Hypoxia prolyl 4 hydroxylases

P. Frohna, S. Milwee, J. Pinkett, T. Lee, K. Moore-Perry, J. Chou, and R. H. Ellison in Results from a Randomized, Single-Blind, Placebo-Controlled Trial of FG-4592, a Novel Hypoxia Inducible Factor Prolyl Hydroxylase Inhibitor, in CKD Anemia, Vol. 2007, American Society of Nephrology Renal Week, San Francisco, November 2-5, 2007, Abstract SU-PO806, as abstracted in N. Agarwal and J. T. Prchal, Semin. Hematol., 2008, 45, 267. 

Nakayama, K., et al., Siah2 regulates stability of prolyl-hydroxylases, controls HlFla abundance and modulates physiological responses to hypoxia. Cell, 2004, 117(7), 941-52. 

AA serves as an important cofactor for enzymes. Lack of AA in food causes scurvy in humans due to inefficient collagen synthesis, caused by the inactivation of the Fe(II)-activating prolyl hydroxylase and lysyl hydroxylase which catalyze the formation of hydroxyproline and hydroxylysine as essential components for collagens. Prolyl hydroxylases can also hydroxylate conserved prolyl residues in the alpha subunit of the hypoxia-inducible transcription factor, which signals for proteasomal degradation of the transcription factor . The proper action of these hydrolases requires dioxygen, thus they can act... 

Appelhoff RJ, Tian YM, Raval RR, Turley H, Hands AL, Pugh CW, Ratcliffe PJ, Gleadle JM. Differential function of the prolyl hydroxylases, PHDl, 2 and 3 in the regulation of hypoxia inducible factor (HIF). J. Biol. Chem. 2004 279 38458-38465. Berra E, Benizri E, Ginouves A, Vofinat V, Roux D, Pouysse-gur J. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-la in normoxia. EMBO J. 2003 22 4082-4090. 

Huang J, Zhao Q, Mooney SM, Lee FS. Sequence determinants in hypoxia-inducible factor-la for hydroxylation by the prolyl hydroxylases PHDl, PHD2 and PHD3. J. Biol. Chem. 105. 2002 277 39792-39800. 

McDonough MA, Li V, Flashman E, Chowdhury R, Mohr C, Lienard BM, Zondlo J, Oldham NJ, Clifton IJ, Lewis J, McNeill LA, Kurzeja RJ, Hewitson KS, Yang E, Jordan S, Syed RS, Schofield CJ. Cellular oxygen sensing crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2). Proc. Natl. Acad. Sci. U.S.A. 2006 103 9814-9819. 

McNeill LA, Flashman E, Buck MR, Hewitson KS, Clifton IJ, Jeschke G, Claridge TD, Ehrismann D, Oldham NJ, Schofield CJ. Hypoxia-inducible factor prolyl hydroxylase 2 has a high affinity for ferrous iron and 2-oxoglutarate. Mol. Biosyst. 2005 1 321-324. 

Ivan M, Haberberger T, Gervasi DC, Michelson KS, Giinzler V, Kondo K, Yang H, Sorokina I, Conaway RC, Conaway JW, Kaelin WG. Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor. Proc. Natl. Acad. Sci. U.S.A. 2002 99 13459-13464. 

Appelhoff, R.J., Tian, Y.M., Raval, R.R., Turley, H., Harris,A.L., Pugh, C.W., Ratcliffe, P.J., and Gleadle, J.M. 2004. Differential function of the prolyl hydroxylases PHDl, PHD2, and PHDS in the regulation of hypoxia-inducible factor. Journal of Biological Chemistry 279 38458-38465... 

It was shown that hydroxylation of prolyl moieties can mark important proteins for proteasomal degradation. This structural modification, accomplished by prolyl hydroxylase enzymes, induces a conformational bias into the protein 3D-structure that is crucial for protein-protein recognition involved in oxygen sensing/ hypoxia response (Figure 11.10). 

Fig. 4.22. Hypoxia-mediated metabolic adaptation for energy preservation. Activation of genes for glucose transporter-1 (GLUT-1 = 1) and glycolytic enzymes yields an increased glycolytic rate. H -ions produced are preferentially exported via a Na /H -antiporter (NHE-1 = 3) and a lactate /H -symporter (monocarboxylate transporter MCT-1 = 2) leading to a drop in extracellular pH (pH.). Low extracellular pH activates the membrane-bound ectoenzyme carbonic anhydrase IX (CA IX = 4). Key mechanism regulating intracellular pH in tumor cells when protons are produced is also shown (Na -depen-dent HCOs" /CL -exchanger = 5). HIF-Ia = hypoxia-inducible factor la, PHDs = prolyl hydroxylases, FIH = asparagyl hydroxylase, lac" = lactic acid...

A prolyl hydroxylase-based oxygen-sensing system fits well with key attributes of the HIF system— notably that iron chelators or cobaltous ions mimic the effect of hypoxia. In particular, iron chelators remove the ferrous iron atom from the active site of the enzyme, preventing the conversion of HIF alpha subunits and thereby mimicking hypoxia. Exposure to cobaltous ions results in substitution of cobalt for the iron at the active center, also disabling the enzyme. Thus, the prolyl hydroxylase sensor accommodates the effects of cobalt and iron chelators without requiring the sensor itself to be turning over rapidly. 

In conclusion, EPO expression is induced by hypoxia via activation of HIF-1. The P02-dependent modulation of ROS levels within the physiological range of PO2 values can modulate EPO gene expression. HIF-1 regulation, however, depends on oxygen-sensitive hydroxylation of prohne residues of HIF-la by prolyl hydroxylases... 

Molidustat (BAY 85-3934) is a novel inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylase (PH), which stimulates erythropoietin (EPO) production and the formation of red blood cells. Phase I data have shown that inhibition of HIF-PH by Molidustat results in an increase in endogenous production of EPO [136]. Molidustat is currently under clinical trials at Bayer for the treatment of patients suffering from renal anemia due to chronic kidney disease. 

Siddiq, A., Ayoub, I.A., Chavez, J.C., et al. (2005) Hypoxia-inducible factor prolyl 4-hydroxylase inhibition. A target for neuroprotection in the central nervous system. J. Biol. Chem., 280,41732-41743. 

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