Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health.

The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell-surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X-ray crystal structure of the lysine-specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis.

Crystal structure of human insulin-regulated aminopeptidase with specificity for cyclic peptides.

Insulin-regulated aminopeptidase (IRAP or oxytocinase) is a membrane-bound zinc-metallopeptidase that cleaves neuroactive peptides in the brain and produces memory enhancing effects when inhibited. We have determined the crystal structure of human IRAP revealing a closed, four domain arrangement with a large, mostly buried cavity abutting the active site. The structure reveals that the GAMEN exopeptidase loop adopts a very different conformation from other aminopeptidases, thus explaining IRAP's unique specificity for cyclic peptides such as oxytocin and vasopressin. Computational docking of a series of IRAP-specific cognitive enhancers into the crystal structure provides a molecular basis for their structure-activity relationships and demonstrates that the structure will be a powerful tool in the development of new classes of cognitive enhancers for treating a variety of memory disorders such as Alzheimer's disease.

Phosphorylation regulates copper-responsive trafficking of the Menkes copper transporting P-type ATPase.

The Menkes copper-translocating P-type ATPase (ATP7A) is a critical copper transport protein functioning in systemic copper absorption and supply of copper to cuproenzymes in the secretory pathway. Mutations in ATP7A can lead to the usually lethal Menkes disease. ATP7A function is regulated by copper-responsive trafficking between the trans-Golgi Network and the plasma membrane. We have previously reported basal and copper-responsive kinase phosphorylation of ATP7A but the specific phosphorylation sites had not been identified. As copper stimulates both trafficking and phosphorylation of ATP7A we aimed to identify all the specific phosphosites and to determine whether trafficking and phosphorylation are linked. We identified twenty in vivo phosphorylation sites in the human ATP7A and eight in hamster, all clustered within the N- and C-terminal cytosolic domains. Eight sites were copper-responsive and hence candidates for regulating copper-responsive trafficking or catalytic activity. Mutagenesis of the copper-responsive phosphorylation site Serine-1469 resulted in mislocalization of ATP7A in the presence of added copper in both polarized (Madin Darby canine kidney) and non-polarized (Chinese Hamster Ovary) cells, strongly suggesting that phosphorylation of specific serine residues is required for copper-responsive ATP7A trafficking to the plasma membrane. A constitutively phosphorylated site, Serine-1432, when mutated to alanine also resulted in mislocalization in the presence of added copper in polarized Madin Darby kidney cells. These studies demonstrate that phosphorylation of specific serine residues in ATP7A regulates its sub-cellular localization and hence function and will facilitate identification of the kinases and signaling pathways involved in regulating this pivotal copper transporter.

AMPK-independent pathways regulate skeletal muscle fatty acid oxidation.

The activation of AMP-activated protein kinase (AMPK) and phosphorylation/inhibition of acetyl-CoA carboxylase 2 (ACC2) is believed to be the principal pathway regulating fatty acid oxidation. However, during exercise AMPK activity and ACC Ser-221 phosphorylation does not always correlate with rates of fatty acid oxidation. To address this issue we have investigated the requirement for skeletal muscle AMPK in controlling aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside (AICAR) and contraction-stimulated fatty acid oxidation utilizing transgenic mice expressing a muscle-specific kinase dead (KD) AMPK alpha2. In wild-type (WT) mice, AICAR and contraction increased AMPK alpha2 and alpha1 activities, the phosphorylation of ACC2 and rates of fatty acid oxidation while tending to reduce malonyl-CoA levels. Despite no activation of AMPK in KD mice, ACC2 phosphorylation was maintained, malonyl-CoA levels were reduced and rates of fatty acid oxidation were comparable between genotypes. During treadmill exercise both KD and WT mice had similar values of respiratory exchange ratio. These studies suggested the presence of an alternative ACC2 kinase(s). Using a phosphoproteomics-based approach we identified 18 Ser/Thr protein kinases whose phosphorylation was increased by greater than 25% in contracted KD relative to WT muscle. Utilizing bioinformatics we predicted that extracellular regulated protein-serine kinase (ERK1/2), inhibitor of nuclear factor (NF)-kappaB protein-serine kinase beta (IKKbeta) and protein kinase D (PKD) may phosphorylate ACC2 at Ser-221 but during in vitro phosphorylation assays only AMPK phosphorylated ACC2. These data demonstrate that AMPK is not essential for the regulation of fatty acid oxidation by AICAR or muscle contraction.

