Biochemical characterization of human HIF hydroxylases using HIF protein substrates that contain all three hydroxylation sites

2011 ◽  
Vol 436 (2) ◽  
pp. 363-369 ◽  
Author(s):  
Melissa B. Pappalardi ◽  
Dean E. McNulty ◽  
John D. Martin ◽  
Kelly E. Fisher ◽  
Yong Jiang ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Transactivation Domain ◽  
Prolyl Hydroxylases ◽  
Proline Residue ◽  
Protein Substrates ◽  
Steady State Kinetic ◽  
Asparaginyl Hydroxylase

The HIF (hypoxia-inducible factor) plays a central regulatory role in oxygen homoeostasis. HIF proteins are regulated by three Fe(II)- and α-KG (α-ketoglutarate)-dependent prolyl hydroxylase enzymes [PHD (prolyl hydroxylase domain) isoenzymes 1–3 or PHD1, PHD2 and PHD3] and one asparaginyl hydroxylase [FIH (factor inhibiting HIF)]. The prolyl hydroxylases control the abundance of HIF through oxygen-dependent hydroxylation of specific proline residues in HIF proteins, triggering subsequent ubiquitination and proteasomal degradation. FIH inhibits the HIF transcription activation through asparagine hydroxylation. Understanding the precise roles and regulation of these four Fe(II)- and α-KG-dependent hydroxylases is of great importance. In the present paper, we report the biochemical characterization of the first HIF protein substrates that contain the CODDD (C-terminal oxygen-dependent degradation domain), the NODDD (N-terminal oxygen-dependent degradation domain) and the CAD (C-terminal transactivation domain). Using LC-MS/MS (liquid chromatography–tandem MS) detection, we show that all three PHD isoenzymes have a strong preference for hydroxylation of the CODDD proline residue over the NODDD proline residue and the preference is observed for both HIF1α and HIF2α protein substrates. In addition, steady-state kinetic analyses show differential substrate selectivity for HIF and α-KG in reference to the three PHD isoforms and FIH.

scholarly journals Hypoxia-inducible factor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the β-carbon of asparagine-803

2002 ◽  
Vol 367 (3) ◽  
pp. 571-575 ◽  
Author(s):  
Luke A. McNEILL ◽  
Kirsty S. HEWITSON ◽  
Timothy D. CLARIDGE ◽  
Jürgen F. SEIBEL ◽  
Louise E. HORSFALL ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Hypoxia Inducible Factor ◽  
Transactivation Domain ◽  
Α Subunit ◽  
Asparaginyl Hydroxylase ◽  
Threo Isomer

Asparagine-803 in the C-terminal transactivation domain of human hypoxia-inducible factor (HIF)-1 α-subunit is hydroxylated by factor inhibiting HIF-1 (FIH-1) under normoxic conditions causing abrogation of the HIF-1α/p300 interaction. NMR and other analyses of a hydroxylated HIF fragment produced in vitro demonstrate that hydroxylation occurs at the β-carbon of Asn-803 and imply production of the threo-isomer, in contrast with other known aspartic acid/asparagine hydroxylases that produce the erythro-isomer.

scholarly journals Molecular and cellular mechanisms of HIF prolyl hydroxylase inhibitors in clinical trials

2017 ◽  
Vol 8 (11) ◽  
pp. 7651-7668 ◽  
Author(s):  
Tzu-Lan Yeh ◽  
Thomas M. Leissing ◽  
Martine I. Abboud ◽  
Cyrille C. Thinnes ◽  
Onur Atasoylu ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Active Site ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Cellular Mechanisms ◽  
Prolyl Hydroxylases ◽  
Hif Prolyl Hydroxylase

Four compounds in clinical trials for anaemia treatment are potent inhibitors of the hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs), but differ in potency and how they interact with HIF at the PHD active site.

scholarly journals Biochemical Characterization of β-Lactamases Bla1 and Bla2 from Bacillus anthracis

2003 ◽  
Vol 47 (6) ◽  
pp. 2040-2042 ◽  
Author(s):  
Isabel C. Materon ◽  
Anne Marie Queenan ◽  
Theresa M. Koehler ◽  
Karen Bush ◽  
Timothy Palzkill
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Bacillus Anthracis ◽  
Biochemical Characterization ◽  
Steady State Kinetic ◽  
Chromosomal Genes ◽  
State Kinetic

ABSTRACT The Sterne and Ames strains of Bacillus anthracis carry chromosomal genes bla1 and bla2, which confer β-lactam resistance when expressed in Escherichia coli. MIC measurements and steady-state kinetic analyses indicate that Bla1 possesses penicillinase activity while Bla2 possesses penicillinase, cephalosporinase, and carbapenem-hydrolyzing activities.

scholarly journals Molecular and Biochemical Characterization of CTX-M-131, a Natural Asp240Gly Variant Derived from CTX-M-2, Produced by a Providencia rettgeri Clinical Strain in São Paulo, Brazil

