Discovery of pyridoxal reductase activity as part of human vitamin B6 metabolism

BackgroundPyridoxal 5′-phosphate (PLP) is the active form of vitamin B6. Mammals cannot synthesize vitamin B6, so they rely on dietary uptake of the different B6 forms, and via the B6 salvage pathway they interconvert them into PLP. Humans possess three enzymes in this pathway: pyridoxal kinase, pyridox(am)ine phosphate oxidase and pyridoxal phosphatase. Besides these, a fourth enzyme has been described in plants and yeast but not in humans: pyridoxal reductase.MethodsWe analysed B6 vitamers in remnant CSF samples of PLP-treated patients and four mammalian cell lines (HepG2, Caco2, HEK293 and Neuro-2a) supplemented with PL as the sole source of vitamin B6.ResultsStrong accumulation of pyridoxine (PN) in CSF of PLP-treated patients was observed, suggesting the existence of a PN-forming enzyme. Our in vitro studies show that all cell lines reduce PL to PN in a time- and dose-dependent manner. We compared the amino acid sequences of known PL reductases to human sequences and found high homology for members of the voltage-gated potassium channel beta subunits and the human aldose reductases. Pharmacological inhibition and knockout of these proteins show that none of the candidates is solely responsible for PL reduction to PN.ConclusionsWe show evidence for the presence of PL reductase activity in humans. Further studies are needed to identify the responsible protein.General significanceThis study expands the number of enzymes with a role in B6 salvage pathway. We hypothesize a protective role of PL reductase(s) by limiting the intracellular amount of free PL and PLP.     

Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1

Background

Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known.

Methodology/Principal Findings

Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus.

Conclusions/Significance

hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.     

Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-binding activities

The leucine-rich repeat kinase 2 (LRRK2) protein has both guanosine triphosphatase (GTPase) and kinase activities, and mutation in either enzymatic domain can cause late-onset Parkinson disease. Nucleotide binding in the GTPase domain may be required for kinase activity, and residues in the GTPase domain are potential sites for autophosphorylation, suggesting a complex mechanism of intrinsic regulation. To further define the effects of LRRK2 autophosphorylation, we applied a technique optimal for detection of protein phosphorylation, electron transfer dissociation, and identified autophosphorylation events exclusively nearby the nucleotide binding pocket in the GTPase domain. Parkinson-disease-linked mutations alter kinase activity but did not alter autophosphorylation site specificity or sites of phosphorylation in a robust in vitro substrate myelin basic protein. Amino acid substitutions in the GTPase domain have large effects on kinase activity, as insertion of the GTPase-associated R1441C pathogenic mutation together with the G2019S kinase domain mutation resulted in a multiplicative increase (∼ 7-fold) in activity. Removal of a conserved autophosphorylation site (T1503) by mutation to an alanine residue resulted in greatly decreased GTP-binding and kinase activities. While autophosphorylation likely serves to potentiate kinase activity, we find that oligomerization and loss of the active dimer species occur in an ATP- and autophosphorylation-independent manner. LRRK2 autophosphorylation sites are overall robustly protected from dephosphorylation in vitro, suggesting tight control over activity in vivo. We developed highly specific antibodies targeting pT1503 but failed to detect endogenous autophosphorylation in protein derived from transgenic mice and cell lines. LRRK2 activity in vivo is unlikely to be constitutive but rather refined to specific responses.     

