Myosin IIb-dependent regulation of actin dynamics is required for NMDA receptor trafficking during synaptic plasticity [Cell Biology]

September 1st, 2015 by

N-methyl-D-aspartate receptor (NMDAR) synaptic incorporation changes the number of NMDARs at synapses and is thus critical to various NMDAR-dependent brain functions. To date, the molecules involved in NMDAR trafficking and the underlying mechanisms are poorly understood. Here, we report that myosin IIb is an essential molecule in NMDAR synaptic incorporation during protein kinase C (PKC) or theta burst stimulation (TBS)-induced synaptic plasticity. Moreover, we demonstrate that myosin light chain kinase(MLCK)-dependent actin reorganization contributes to NMDAR trafficking. The findings from additional mutual occlusion experiments demonstrate that PKC and MLCK share a common signaling pathway in NMDAR-mediated synaptic regulation. Because myosin IIb is the primary substrate of MLCK and can regulate actin dynamics during synaptic plasticity, we propose that the MLCK- and myosin IIb-dependent regulation of actin dynamics is required for NMDAR trafficking during synaptic plasticity. This study provides important insights into a mechanical framework for understanding NMDAR trafficking associated with synaptic plasticity.

Disruption of Rhodopsin Dimerization with Synthetic Peptides Targeting an Interaction Interface [Membrane Biology]

September 1st, 2015 by

Though homo- and heterodimerization of G protein-coupled receptors (GPCRs) are well documented, GPCR monomers could assemble in different ways thus causing variations in the interactive interface between receptor monomers among different GPCRs. Moreover, the functional consequences of this phenomenon remain to be clarified and could be specific for different GPCRs. Synthetic peptides derived from transmembrane (TM) domains can interact with a full length GPCR, blocking dimer formation and affecting its function. Here we used peptides corresponding to TM helices of bovine rhodopsin (Rho) to investigate the Rho dimer interface and functional consequences of its disruption. Incubation of Rho with TM1, TM2, TM4 and TM5 peptides in rod outer segment (ROS) membranes shifted the resulting detergent-solubilized protein migration through a gel filtration column towards smaller molecular masses with a reduced propensity for dimer formation in a crosslinking reaction. Binding of these TM peptides to Rho was characterized by both mass spectrometry and a label-free assay, from which dissociation constants were calculated. A bioluminescence resonance energy transfer (BRET) assay revealed that the physical interaction between Rho molecules expressed in membranes of living cells was blocked by the same four TM peptides identified in our in vitro experiments. Though disruption of the Rho dimer/oligomer had no effect on the rates of G protein activation, binding of Gt to the activated receptor stabilized the dimer. However, TM peptide-induced disruption of dimer/oligomer decreased receptor stability, suggesting that Rho supramolecular organization could be essential for ROS stabilization and receptor trafficking.

The Cell Division Protein FtsZ from Streptococcus pneumoniae Exhibits a GTPase Activity Delay [Cell Biology]

September 1st, 2015 by

The cell division protein FtsZ assembles in vitro by a mechanism of cooperative association dependent on GTP, monovalent cations and Mg2+. We have analyzed the GTPase activity and assembly dynamics of Streptococcus pneumoniae FtsZ (SpnFtsZ). SpnFtsZ assembled in an apparently cooperative process, with a higher critical concentration than values reported for other FtsZ proteins. It sedimented in the presence of GTP as a high molecular mass polymer with a well-defined size and tended to form double-stranded filaments in electron microscope preparations. GTPase activity depended on K+ and Mg2+ and was inhibited by Na+. GTP hydrolysis exhibited a delay that included a lag phase followed by a GTP hydrolysis activation step, untill reaction reached the GTPase rate. The lag phase was not found in polymer assembly, suggesting a transition from an initial non-GTP-hydrolyzing polymer that switches to a GTP-hydrolyzing polymer, supporting models that explain FtsZ polymer cooperativity.

Bacteriophage-mediated glucosylation can modify lipopolysaccharide O antigens synthesized by an ABC transporter-dependent assembly mechanism [Glycobiology and Extracellular Matrices]

September 1st, 2015 by Mann, E., Ovchinnikova, O. G., King, J. D., Whitfield, C.