2015 ◽  
Vol 59 (3) ◽  
pp. 1815-1817 ◽  
Author(s):  
Milena Dropa ◽  
Barbara Ghiglione ◽  
Maria Helena Matté ◽  
Livia Carminato Balsalobre ◽  
Nilton Lincopan ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Clinical Strain ◽  
Class 1 Integron ◽  
Providencia Rettgeri ◽  
Content Type ◽  
Steady State Kinetic ◽  
Class 1 ◽  
Encoding Genes ◽  
State Kinetic

ABSTRACTCTX-M-131 is a natural Asp240Gly variant from the CTX-M-2 group detected in aProvidencia rettgericlinical strain from Brazil. Molecular analysis showed thatblaCTX-M-131was inserted in a complex class 1 integron harbored by a 112-kb plasmid, which has not been previously described as a platform for CTX-M-encoding genes with the Asp240Gly mutation. Steady-state kinetic parameters showed that the enzyme has a typical cefotaximase catalytic profile and an enhanced activity against ceftazidime.

scholarly journals Characterization of different isoforms of the HIF prolyl hydroxylase PHD1 generated by alternative initiation

2006 ◽  
Vol 397 (1) ◽  
pp. 179-186 ◽  
Author(s):  
Ya-Min Tian ◽  
David R. Mole ◽  
Peter J. Ratcliffe ◽  
Jonathan M. Gleadle
Keyword(s):  
Regulation Of Gene Expression ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Biologically Active ◽  
Genetic Studies ◽  
Translational Initiation ◽  
Von Hippel Lindau ◽  
Seven In Absentia ◽  
Hif Prolyl Hydroxylase

The heterodimeric transcription factor HIF (hypoxia-inducible factor) is central to the regulation of gene expression by oxygen. Three oxygen-dependent prolyl hydroxylase enzymes [PHD1 (prolyl hydroxylase domain 1), PHD2 and PHD3] control the abundance of HIF. In the presence of oxygen, they hydroxylate specific proline residues in HIF-α, allowing recognition by pVHL (von Hippel-Lindau protein) and subsequent ubiquitylation and proteasomal destruction. The precise roles and regulation of these enzymes are therefore of particular importance in understanding the physiological and pathological responses to hypoxia. In the present study, we define the existence of two species of PHD1 and provide evidence that they are generated by alternative translational initiation. We demonstrate that these alternative forms are both biologically active with similar HIF prolyl hydroxylase activity but that they differ in their responses to oestrogen, cell confluence and proteasomal inhibition. We show that the two PHD1 species are subject to proteolytic regulation but differ markedly in their protein stability. Though each isoform has the potential to interact with members of the Siah (seven in absentia homologue) ubiquitin ligase family, genetic studies indicated that other proteolytic mechanisms are responsible for control of stability under the conditions examined. The data define the existence of a further level of control in the pathway that regulates cellular responses to hypoxia.

Identification and biochemical characterization of two novel peroxiredoxins in a liver fluke,Clonorchis sinensis

Parasitology ◽  
2011 ◽  
Vol 138 (9) ◽  
pp. 1143-1153 ◽  
Author(s):  
Y.-A. BAE ◽  
S.-H. KIM ◽  
E.-G. LEE ◽  
W.-M. SOHN ◽  
Y. KONG
Keyword(s):  
Biochemical Characterization ◽  
Expression Patterns ◽  
Clonorchis Sinensis ◽  
Type Equation ◽  
Protein Product ◽  
Steady State Kinetic ◽  
Genes Encoding ◽  
Secretory Products ◽  
Almost All

SUMMARYWe identified 2 novel genes encoding different 2-Cys peroxiredoxins (PRxs), designated CsPRx2 and CsPRx3, inClonorchis sinensis, which invades the human hepatobiliary tracts. TheCsPRx2gene expression was temporally increased along with the parasite's development and its protein product was detected in almost all parts of adult worms including subtegument, as well as excretory-secretory products. Conversely,CsPRx3expression was temporally maintained at a basal level and largely restricted within interior parts of various tissues/organs. The recombinant forms of CsPRx proteins exhibited reducing activity against various hydroperoxides in the presence of either thioredoxin or glutathione (GSH) as a reducing equivalent, although they preferred H2O2and GSH as a catalytic substrate and electron donor, respectively. A steady-state kinetic study demonstrated that the CsPRx proteins followed a saturable, Michaelis-Menten-type equation with the catalytic efficiencies (kcat/Km) ranging from 103to 104M−1s−1, somewhat lower than those for other PRxs studied (104–105M−1s−1). The expression patterns and histological distributions specific to CsPRx2 and CsPRx3 might suggest different physiological functions of the antioxidant enzymes in protecting the worms against oxidative damage.

Biochemical Characterization of Human Prolyl Hydroxylase Domain Protein 2 Variants Associated with Erythrocytosis

Biochemistry ◽  
2008 ◽  
Vol 47 (43) ◽  
pp. 11165-11167 ◽  
Author(s):  
Melissa B. Pappalardi ◽  
John D. Martin ◽  
Yong Jiang ◽  
Matthew C. Burns ◽  
Huizhen Zhao ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Prolyl Hydroxylase ◽  
Domain Protein ◽  
Prolyl Hydroxylase Domain
Sign in / Sign up

Export Citation Format

Share Document