Reidar Wallin

Matrix Gla protein (MGP) and bone morphogenetic protein-2 in aortic calcified lesions of aging rats.

Summary.  The vitamin K‐dependent protein, matrix Gla protein (MGP) is a binding protein for bone morphogenetic protein‐2 (BMP‐2). Here we present additional evidence that the Ca2+‐induced conformer of the vitamin K‐dependent Gla region in MGP is involved in BMP‐2 binding. Recombinant BMP‐2 binds to the Gla‐containing region of MGP in the presence of Ca2+. Immunohistochemistry showed that calcified lesions in the aortic wall of aging rats contained elevated concentrations of MGP that was poorly γ‐carboxylated and did not bind BMP‐2. In contrast, we were able to identify glandular structures in the mucosa of the rat nasal septum that gave bright fluorescent signals with both antigens; confocal microscopy confirmed their colocalization. These results demonstrate that the BMP‐2/MGP complex exists in vivo, consistent with a role for MGP as a BMP‐2 inhibitor. Age‐related arterial calcification may be a consequence of under‐γ‐carboxylation of MGP, allowing unopposed BMP‐2 activity.

Phosphatidic Acid-mediated Phosphorylation of the NADPH Oxidase Component p47-phox EVIDENCE THAT PHOSPHATIDIC ACID MAY ACTIVATE A NOVEL PROTEIN KINASE

Phosphatidic acid (PA), generated by phospholipase D activation, has been linked to the activation of the neutrophil respiratory burst enzyme, NADPH oxidase; however, the intracellular enzyme targets for PA remain unclear. We have recently shown (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995)Proc. Natl. Acad. Sci. U. S. A. 92, 7931–7935) that a PA-activated protein kinase is involved in the activation of NADPH oxidase in a cell-free system. This protein kinase phosphorylates numerous endogenous proteins, including p47-phox, a component of the NADPH oxidase complex. Phospholipids other than PA were less effective at inducing endogenous protein phosphorylation. Several of these endogenous substrates were also phosphorylated during stimulation of intact cells by opsonized zymosan, an agonist that induces phospholipase D activation. We sought to identify the PA-activated protein kinase that phosphorylates p47-phox. The PA-dependent protein kinase was shown to be cytosolic.cis-Unsaturated fatty acids were poor inducers of protein kinase activity, suggesting that the PA-activated protein kinase is not a fatty acid-regulated protein kinase (e.g. protein kinase N). Chromatographic techniques separated the PA-activated protein kinase from a number of other protein kinases known to be activated by PA or to phosphorylate p47-phox. These included isoforms of protein kinase C, p21 (Cdc42/Rac)-activated protein kinase, and mitogen-activated protein kinase. Gel filtration chromatography indicated that the protein kinase has an apparent molecular size of 125 kDa. Screening of cytosolic fractions from several cell types and rat brain suggested the enzyme has widespread cell and tissue distribution. The partially purified protein kinase was sensitive to the same protein kinase inhibitors that diminished NADPH oxidase activation and was independent of guanosine 5′-3-O-(thio)triphosphate and Ca2+. Phosphoamino acid analysis showed that serine and tyrosine residues were phosphorylated on p47-phox by this kinase(s). These data indicate that one or more potentially novel protein kinases are targets for PA in neutrophils and other cell types. Furthermore, a PA-activated protein kinase is likely to be an important regulator of the neutrophil respiratory burst by phosphorylation of the NADPH oxidase component p47-phox.

Assembly of the Warfarin-sensitive Vitamin K 2,3-Epoxide Reductase Enzyme Complex in the Endoplasmic Reticulum Membrane

