P4Hs than the HIF-P4Hs, but it lacks the peptide substrate-binding domain of the C-P4Hs. P4H-TM levels in cultured cells are increased by hypoxia, and P4H-TM is N-glycosylated and is located in endoplasmic reticulum membranes with its catalytic site inside the lumen, a location differing from those of the HIF-P4Hs. Despite this, P4H-TM overexpression in cultured neuroblastoma cells reduced HIF-alpha ODDD reporter construct levels, and its small interfering RNA increased HIF-1alpha protein level, in the same way as those of HIF-P4Hs. Furthermore, recombinant P4H-TM hydroxylated the two critical prolines in HIF-1alpha ODDD in vitro, with a preference for the C-terminal proline, whereas it did not hydroxylate any prolines in recombinant type I procollagen chains. 相似文献
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HIF hydroxylation and cellular oxygen sensing 总被引:7,自引:0,他引:7
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Quintuple mutants of Escherichia coli deficient in the C(4)-dicarboxylate carriers of aerobic and anaerobic metabolism (DctA, DcuA, DcuB, DcuC, and the DcuC homolog DcuD, or the citrate/succinate antiporter CitT) showed only poor growth on succinate (or other C(4)-dicarboxylates) under oxic conditions. At acidic pH (pH 6) the mutants regained aerobic growth on succinate, but not on fumarate. Succinate uptake by the mutants could not be saturated at physiological succinate concentrations (< or =5 mM), in contrast to the wild-type, which had a K(m) for succinate of 50 microM and a V(max) of 35 U/g dry weight at pH 6. At high substrate concentrations, the mutants showed transport activities (32 U/g dry weight) comparable to that of the wild-type. In the wild-type using DctA as the carrier, succinate uptake had a pH optimum of 6, whereas succinate uptake in the mutants was maximal at pH 5. In the mutants succinate uptake was inhibited competitively by monocarboxylic acids. Diffusion of succinate or fumarate across phospholipid membranes (liposomes) was orders of magnitude slower than the transport in the wild-type or the mutants. The data suggest that mutants deficient in DctA, DcuA, DcuB, DcuC, DcuD (or CitT) contain a carrier, possibly a monocarboxylate carrier, which is able to transport succinate, but not fumarate, at acidic pH, when succinate is present as a monoanion. Succinate uptake by this carrier was inhibited by addition of an uncoupler. Growth by fumarate respiration (requiring fumarate/succinate antiport) was also lost in the quintuple mutants, and growth was not restored at pH 6. In contrast, the efflux of succinate produced during glucose fermentation was not affected in the mutants, demonstrating that, for succinate efflux, a carrier different from, or in addition to, the known Dcu and CitT carriers is used. 相似文献