Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay

Abstract

cMyBP-C [cardiac (MyBP-C) myosin-binding protein-C)] is a sarcomeric protein involved both in thick filament structure and in the regulation of contractility. It is composed of eight IgI-like and three fibronectin-3-like domains (termed C0-C10). Mutations in the gene encoding cMyBP-C are a principal cause of HCM (hypertrophic cardiomyopathy). cMyBP-C binds to the LMM (light meromyosin) portion of the myosin rod via its C-terminal domain, C10. We investigated this interaction in detail to determine whether HCM mutations in beta myosin heavy chain located within the LMM portion alter the binding of cMyBP-C, and to define the precise region of LMM that binds C10 to aid in developing models of the arrangement of MyBP-C on the thick filament. In co-sedimentation experiments recombinant C10 bound full-length LMM with a K(d) of 3.52 microM and at a stoichiometry of 1.14 C10 per LMM. C10 was also shown to bind with similar affinity to LMM containing either the HCM mutations A1379T or S1776G, suggesting that these HCM mutations do not perturb C10 binding. Using a range of N-terminally truncated LMM fragments, the cMyBP-C-binding site on LMM was shown to lie between residues 1554 and 1581. Since it had been reported previously that acidic residues on myosin mediate the C10 interaction, three clusters of acidic amino acids (Glu1554/Glu1555, Glu1571/Glu1573 and Glu1578/Asp1580/Glu1581/Glu1582) were mutated in full-length LMM and the proteins tested for C10 binding. No effect of these mutations on C10 binding was however detected. We interpret our results with respect to the localization of the proposed trimeric collar on the thick filament.

Turn me on: regulating HIF transcriptional activity

“…34 However, more recent studies using larger peptides suggest that the K m of FIH-1 for oxygen in vitro is closer to 250 mM like the PHDs, depending on the length of peptide used. 35 More importantly, cell-based assays examining the activity of endogenous FIH-1 and the PHDs indicate that FIH-1 was less sensitive to decreasing oxygen levels than the PHDs in some cell types, whereas in other cells types it was more sensitive, thus demonstrating that the oxygen-dependent sensitivity of FIH-1 can vary between different cell types independently of the PHDs. 36 These cell-based results do not invalidate the in vitro results, but rather reflect the intrinsic differences between proteins in a complex cellular environment compared with isolated proteins and peptides.…”

Targeting the hypoxic response in bone tissue engineering: A balance between supply and consumption to improve bone regeneration

“…Indeed, a recent study has shown that in bone, physiological oxygen concentrations can be as low as 1.3% or 10 mmHg (Spencer et al, 2014), which are much lower than the ambient oxygen levels that we breathe (21% or 160 mmHg). Moreover, activation of the hypoxia signalling pathway is known to increase exponentially as oxygen concentrations fall below 8% or 60 mmHg (Ehrismann et al, 2007). A better understanding of the processes activated by the hypoxia signalling pathway in skeletal cells may therefore provide novel options to improve bone tissue engineering strategies.…”

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies

“…Such studies have also defined apparent Michaelis constant (K m ) values for Fe(II), ascorbate, 2-OG, and oxygen. In keeping with predictions from kinetic analyses of other members of the 2-OG oxygenase superfamily that it is an E.2-OG substrate species that interacts with molecular oxygen, the apparent K mO2 varies with the HIF polypeptide substrate used in the assay 36,37 . 2 , and (given that oxygen is not produced by animal cells) they can be predicted to be above the concentration available to the enzymes within their subcellular compartment.…”