γ-Carboxylation of vitamin K-dependent proteins requires a functional vitamin K cycle to produce the active vitamin K cofactor for the γ-carboxylase which posttranslationally modifies precursors of these proteins to contain γ-carboxyglutamic acid residues. The warfarin-sensitive enzyme vitamin K epoxide reductase (VKOR) of the cycle reduces vitamin K 2,3-epoxide to the active vitamin K hydroquinone cofactor. Because of the importance of warfarin as an anticoagulant in prophylactic medicine and as a poison in rodent pest control, numerous attempts have been made to understand the molecular mechanism underlying warfarin-sensitive vitamin K 2,3-epoxide reduction. In search for protein components that could be involved in this reaction we designed an in vitro γ-carboxylation test system where the warfarin-sensitive VKOR produces the cofactor for the γ-carboxylase. Dissection of this system by chromatographic techniques has identified a member(s) of the glutathione S-transferase gene family as one component of the VKOR enzyme complex in the endoplasmic reticulum membrane. The affinity-purified glutathioneS-transferase(s) was sensitive to warfarin but lost its warfarin sensitivity and glutathione S-transferase activity upon association with lipids in the presence of Mn2+ or Ca2+. In the γ-carboxylation test system, loss of warfarin-sensitive glutathione S-transferase activity coincided with formation of the VKOR enzyme complex. It is proposed that formation of VKOR in the endoplasmic reticulum membrane resembles formation of the lipoxygenase enzyme complex where the glutathioneS-transferase-related FLAP protein binds cytosolic lipoxygenase to form a membrane enzyme complex.

A novel protein kinase target for the lipid second messenger phosphatidic acid

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.

A molecular mechanism for genetic warfarin resistance in the rat

Warfarin targets vitamin K 2,3‐epoxide reductase (VKOR), the enzyme that produces reduced vitamin K, a required cofactor for γ‐carboxylation of vitamin K‐dependent proteins. To identify VKOR, we used 4′‐azido‐warfarin‐3H‐alcohol as an affinity label. When added to a partially purified preparation of VKOR, two proteins were identified by mass spectrometry as calumenin and cytochrome B5. Rat calumenin was cloned and sequenced and the recombinant protein was produced. When added to an in vitro test system, the 47 kDa recombinant protein was found to inhibit VKOR activity and to protect the enzyme from warfarin inhibition. Calumenin was also shown to inhibit the overall activity of the complete vitamin K‐dependent γ‐carboxylation system. The results were repeated in COS‐1 cells overexpressing recombinant calumenin. By comparing calumenin mRNA levels in various tissues from normal rats and warfarin‐resistant rats, only the livers from resistant rats were different from normal rats by showing increased levels. Partially purified VKOR from resistant and normal rat livers showed no differences in Km‐values, specific activity, and sensitivity to warfarin. A novel model for genetic warfarin resistance in the rat is proposed, whereby the concentration of calumenin in liver determines resistance.

A Novel Branched-chain Amino Acid Metabolon PROTEIN-PROTEIN INTERACTIONS IN A SUPRAMOLECULAR COMPLEX

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B6-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain α-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain α-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain α-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a KD of 2.8 μm. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, kcat values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.

A phosphatidic acid-activated protein kinase and conventional protein kinase C isoforms phosphorylate p22(phox), an NADPH oxidase component.

Using a phosphorylation-dependent cell-free system to study NADPH oxidase activation (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931–7935), we previously showed that p47 phox , a cytosolic NADPH oxidase component, is phosphorylated. Now, we show that p22 phox , a subunit of the NADPH oxidase component flavocytochrome b 558, also is phosphorylated. Phosphorylation is selectively activated by phosphatidic acid (PA) versus other lipids and occurs on a threonine residue in p22 phox . We identified two protein kinase families capable of phosphorylating p22 phox : 1) a potentially novel, partially purified PA-activated protein kinase(s) known to phosphorylate p47 phox and postulated to mediate the phosphorylation-dependent activation of NADPH oxidase by PA and 2) conventional, but not novel or atypical, isoforms of protein kinase C (PKC). In contrast, all classes of PKC isoforms could phosphorylate p47 phox . In a gel retardation assay both the phosphatidic acid-dependent kinase and conventional PKC isoforms phosphorylated all molecules of p22 phox . These findings suggest that phosphorylation of p22 phox by conventional PKC and/or a novel PA-activated protein kinase regulates the activation/assembly of NADPH oxidase.

