ACS DIVISION OF BIOLOGICAL CHEMISTRY

May 18th, 2012 by Benziane, B., Bjornholm, M., Pirkmajer, S., Austin, R. L., Kotova, O., Violett, B., Zierath, J. R., Chibalin, A. V.

Contraction stimulates Na+,K+-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na+,K+-ATPase activity in skeletal muscle remains to be determined. Short-term stimulation of rat L6 myotubes with the AMPK activator AICAR, activates AMPK and promotes translocation of the Na+,K+-ATPase α1-subunit to the plasma membrane and increases Na+,K+-ATPase activity as assessed by ouabain-sensitive 86Rb+-uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increases AMPK phosphorylation and Na+,K+-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na+,K+-ATPase activity. The effect of AICAR on Na+,K+-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na+,K+-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na+,K+-ATPase α1-subunit dephosphorylation at Ser18, which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na+,K+-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A/PME-1 complex in AMPK-mediated regulation of Na+,K+-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.

May 18th, 2012 by Bogan, J. S., Rubin, B. R., Yu, C., Loffler, M. G., Orme, C. M., Belman, J. P., McNally, L. J., Hao, M., Cresswell, J. A.

To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells, and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes, and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18 kD ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kD, and also as a 54 kD form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST, and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake.

May 18th, 2012 by Peralta, S., Clemente, P., Sanchez-Martinez, A., Calleja, M., Hernandez-Sierra, R., Matsushima, Y., Adan, C., Ugalde, C., Fernandez-Moreno, M. A., Kaguni, L. S., Garesse, R.

In Drosophila melanogaster, the mitochondrial transcription factor B1 (d-mtTFB1) transcript contains in its 5-untranslated region a conserved upstream Open Reading Frame (uORF) denoted as CG42630 in FlyBase. We demonstrate that CG42630 encodes a novel protein: the Coiled-Coil Domain Containing protein 56, CCDC56, conserved in metazoans. We show that Drosophila CCDC56 protein localizes to mitochondria and contains 87 amino acids in flies and 106 in humans, with the two proteins sharing 42% amino acid identity. We show by Rapid Amplification of cDNA Ends (RACE) and Northern-blotting that Drosophila CCDC56 protein and mtTFB1 are encoded on a bona fide bicistronic transcript. We report the generation and characterization of two ccdc56 knockout lines in Drosophila carrying the ccdc56D6 and ccdc56D11 alleles. Lack of the CCDC56 protein in flies induces a developmental delay and 100% lethality by arrest of larval development at the third instar. ccdc56 knockout larvae show a significant decrease in the level of fully-assembled cytochrome c oxidase (COX) and in its activity, suggesting a defect in complex assembly; the activity of the other Oxidative Phosphorylation (OXPHOS) complexes remained either unaffected or increased in the ccdc56 knockout larvae. The lethal phenotype and the decrease in COX were rescued partially by reintroduction of a wild-type UAS-CCDC56 transgene. These results indicate an important role for CCDC56 in the OXPHOS system and in particular in COX function, required for proper development in Drosophila melanogaster. We propose CCDC56 as a candidate factor required for COX biogenesis/ assembly.

May 18th, 2012 by Amy Rhoden Smith, Brian A. Ikkanda, Garen G. Holman and Brent L. Iverson

May 18th, 2012 by Manuel Tsiang, Anita Niedziela-Majka, Magdeleine Hung, Debi Jin, Eric Hu, Stephen Yant, Dharmaraj Samuel, Xiaohong Liu and Roman Sakowicz

May 18th, 2012 by Ng, K.-S., Leung, H.-W., Wong, P. T.-H., Low, C.-M.

Thrombolysis using tissue plasminogen activator (tPA) has been the key treatment for patients with acute ischemic stroke for the past decade. Recent studies, however, suggest that this clot-busting protease also plays various roles in brain physiological and pathophysiological glutamatergic-dependent processes, such as synaptic plasticity and neurodegeneration. In addition, increasing evidence implicate tPA as an important neuromodulator of the N-methyl-D-aspartate (NMDA) receptors. Here, we demonstrate that recombinant human tPA cleaves the NR2B subunit of NMDA receptor. Analysis of NR2B in rat brain lysates and cortical neurons treated with tPA revealed concentration- and time-dependent degradation of NR2B proteins. Peptide sequencing studies performed on the cleaved-off products obtained from the tPA treatment on a recombinant fusion protein of the amino-terminal domain (ATD) of NR2B revealed that tPA-mediated cleavage occurred at arginine 67 (Arg67). This cleavage is tPA-specific, plasmin-independent and removes a predicted ~4 kDa fragment (Arg27-Arg67) from the ATD of the NR2B protein. Site-directed mutagenesis of putative cleavage site Arg67 to Ala67 impeded tPA-mediated degradation of recombinant protein. This analysis revealed that NR2B is a novel substrate of tPA and suggested that an Arg27-Arg67 truncated NR2B-containing NMDA receptor could be formed. Heterologous expression of NR2B with Gln29-Arg67 deleted is functional but exhibits reduced ifenprodil inhibition and increased glycine EC50 with no change in glutamate EC50. Our results confirmed NR2B as a novel proteolytic substrate of tPA where tPA may directly interact with NR2B subunits leading to a change in pharmacological properties of NR2B-containing NMDA receptors.

