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.

MEF2D Deficiency in Neonatal Cardiomyocytes Triggers Cell Cycle Re-entry and Programmed Cell Death in vitro [Cell Biology]

August 20th, 2015 by

The cardiomyocyte cell cycle is a poorly understood process. Mammalian cardiomyocytes permanently withdraw from the cell cycle shortly after birth but can re-enter the cell cycle and proliferate when subjected to injury within a brief temporal window in the neonatal period. Thus, investigating the mechanisms of cell cycle regulation in neonatal cardiomyocytes may provide critical insight into the molecular events that prevent adult myocytes from proliferating in response to injury or stress. MEF2D is a key transcriptional mediator of pathological remodeling in the adult heart downstream of various stress-promoting insults. However, the specific gene programs regulated by MEF2D in cardiomyocytes are unknown. By performing genome-wide transcriptome analysis using MEF2D-depleted neonatal cardiomyocytes we found a significant impairment in the cell cycle, characterized by the upregulation of numerous positive cell cycle regulators. Expression of PTEN, the primary negative regulator of PI3K/Akt, was significantly reduced in MEF2D-deficient cardiomyocytes and found to be a direct target gene of MEF2D. Consistent with these findings mutant cardiomyocytes showed activation of the PI3K/Akt survival pathway. Paradoxically, prolonged deficiency of MEF2D in neonatal cardiomyocytes did not trigger proliferation but instead resulted in programmed cell death, which is likely mediated by the E2F transcription factor. These results demonstrate a critical role for MEF2D in cell cycle regulation of post-mitotic, neonatal cardiomyocytes in vitro.

Structure of the Legionella Effector, lpg1496, Suggests a Role in Nucleotide Metabolism [Protein Structure and Folding]

August 20th, 2015 by Wong, K., Kozlov, G., Zhang, Y., Gehring, K.

Pathogenic gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. Lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaire's disease. Here, we crystallized three nucleotide-binding domains from lpg1496. The C-terminal domain, which is conserved among the SidE family of effectors, is formed of two largely α-helical lobes with a nucleotide-binding cleft. A structural homology search has shown similarity to phosphodiesterases involved in cleavage of cyclic nucleotides. We have also crystallized a novel domain that occurs twice in the N-terminal half of the protein that we term KLAMP domains due to the presence of homologous domains in bacterial histidine kinase-like ATP-binding region-containing proteins and S-adenosylmethionine-dependent methyltransferase proteins. Both KLAMP structures are very similar but selectively bind 3′,5′-cAMP and ADP. A co-crystal of the KLAMP1 domain with 3′,5′-cAMP reveals the contribution of Tyr61 and Tyr69 that produce π-stacking interactions with the adenine ring of the nucleotide. Our study provides the first structural insights into two novel nucleotide-binding domains associated with bacterial virulence.

Two-step Ligand Binding in a ({beta}{alpha})8 Barrel Enzyme – Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA [Enzymology]

August 20th, 2015 by

HisA is a (βα)8 barrel enzyme that catalyzes the Amadori rearrangement of ProFAR to PRFAR in the histidine biosynthesis pathway and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp7 acts as the catalytic base and Asp176 as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure, and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen-bond network including His47, Asp129, Thr171 and Ser202, all shown to be functionally important. The closed-conformation structure is highly similar to the bi-functional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.
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Recapitulating the Structural Evolution of Redox-Regulation in Adenosine-5′-Phosphosulfate Kinase from Cyanobacteria to Plants [Protein Structure and Folding]

August 20th, 2015 by Herrmann, J., Nathin, D., Lee, S. G., Sun, T., Jez, J. M.

In plants, adenosine 5'-phosphosulfate (APS) kinase (APSK) is required for reproductive viability and the production of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as a sulfur-donor in specialized metabolism. Previous studies of the APSK from Arabidopsis thaliana (AtAPSK) identified a regulatory disulfide bond formed between the N-terminal domain (NTD) and a cysteine on the core scaffold. This thiol-switch is unique to mosses, gymnosperms, and angiosperms. To understand the structural evolution of redox-control of APSK, we investigated the redox-insensitive APSK from the cyanobacterium Synechocystis sp. PCC 6803 (SynAPSK). Crystallographic analysis of SynAPSK in complex with either APS and a non-hydrolyzable ATP analog or APS and sulfate reveals the overall structure of the enzyme, which lacks the NTD found in homologs from mosses and plants. A series of engineered SynAPSK variants reconstruct the structural evolution of the plant APSK. Biochemical analyses of SynAPSK, SynAPSK H23C mutant, SynAPSK fused to the AtAPSK NTD, and the fusion protein with the H23C mutation show that addition of the NTD and cysteines recapitulates thiol-based regulation. These results reveal the molecular basis for structural changes leading to the evolution of redox-control in APSK of the green lineage from cyanobacteria to plants.
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