AMP-activated protein kinase subunit interactions: beta1:gamma1 association requires beta1 Thr-263 and Tyr-267.

AMP-activated protein kinase (AMPK) plays multiple roles in the body's overall metabolic balance and response to exercise, nutritional stress, hormonal stimulation, and the glucose-lowering drugs metformin and rosiglitazone. AMPK consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, each with multiple isoforms that form active 1:1:1 heterotrimers. Here we show that recombinant human AMPK alpha1beta1gamma1 expressed in insect cells is monomeric and displays specific activity and AMP responsiveness similar to rat liver AMPK. The previously determined crystal structure of the core of mammalian alphabetagamma complex shows that beta binds alpha and gamma. Here we show that a beta1(186-270)gamma1 complex can form in the absence of detectable alpha subunit. Moreover, using alanine mutagenesis we show that beta1 Thr-263 and Tyr-267 are required for betagamma association but not alphabeta association.

Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling.

Elevated levels of tumor necrosis factor (TNFalpha) are implicated in the development of insulin resistance, but the mechanisms mediating these chronic effects are not completely understood. We demonstrate that TNFalpha signaling through TNF receptor (TNFR) 1 suppresses AMPK activity via transcriptional upregulation of protein phosphatase 2C (PP2C). This in turn reduces ACC phosphorylation, suppressing fatty-acid oxidation, increasing intramuscular diacylglycerol accumulation, and causing insulin resistance in skeletal muscle, effects observed both in vitro and in vivo. Importantly even at pathologically elevated levels of TNFalpha observed in obesity, the suppressive effects of TNFalpha on AMPK signaling are reversed in mice null for both TNFR1 and 2 or following treatment with a TNFalpha neutralizing antibody. Our data demonstrate that AMPK is an important TNFalpha signaling target and is a contributing factor to the suppression of fatty-acid oxidation and the development of lipid-induced insulin resistance in obesity.

Differential calcineurin signalling activity and regeneration efficacy in diaphragm and limb muscles of dystrophic mdx mice.

Calcineurin activity is essential for successful skeletal muscle regeneration in young mdx mice and in wild type mice following myotoxic injury and cryodamage. In mature myofibres of adult mdx mice, calcineurin stimulation can ameliorate the dystrophic pathology. The aim of this study was to test the hypothesis that the more severe dystrophic pathology of the diaphragm compared with hindlimb muscles of mdx mice could be attributed to aberrant calcineurin signalling and that due to ongoing regeneration calcineurin activity would be greater in muscles of adult mdx than wild type mice. Differences in markers of regeneration between tibialis anterior and diaphragm muscles were also characterised, to determine whether there was an association between regeneration efficacy and calcineurin activity in dystrophic muscles. In diaphragm muscles of adult mdx mice, the proportion of centrally nucleated fibres and developmental myosin heavy chain protein expression was lower and myogenin protein expression was higher than in tibialis anterior muscles. Calcineurin and activated NFATc1 protein content and calcineurin phosphatase activity were higher in muscles from mdx than wild type mice and calcineurin activation was greater in diaphragm than tibialis anterior muscles of mdx mice. Thus, despite greater calcineurin activity in diaphragm compared to hindlimb muscles, regeneration events downstream of myoblast differentiation and mediated by the injured myofibre were severely compromised.

AMP-activated protein kinase beta subunit tethers alpha and gamma subunits via its C-terminal sequence (186-270).

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable alphabetagamma heterotrimer comprising a catalytic alpha and two non-catalytic subunits, beta and gamma. The beta subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here we find that the conserved C-terminal 85-residue sequence of the beta subunit, beta1-(186-270), is sufficient to form an active AMP-dependent heterotrimer alpha1beta1-(186-270)-gamma1, whereas the 25-residue beta1 C-terminal (246-270) sequence is sufficient to bind gamma1, gamma2, or gamma3 but not the alpha subunit. Deletion of the beta C-terminal Ile-270 precludes betagamma association in the absence of the alpha subunit, but the presence of the alpha subunit or substitution of Ile-270 with Ala or Glu restores betagamma binding. Truncation of the alpha subunit reveals that beta1 binding requires the alpha1-(313-473) sequence. The conserved C-terminal 85-residue sequence of the beta subunit (90% between beta1 and beta2) is the primary alphagamma binding sequence responsible for the formation of the AMPK alphabetagamma heterotrimer.