Branched-chain amino acids and neurotransmitter metabolism: Expression of cytosolic branched-chain aminotransferase (BCATc) in the cerebellum and hippocampus

In the brain, catabolism of the branched‐chain amino acids (BCAAs) provides nitrogen for the synthesis of glutamate and glutamine. Glutamate is formed through transfer of an amino group from BCAA to α‐ketoglutarate in reaction catalyzed by branched‐chain aminotransferases (BCAT). There are two isozymes of BCAT: cytosolic BCATc, which is found in the nervous system, ovary, and placenta, and mitochondrial BCATm, which is found in all organs except rat liver. In cell culture systems, BCATc is found only in neurons and developing oligodendrocytes, whereas BCATm is the isoform in astroglia. In this study, we used immunohistochemistry to examine the distribution of BCATc in the rat brain, focusing on the well‐known neural architecture of the cerebellum and hippocampus. We show that BCATc is expressed only in neurons in the adult rat brain. In glutamatergic neurons such as granule cells of the cerebellar cortex and of the dentate gyrus, BCATc is localized to axons and nerve terminals. In contrast, in GABAergic neurons such as cerebellar Purkinje cells and hippocampal pyramidal basket cells, BCATc is concentrated in cell bodies. A common function for BCATc in these neurotransmitter systems may be to modulate amounts of glutamate available either for release as neurotransmitter or for use as precursor for synthesis of GABA. Particularly striking in our findings is the strong expression of BCATc in the mossy fiber pathway of the hippocampal formation. This result is discussed in light of the effectiveness of the anticonvulsant drug gabapentin, which is a specific inhibitor of BCATc. J. Comp. Neurol. 477:360–370, 2004.

Vitamin K 2,3-epoxide reductase and the vitamin K-dependent γ-carboxylation system

Vitamin K is an essential cofactor for post translational gamma-carboxylation of vitamin K-dependent coagulation factors. The modification is carried out by a system of integral proteins of the endoplasmic reticulum (ER) membrane where the warfarin sensitive vitamin K 2,3-epoxide reductase (VKOR) produces the reduced hydroquinone form of vitamin K (vit.KH(2)) needed by the gamma-carboxylase as the active cofactor. Currently, we have only limited knowledge about how the system functions at the molecular level. VKOR harbors a thiol red/ox center that is essential for electron transfer leading to vitamin K reduction. Reduction of this center with hydrophilic and hydrophobic trialkylphosphines shows that it is located in a hydrophobic environment which must be accessible by an as yet unidentified in vivo reductant of the center. Furthermore, we have addressed the question of whether VKOR or the gamma-carboxylase is the rate-limiting step in the vitamin K-dependent gamma-caboxylation system. A detailed kinetic analysis of an in vitro preparation of the system was undertaken in which gamma-carboxylation of the carboxylase peptide substrate FLEEL was measured as the gamma-carboxylation capacity of the system. Adding VKOR to the test system increased the gamma-carboxylation capacity of the system suggesting that VKOR is the rate-limiting step in the system. This finding puts VKOR in a central position to regulate biosynthesis of biologically active vitamin K-dependent proteins.

Localization of Tissue Transglutaminase in Human Carotid and Coronary Artery Atherosclerosis: Implications for Plaque Stability and Progression

Although atherosclerosis progresses in an indolent state for decades, the rupture of plaques creates acute ischemic syndromes that may culminate in myocardial infarction and stroke. Mechanical forces and matrix metalloproteinase activity initiate plaque rupture, whereas tissue inhibitors of metalloproteinases have an important (albeit indirect) role in plaque stabilization. In this paper, an enzyme that could directly stabilize the plaque is described. Tissue transglutaminase (TG) catalyzes the formation of ε(γ-glutamyl)lysine isopeptide bonds that are resistant to enzymatic, mechanical, and chemical degradation. We performed immunohistochemistry for TG in atherosclerotic human coronary and carotid arteries. TG was most prominent along the luminal endothelium and in the medium of the vessels with a distribution mirroring that of smooth muscle cells. Variable, often prominent, immunoreactivity for TG was also seen in the intima, especially in regions with significant neovascularization. Additionally, TG was detected in fibrous caps and near the “shoulder regions” of some plaques. A monoclonal antibody to the transglutaminase product ε(γ-glutamyl)lysine isopeptide demonstrated co-localization with TG antigen. Transglutaminase activity was found in 6 of 14 coronary artery atherectomy samples. Cross-linking of TG substrates such as fibrinogen, fibronectin, vitronectin, collagen type I, and protease inhibitors stabilized the plaque. Furthermore, the activation of transforming growth factor–beta-1 by TG might be an additional mechanism for the promotion of plaque stabilization and progression by increasing the synthesis of extracellular matrix components.