Dephosphorylation of skeletal muscle phosphorylase,glycogen synthase,and phosphorylase kinase β-subunit by a Mn2+-activated protein phosphatase

An Mn²⁺-activated phosphoprotein phosphatase of M_r = 80,000 from rabbit muscle catalyzes the dephosphorylation of skeletal muscle proteins that are phosphorylated by either phosphorylase kinase or cAMP-dependent protein kinase. Phosphorylase or glycogen synthase labeled by phosphorylase kinase at seryl residues 14 or 7, respectively, are both dephosphorylated by the phosphatase. Phosphorylase a and glycogen synthase compete with one another for the phosphatase. The phosphatase discriminates between different sites labeled by the cAMP-dependent protein kinase: glycogen synthase phosphorylated either to 1.0 or 1.8 mol phosphate/mol, or phosphorylase kinase phosphorylated on its β-subunit serve as substrates for the phosphatase, but the phosphorylase kinase α-subunit, the phosphorylated phosphatase inhibitor 1, or casein do not. Histone fraction IIA, phosphorylated by the catalytic subunit, was a poor substrate even at a concentration of 100 μm. Phosphorylation of the α-subunit of phosphorylase kinase had no influence on the kinetics of dephosphorylation of the β-subunit. Thus, the M_r = 80,000 phosphatase meets the functional definition of a protein phosphatase 1 [Cohen, P. (1978) Curr. Top. Cell. Regul.14, 117–196]. Furthermore, from a comparison of the known phosphorylated sites of these proteins, it appears that the phosphatase discriminates between different sites present in the phosphoproteins tested on the basis of the K_m values for the reactions. It displays a preferential activity toward proteins with a primary structure wherein basic residues are two positions amino-terminal from the phosphoserine, AgrLysX-YSer(P) or LysArgX-YSer(P), rather and one residue away, ArgArgX-Ser(P).     

A search for a mammalian homologue of the Drosophila photoreceptor development gene glass yields Zfp64, a zinc finger encoding gene which maps to the distal end of mouse chromosome 2

Whilst searching for a mammalian homologue of the Drosophila glass gene we cloned a mouse cDNA whose deduced sequence encodes a 614 amino acid (aa) protein with ten Cys2-His2 (C2H2) zinc finger (Zf) motifs. Zfp64 is expressed in all developing and mature mouse tissues examined, except the mouse erythroleukemia (MEL) cell line. Zfp64 maps to the distal region of mouse chromosome 2 close to lens opacity 4 (Lop4), a semidominant cataract mutation. Sequence analysis shows that Zfp64 has multiple potential phosphorylation sites for casein kinase II (CK II), protein kinase C (PKC), tyrosine kinase (TK) and c-AMP- and c-GMP-dependent protein kinase (cA/GMPDPK).     

Integrin-linked kinase can facilitate syncytialization and hormonal differentiation of the human trophoblast-derived BeWo cell line

Background  

In the fusion pathway of trophoblast differentiation, stem villous cytotrophoblast cells proliferate and daughter cells differentiate and fuse with existing syncytiotrophoblast to maintain the multi-nucleated layer. Integrin-linked kinase (ILK) is highly expressed in 1st and 2nd trimester villous cytotrophoblast cells, yet barely detectable in syncytiotrophoblast, thus we examined the potential role of ILK in aiding trophoblast fusion.     

Reevaluation of Phosphorylation Sites in the Parkinson Disease-associated Leucine-rich Repeat Kinase 2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson disease and contribute to sporadic Parkinson disease. LRRK2 is a large complex protein with multiple functional domains, including a Roc-GTPase, protein kinase, and multiple protein-protein interaction domains. Previous studies have suggested an important role for kinase activity in LRRK2-induced neuronal toxicity and inclusion body formation. Disease-associated mutations in LRRK2 also tend to increase kinase activity. Thus, enhanced kinase activity may therefore underlie LRRK2-linked disease. Similar to the closely related mixed-lineage kinases, LRRK2 can undergo autophosphorylation in vitro. Three putative autophosphorylation sites (Thr-2031, Ser-2032, and Thr-2035) have been identified within the activation segment of the LRRK2 kinase domain based on sequence homology to mixed-lineage kinases. Phosphorylation at one or more of these sites is critical for the kinase activity of LRRK2. Sensitive phopho-specific antibodies to each of these three sites have been developed and validated by ELISA, dot-blot, and Western blot analysis. Using these antibodies, we have found that all three putative sites are phosphorylated in LRRK2, and Ser-2032 and Thr-2035 are the two important sites that regulate LRRK2 kinase activity.     