Effect of exercise on protein kinase C activity and localization in human skeletal muscle.

To investigate the effect of exercise on protein kinase C (PKC) activity and localization in human skeletal muscle, eight healthy men performed cycle ergometer exercise for 40 min at 76 +/- 1% the peak pulmonary O(2) uptake , with muscle samples obtained at rest and after 5 and 40 min of exercise. PKC expression, phosphorylation and activities were examined by immunoblotting and in vitro kinase assays of fractionated and whole tissue preparations. In response to exercise, total PKC activity was slightly higher at 40 min in an enriched membrane fraction, and using a pSer-PKC-substrate motif antibody it was revealed that exercise increased the serine phosphorylation of an approximately 50 kDa protein. There were no changes in conventional PKC (cPKC) or PKC activities; however, atypical PKC (aPKC) activity was approximately 70% higher at 5 and 40 min, and aPKC expression and Thr(410/403) phosphorylation were unaltered by exercise. There were no effects of exercise on the abundance of PKCalpha, PKCdelta, PKC and aPKC within cytosolic or enriched membrane fractions of skeletal muscle. These data indicate that aPKC, but not cPKC or PKC, are activated by exercise in contracting muscle suggesting a potential role for aPKC in the regulation of skeletal muscle function and metabolism during exercise in humans.

AMPK beta subunit targets metabolic stress sensing to glycogen.

AMP-activated protein kinase (AMPK) is a multisubstrate enzyme activated by increases in AMP during metabolic stress caused by exercise, hypoxia, lack of cell nutrients, as well as hormones, including adiponectin and leptin. Furthermore, metformin and rosiglitazone, frontline drugs used for the treatment of type II diabetes, activate AMPK. Mammalian AMPK is an alphabetagamma heterotrimer with multiple isoforms of each subunit comprising alpha1, alpha2, beta1, beta2, gamma1, gamma2, and gamma3, which have varying tissue and subcellular expression. Mutations in the AMPK gamma subunit cause glycogen storage disease in humans, but the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. We show that the AMPK beta subunit contains a functional glycogen binding domain (beta-GBD) that is most closely related to isoamylase domains found in glycogen and starch branching enzymes. Mutation of key glycogen binding residues, predicted by molecular modeling, completely abolished beta-GBD binding to glycogen. AMPK binds to glycogen but retains full activity. Overexpressed AMPK beta1 localized to specific mammalian subcellular structures that corresponded with the expression pattern of glycogen phosphorylase. Glycogen binding provides an architectural link between AMPK and a major cellular energy store and juxtaposes AMPK to glycogen bound phosphatases.

Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism.

Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser(1179) in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser(116), Thr(497), and Ser(635). Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser(635), which was consistently slower than that at Ser(1179). Thr(497) was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser(116). Whereas shear-stimulated Ser(635) phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser(635) may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.

Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635.

Endothelial nitric-oxide synthase (eNOS) is regulated by signaling pathways involving multiple sites of phosphorylation. The coordinated phosphorylation of eNOS at Ser(1179) and dephosphorylation at Thr(497) activates the enzyme, whereas inhibition results when Thr(497) is phosphorylated and Ser(1179) is dephosphorylated. We have identified two further phosphorylation sites, at Ser(617) and Ser(635), by phosphopeptide mapping and matrix-assisted laser desorption ionization time of flight mass spectrometry. Purified protein kinase A (PKA) phosphorylates both sites in purified eNOS, whereas purified Akt phosphorylates only Ser(617). In bovine aortic endothelial cells, bradykinin (BK), ATP, and vascular endothelial growth factor stimulate phosphorylation of both sites. BK-stimulated phosphorylation of Ser(617) is Ca(2+)-dependent and is partially inhibited by LY294002 and wortmannin, phosphatidylinositol 3-kinase inhibitors, suggesting signaling via Akt. BK-stimulated phosphorylation of Ser(635) is Ca(2+)-independent and is completely abolished by the PKA inhibitor, KT5720, suggesting signaling via PKA. Activation of PKA with isobutylmethylxanthine also causes Ser(635), but not Ser(617), phosphorylation. Mimicking phosphorylation at Ser(635) by Ser to Asp mutation results in a greater than 2-fold increase in activity of the purified protein, whereas mimicking phosphorylation at Ser(617) does not alter maximal activity but significantly increases Ca(2+)-calmodulin sensitivity. These data show that phosphorylation of both Ser(617) and Ser(635) regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process.