Oxidation of the diphosphoinositol polyphosphate phosphohydrolase-like Nudix hydrolase Aps from Drosophila melanogaster induces thermolability--A possible regulatory switch?
ABSTRACT Unlike mammalian cells, Drosophila melanogaster contains only a single member of the diphosphoinositol polyphosphate phosphohydrolase subfamily of the Nudix hydrolases, suggesting that functional specialisation has not occurred in this organism. In order to evaluate its function, Aps was cloned and characterized. It hydrolyses a range of (di)nucleoside polyphosphates, the most efficient being guanosine 5'-tetraphosphate (K(m)=11 microM, k(cat)=0.79 s(-1)). However, it shows a 5-fold preference for the hydrolysis of diphosphoinositol pentakisphosphate (PP-InsP(5), K(m)=0.07 microM, k(cat)=0.024 s(-1)). Assayed at 26 degrees C, Aps had an alkaline pH optimum and required a divalent ion: Mg(2+) (10-20 mM) or Mn(2+) (1 mM) were preferred for nucleotide hydrolysis and Mg(2+) (0.5-1 mM) or Co(2+) (1-100 microM) for PP-InsP(5) hydrolysis. GFP-fusions showed that Aps was predominantly cytoplasmic, with some nuclear localization. In the absence of dithiothreitol Aps was heat labile, rapidly losing activity even at 36 degrees C, while in the presence of dithiothreitol, Aps was heat stable, surviving for 5 min at 76 degrees C. Heat lability was restored by H(2)O(2) and mass spectrometric analysis suggested that this was due to reversible dimerisation involving two inter-molecular disulphides between Cys23 and Cys25. Aps expression was highest in embryos and declined throughout development. The ratio of PP-InsP(5) to inositol hexakisphosphate also decreased throughout development, with the highest level of PP-InsP(5) found in embryos. These data suggest that the redox state of Aps may play a role in controlling its activity by altering its stability, something that could be important for regulating PP-InsP(5) during development.
- [Show abstract] [Hide abstract]
ABSTRACT: Many members of the nudix hydrolase family exhibit considerable substrate multispecificity and ambiguity, which raises significant issues when assessing their functions in vivo and gives rise to errors in database annotation. Several display low antimutator activity when expressed in bacterial tester strains as well as some degree of activity in vitro towards mutagenic, oxidized nucleotides such as 8-oxo-dGTP. However, many of these show greater activity towards other nucleotides such as ADP-ribose or diadenosine tetraphosphate (Ap(4)A). The antimutator activities have tended to gain prominence in the literature, whereas they may in fact represent the residual activity of an ancestral antimutator enzyme that has become secondary to the more recently evolved major activity after gene duplication. Whether any meaningful antimutagenic function has also been retained in vivo requires very careful assessment. Then again, other examples of substrate ambiguity may indicate as yet unexplored regulatory systems. For example, bacterial Ap(4)A hydrolases also efficiently remove pyrophosphate from the 5' termini of mRNAs, suggesting a potential role for Ap(4)A in the control of bacterial mRNA turnover, while the ability of some eukaryotic mRNA decapping enzymes to degrade IDP and dIDP or diphosphoinositol polyphosphates (DIPs) may also be indicative of new regulatory networks in RNA metabolism. DIP phosphohydrolases also degrade diadenosine polyphosphates and inorganic polyphosphates, suggesting further avenues for investigation. This article uses these and other examples to highlight the need for a greater awareness of the possible significance of substrate ambiguity among the nudix hydrolases as well as the need to exert caution when interpreting incomplete analyses.Cellular and Molecular Life Sciences CMLS 11/2012; · 5.62 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The past ten years have seen a contained explosion of interest in inositol pyrophosphates. The early cloning of the IP6Ks and the more recent identification of the PP-IP5Ks have allowed the development of essential experimental tools to investigate the physiological role of inositol pyrophosphates. However, for this exciting field of research to gain momentum, simpler and more reliable research protocols need to be further developed. The ability to resolve and quantify inositol pyrophosphates using gel electrophoresis (Losito et al., 2009) has dramatically altered the way we are studying this class of molecules, opening new avenues for research. The use of this technology to resolve, detect and characterize inositol pyrophosphates extracted from cells certainly represents one desirable aim. The most crucial objective, however, is to obtain definite proof of the new mechanism of post-translational modification by identifying with biophysical methods the presence in vivo of pyrophosphorylated serines. This will hopefully precipitate the development of new ways to detect this modification, for example through the production of antibodies that specifically recognize pyrophosphorylated serines.Advances in enzyme regulation 10/2010; 51(1):74-82.
- [Show abstract] [Hide abstract]
ABSTRACT: Polygenetic effect has rarely been addressed in the genetic studies of coronary artery disease (CAD). We used the largest and ethnically homogeneous angiographic cohort to analyze multilocus data in renin-angiotensin system genes, and provide an explicit demonstration of gene-gene interactions. A total of 1254 consecutive patients who underwent cardiac catheterization (735 with coronary artery disease and 519 without) were recruited. Angiotensin converting enzyme(ACE) gene I/D polymorphism; T174M, M235T, G-6A, A-20C, G-152A, and G-217A polymorphisms of the angiotensinogen (AGT) gene; and A1166C polymorphism of the angiotensin II type I receptor (AT1R) gene were genotyped. We used a regression approach based on a generalized linear model to evaluate haplotype effects, adjust non-genetic confounding effects and detect gene-gene interaction between ACE and AT1R genes. We found significant differences in global AGT gene haplotype profile and individual haplotypes between cases and controls. Significant two-way and three-way gene-gene interactions between ACE I/D, AT1R A1166C polymorphisms and AGT gene haplotypes were detected. However, subjects carrying both D allele and GGCATC haplotype had an increased risk of CAD (odds ratio=1.63 [1.16-2.29]; P=0.004). We also used haplotype counting to directly estimate the odds ratio of each specific AGT gene haplotype, and found that the effects of haplotypes were markedly different in subgroups with different ACE or AT1R gene genotype. The regression-based haplotype analyses permits simultaneous dectection of multi-locus and multi-gene effects in determining the risk of CAD. We provide the paradigm for genetic studies of complex-trait diseases using candidate genes based on biological pathways.Clinica chimica acta; international journal of clinical chemistry 03/2011; 412(7-8):619-24. · 2.54 Impact Factor