Lysogenic bacteriophages may encode enzymes that modify the structures of lipopolysaccharide O antigen glycans, altering the structure of the bacteriophage receptor, and resulting in serotype-conversion. This can enhance virulence and has implications for antigenic diversity and vaccine development. Side-chain glucosylation is a common modification strategy found in a number of bacterial species. To date, glucosylation has only been observed in O antigens synthesized by Wzy-dependent pathways, one of the two most prevalent O-antigen synthesis systems. Here we exploited a heterologous system to study the glucosylation-potential of a model O antigen produced in an ABC transporter-dependent system. Although O-antigen production is cryptic in E. coli K-12, due to a mutation in the synthesis genes, it possesses a prophage-glucosylation cluster, which modifies the GlcNAc residue in an α-L-Rha-(1→3)-D-GlcNAc motif found in the original O16 antigen. Raoultella terrigena ATCC 33257 produces an O antigen possessing the same disaccharide motif but its assembly uses an ABC transporter-dependent system. E. coli harboring the R. terrigena O-antigen biosynthesis genes produced an O antigen displaying reduced reactivity towards antisera raised against the native R. terrigena repeat structure, indicative of an altered chemical structure. Structural determination using NMR revealed the addition of glucose side-chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from the E. coli chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways.
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S-Nitrosylation induces both autonomous activation and inhibition of calciumcalmodulin dependent protein kinase II delta [Cell Biology]

August 27th, 2015 by

Nitric oxide (NO) is known to modulate calcium handling and cellular signaling in the myocardium, but key targets for NO in the heart remain unidentified. Recent reports have implicated that NO can activate calcium/calmodulin (Ca2+/CaM) dependent protein kinase II (CaMKII) in neurons and heart. Here, we use our novel sensor of CaMKII activation, Camui, to monitor changes in conformation and activation of cardiac CaMKII (CaMKIIδ) activity after treatment with the NO donor S-nitrosoglutathione (GSNO). We demonstrate that exposure to NO after Ca2+/CaM binding to CaMKIIδ results in autonomous kinase activation, which is abolished by mutation of the C290 site. However, exposure of CaMKIIδ to GSNO prior to Ca2+/CaM exposure strongly suppresses kinase activation and conformational change by Ca2+/CaM. This NO-induced inhibition was ablated by mutation of the C273 site. We found parallel effects of GSNO on CaM/CaMKIIδ binding and CaMKIIδ-dependent ryanodine receptor activation in adult cardiac myocytes. We conclude that NO can play a dual role in regulating cardiac CaMKIIδ activity.

Host cell-catalyzed S-palmitoylation mediates Golgi targeting of the Legionella ubiquitin ligase GobX [Cell Biology]

August 27th, 2015 by

The facultative intracellular pathogen Legionella pneumophila, the causative agent of Legionnaires' disease, infects and replicates within human alveolar macrophages. L. pneumophila delivers almost 300 effector proteins into the besieged host cell that alter signaling cascades and create conditions that favor intracellular bacterial survival. In order for the effectors to accomplish their intracellular mission, their activity needs to be specifically directed towards the correct host cell protein or target organelle. Here, we show that the L. pneumophila effector GobX possesses E3 ubiquitin ligase activity that is mediated by a central region homologous to mammalian U-box domains. Furthermore, we demonstrate that GobX exploits host cell S-palmitoylation in order to specifically localize to Golgi membranes. The hydrophobic palmitate moiety is covalently attached to a cysteine residue at position 175, which is part of an amphipathic alpha helix within the carboxy-terminal region of GobX. Site-directed mutagenesis of cysteine-175 or residues on the hydrophobic face of the amphipathic helix strongly attenuated palmitoylation and Golgi localization of GobX. Together, our study provides evidence that the L. pneumophila effector GobX exploits two post-translational modification pathways of host cells, ubiquitination and S-palmitoylation.