Mono-(2-Ethylhexyl) Phthalate Promotes Pro-Labor Gene Expression in the Human Placenta

Women exposed to phthalates during pregnancy are at increased risk for delivering preterm, but the mechanism behind this relationship is unknown. Placental corticotropin-releasing hormone (CRH) and cyclooxygenase-2 (COX-2) are key mediators of parturition and are regulated by the non-canonical NF-kB (RelB/p52) signaling pathway. In this study, we demonstrate that one of the major phthalate metabolites, mono-(2-ethylhexyl)-phthalate (MEHP), increased CRH and COX-2 mRNA and protein abundance in a dose-dependent manner in primary cultures of cytotrophoblast. This was coupled with an increase in nuclear import of RelB/p52 and its association with the CRH and COX-2 promoters. Silencing of NF-kB inducing kinase, a central signaling component of the non-canonical NF-kB pathway, blocked MEHP-induced upregulation of CRH and COX-2. These results suggest a potential mechanism mediated by RelB/p52 by which phthalates could prematurely induce pro-labor gene activity and lead to preterm birth.     

Binding of thiamin thiazolone pyrophosphate to mammalian pyruvate dehydrogenase and its effects of kinase and phosphatase activities.

The pyruvate dehydrogenase component of the bovine kidney pyruvate dehydrogenase complex has two thiamin-PP binding sites per α₂β₂ tetramer. Titration of these binding sites with the transition state analog, thiamin thiazolone pyrophosphate, strongly inhibits phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase and ATP. The analog has little effect, if any, on dephosphorylation of phosphorylated pyruvate dehydrogenase by pyruvate dehydrogenase phosphatase. Phosphorylation of pyruvate dehydrogenase inactivates the enzyme, but does not significantly affect the thiamin-PP binding sites. It appears that phosphorylation produces a conformational change in pyruvate dehydrogenase that displaces a catalytic group (or groups) at the active center.     

Effects of insulin,glucagon and dexamethasone on pyruvate kinase in cultured hepatocytes

Long-term (24–48 h) and short-term (10–30 min) regulation by hormones of hepatic pyruvate kinase activity was investigated in adult rat hepatocytes cultured under serum-free conditions. In the absence of hormones, pyruvate kinase total activity decreased to 83%, 67% and 39% of the initial level at 24, 48 and 72 h of culture. Insulin (100 nM) maintained total activity significantly above control levels throughout this period. In contrast, glucagon (100 nM) and dexamethasone (100 nM) accelerated the gradual decrease within 24 h (glucagon) or 48 h (dexamethasone) of culture. In these long-term experiments, activity at non-saturating concentrations of phosphoenolpyruvate was decreased by glucagon and dexamethasone but not directly modulated by insulin. However, insulin increased the cellular content of the pyruvate kinase activator fructose-1,6-diphosphate. In short-term experiments on cells cultured under serum- and hormone-free conditions for 48 h, both glucagon and dexamethasone independently caused a rapid, dose-dependent increase of the K_0.5 for phosphoenolpyruvate within 10 min, while V_max was not affected. Insulin inhibited this action of glucagon and dexamethasone and, in their absence, significantly increased substrate affinity for phosphoenolpyruvate within 30 min. Cellular fructose-1,6-diphosphate contents remained unchanged under these conditions. The data identify glucocorticoids and insulin - in addition to glucagon - as short-term regulators of the catalytic properties of pyruvate kinase. All three hormones are effective in the long-term control of total enzyme activity.     