May 18th, 2012 by Nisar, S. P., Cunningham, M., Saxena, K., Pope, R. J., Kelly, E., Mundell, S. J.

We have recently shown in a patient with mild bleeding that the PDZ- binding motif of the platelet G protein-coupled P2Y12 receptor (P2Y12R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ-motif binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y12R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y12R internalization. In vitro and prior to agonist stimulation P2Y12R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR in order to facilitate effective NHERF1-dependent receptor internalization.

May 18th, 2012 by Peters, S. J., Smales, C. M., Henry, A. J., Stephens, P. E., West, S., Humphreys, D. P.

The integrity of antibody structure, stability and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulphide bond arrangement of an IgG4 molecule by mutation of the Cys at the N-terminus of the CH1 domain (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227-230) at the C-terminus of the CH1 domain. An inter LC-CH1 disulphide bond is thus formed which mimics the disulphide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity and purified product was analysed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter LC-CH1 disulphide bond with a Cys when present at several positions on the upper hinge (positions 227-230) and that such engineered disulphide bonds can consequently increase the Fab domain thermal stability between 3oC and 6.8oC. The IgG4 disulphide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulphide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.

May 18th, 2012 by Bena, S., Brancaleone, V., Wang, J. M., Perretti, M., Flower, R. J.

Understanding how pro-resolving agonists selectively activate FPR2/ALX is a crucial step in the clarification of pro-resolution molecular networks that can be harnessed for the design of novel therapeutics for inflammatory disease. FPR2/ALX, a G protein coupled receptor (GPCR) belonging to the formyl peptide receptor (FPR) family conveys the biological functions of a variety of ligands including the pro-resolution mediators Annexin A1 (AnxA1) and lipoxin A4 as well as the activating and pro-inflammatory protein serum amyloid A (SAA). FPR2/ALX is the focus of intense screening for novel anti-inflammatory therapeutics, and the small molecule compound 43 (C43) was identified as a receptor ligand. Here we used chimaeric FPR1 and FPR2/ALX clones, stably transfected in HEK293 cells, to identify the N-terminal region and II extracellular loop as the FPR2/ALX domain required for AnxA1-mediated signalling. Genomic responses were also assessed with domain-specific effects emerging, so that the N-terminal region is required for AnxA1-induction of JAG1 and JAM3, whereas it is dispensable for modulation of SGPP2. By comparison, SAA non-genomic responses were reliant on extracellular loops I and II, while the small molecule C43 activated the I extracellular loop with downstream signalling dependent on II trans-membrane region. In desensitization experiments, the N-terminal region was dispensable for AnxA1-induced FPR2/ALX downregulation both in homo- and hetero-desensitization mode.

May 18th, 2012 by van der Veen, J. N., Lingrell, S., Vance, D. E.

The increased prevalence of obesity and diabetes in human populations can induce the deposition of fat (triacylglycerol) in the liver (steatosis). The current view is that most hepatic triacylglycerols are derived from fatty acids released from adipose tissue. In this article we show that phosphatidylcholine (PC), an important structural component of cell membranes and plasma lipoproteins, can be a precursor of ~65% of the triacylglycerols in liver. Mice were injected with [3H]PC-labeled high density lipoproteins (HDL). Hepatic uptake of HDL-PC was ~10 micromol/day, similar to the rate of hepatic de novo PC synthesis. Consistent with this finding, measurement of the specific radioactivity of PC in plasma and liver indicated that 50% of hepatic PC is derived from the circulation. Moreover, one-third of HDL-derived PC was converted into triacylglycerols. Importantly, ~65% of the total hepatic pool of triacylglycerol appears to be derived from hepatic PC, half of which is derived from HDL. Thus, lipoprotein-associated PC should be considered a quantitatively significant source of triacylglycerol for the etiology of hepatic steatosis.