Modelling of the N-terminal section and the lumenal loop of trimeric light harvesting complex II (LHCII) by using EPR [Plant Biology]

August 27th, 2015 by

The major light harvesting complex II (LHCII) of green plants plays a key role in the absorption of sunlight, the regulation of photo-synthesis, and in preventing photo-damage by excess light. The latter two functions are thought to involve the lumenal loop and the N-terminal domain. Their structure and mobility in an aqueous environment is only partially known. Electron paramagnetic resonance (EPR) has been used to measure the structure of these hydrophilic protein domains in detergent-solubilized LHCII. A new technique is intro-duced to prepare LHCII trimers in which only one monomer is spin-labelled. These heterogeneous trimers allow to measure intra-molecular distances within one LHCII monomer in the context of a trimer by using double electron-electron resonance (DEER). These data together with data from electron-spin-echo-envelope-modulation (ESEEM) allowed to model the N-terminal protein section, which has not been resolved in current crystal structures, and the lumenal loop domain. The N-terminal domain covers only a restricted area above the superhelix in LHCII, which is consistent with the "Velcro" hypothesis to explain thylakoid grana stacking (Standfuss, J., van Terwisscha Scheltinga, A. C., Lamborghini, M., and Kuehlbrandt, W. (2005) Mechanisms of photo-protection and nonphotochemical quenching in pea light-harvesting complex at 2.5 Angstrom (0.25 nm) resolution. EMBO J. 24, 919-928). The conformation of the lumenal loop domain is surprisingly different between LHCII monomers and trimers but not between complexes with and without neoxanthin bound.

Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase [Enzymology]

August 21st, 2015 by Wu, S.-Y., Rothery, R. A., Weiner, J. H.

We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining Mo active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The Mo atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates which are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser719, NarG-His1163, and NarG-His1184); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His1092 and NarG-His1098). A Ser719Ala variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the His1163Ala and His1184Ala variants elicit large effects (deltaEm values of -88 mV and -36 mV, respectively). Ala variants of His1092 and His1098 also elicit large deltaEm values of -143 mV and -101 mV, respectively. An Arg variant of His1092 elicits a small deltaEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the Mo Em value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis.

Protein N-terminal Acetylation by NatA Is Critical for Selective Mitochondria Degradation [Membrane Biology]

August 21st, 2015 by Eiyama, A., Okamoto, K.

Mitophagy is an evolutionarily conserved autophagy pathway that selectively degrades mitochondria. Although it is well established that this degradation system contributes to mitochondrial quality and quantity control, mechanisms underlying mitophagy remain largely unknown. Here we report that protein N-terminal acetyltransferase A (NatA), an enzymatic complex composed of the catalytic subunit Ard1 and the adaptor subunit Nat1, is crucial for mitophagy in yeast. NatA is associated with the ribosome via Nat1 and acetylates the second amino acid residues of nascent polypeptides. Mitophagy, but not bulk autophagy, is strongly suppressed in cells lacking Ard1, Nat1, or both proteins. In addition, loss of NatA enzymatic activity causes impairment of mitochondrial degradation, suggesting that protein N-terminal acetylation by NatA is important for mitophagy. Ard1 and Nat1 mutants exhibited defects in induction of Atg32, a protein essential for mitophagy, and formation of mitochondria-specific autophagosomes. Notably, overexpression of Atg32 partially recovered mitophagy in NatA-null cells, implying that this acetyltransferase participates in mitophagy at least in part via Atg32 induction. Together, our data implicate NatA-mediated protein modification as an early regulatory step crucial for efficient mitophagy.

APP metabolites AICD, A{beta}42 and Tau in nuclear roles [Gene Regulation]

August 21st, 2015 by Multhaup, G., Huber, O., Buee, L., Galas, M.-C.

APP metabolites (Aβ peptides) and Tau are the main components of senile plaques and neurofibrillary tangles, the two histopathological hallmarks of Alzheimer disease (AD). Consequently, intense research has focused upon deciphering their physiological roles to understand their altered state in AD pathophysiology. Recently, the impact of APP metabolites (AICD, Aβ) and Tau on the nucleus has emerged as an important, new topic. Here we discuss (i) how AICD, Aβ, and Tau reach the nucleus and control protein expression at the transcriptional level, (ii) post-translational modifications of AICD, Aβ, and Tau, and (iii) what these three molecules have in common.