Composites of peptide nucleic acids with titanium dioxide nanoparticles. II. Dissociation of DNA/PNA duplexes within TiO2 · polylysine · DNA/PNA nanocomposites and in solution. Effect of polylysine

When creating effective drugs, it is important not only to transport them into cells, but also allow them to be released from the “transporter” after the delivery. It was shown that the dissociation of peptide nucleic acids (PNA) from TiO₂ · PL · DNA/PNA nanocomposites occurred according to a typical thermal denaturation, and polylysine (PL) in the nanocomposite has almost no effect on the dissociation. These data suggest that the immobilization of PNA in the TiO₂ · PL · DNA/PNA nanocomposite is reversible and PNA can be easily released from TiO₂ carrier into solution. In contrast to that, the dissociation of DNA/DNA and DNA/PNA duplexes in physiological solution in the presence of PL was not observed. PL in solution dramatically influences the dependence of the optical density on temperature and time for DNA/DNA duplexes and to a lesser degree for DNA/PNA duplexes. It has been assumed that PL and DNA/DNA duplexes in physiological solutions form triple polycomplexes (DNA/DNA · PL) _m , which can aggregate and precipitate. PL in solution can also interact with DNA/PNA duplexes to form monocomplexes PL · (DNA/PNA) _n consisting of one PL chain and one or more (n) DNA/PNA duplexes. Although these monocomplexes do not precipitate, the dissociation of DNA/PNA duplexes from them is complicated.     

Preparation of poly(ε-lysine) adsorbents and application to selective removal of lipopolysaccharides

To remove endotoxins (lipopolysaccharides; LPS) from cell products used as drugs, water-insoluble poly(ε-lysine) (PL) particles were prepared by cross-linking with PL originating from Streptomyces albulus and chloromethyloxirane (CMO). The apparent pK_a (pK_a,app) and the anion-exchange capacity of the particles were easily adjusted by changing the PL ratio and the CMO ratio. The higher the pK_a,app, the greater the LPS-adsorption capacity of the particles. On the other hand, when the PL ratio (in the particles) increased to 75 unit-mol% or higher, the adsorption of bovine serum albumin by the particles also increased, but decreased with increasing ionic strength of the buffer to μ=0.2 or higher. The adsorption of γ-globulin increased with decreasing PL ratio to 65 unit-mol% or lower. As a result, when the PL ratio was 70 unit-mol% and the pK_a,app was 6.7, the PL/CMO particles selectively removed LPS from various protein solutions that were naturally contaminated with LPS, at pH 6.0 and μ=0.05.     

Autophosphorylated residues involved in the regulation of human chk2 in vitro

Human checkpoint kinase 2 is a major actor in checkpoint activation through phosphorylation by ataxia telangiectasia mutated in response to DNA double-strand breaks. In the absence of de novo DNA damage, its autoactivation, reported in the event of increased Cds1/checkpoint kinase 2 (Chk2) expression, has been attributed to oligomerization. Here we report a study performed on autoactivated recombinant Chk2 proteins that aims to correlate kinase activity and phosphorylation status. Using a fluorescence-based technique to assay human checkpoint kinase 2 catalytic activity, slight differences in the ability to phosphorylate Cdc25C were observed, depending on the recombinant system used. Using mass spectrometry, the phosphorylation sites were mapped to identify sites potentially involved in the kinase activity. Five phosphorylated positions, at Ser120, Ser260, Thr225, Ser379 and Ser435, were found to be common to bacteria and insect cells expression systems. They were present in addition to the six known phosphorylation sites induced by ionizing radiation (Thr68, Thr432, Thr387, Ser516, Ser33/35 and Ser19) detected by immunoblotting. After phosphatase treatment, Chk2 regained activity via autorephosphorylation. The determination of the five common sites and ionizing-radiation-inducible positions as rephosphorylated confirms that they are potential positive regulators of Chk2 kinase activity. For Escherichia coli's most highly phosphorylated 6His-Chk2, 13 additional phosphorylation sites were assigned, including 7 novel sites on top of recently reported phosphorylation sites.