Structural Basis for Ligand Recognition and Functional Selectivity at Angiotensin Receptor [Signal Transduction]

September 29th, 2015 by

Angiotensin II type 1 receptor (AT1R) is the primary blood pressure regulator. AT1R blockers (ARBs) have been widely used in clinical settings as anti-hypertensive drugs, and share a similar chemical scaffold, although even minor variations can lead to distinct therapeutic efficacies towards cardiovascular etiologies. The structural basis for AT1R modulation by different peptide and non-peptide ligands has remained elusive. Here we report the crystal structure of the human AT1R in complex with an inverse agonist olmesartan (BenicarTM), a highly potent anti-hypertensive drug. Olmesartan is anchored to the receptor primarily by the residues Tyr351.39, Trp842.60, and Arg167ECL2, similar to the antagonist ZD7155, corroborating a common binding mode of different ARBs. Using docking simulations and site-directed mutagenesis we identified specific interactions between AT1R and different ARBs, including olmesartan derivatives with inverse agonist, neutral antagonist or agonist activities. We further observed that the mutation Asn1113.35Ala in the putative sodium-binding site affects binding of the endogenous peptide agonist Angiotensin II, but not the β-arrestin-biased peptide TRV120027.

Ethosuximide induces hippocampal neurogenesis and reverses cognitive deficits in amyloid-{beta} toxin induced Alzheimer’s rat model via PI3K/Akt/Wnt/{beta}-catenin pathway [Molecular Bases of Disease]

September 29th, 2015 by

Neurogenesis involves generation of new neurons through finely tuned multistep processes such as neural stem cell's (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and sub-ventricular zone (SVZ). Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders including Alzheimer's disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizure. However, the effects of ETH on adult hippocampal neurogenesis and underlying cellular and molecular mechanism(s) are still elusive. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation, and adult hippocampal neurogenesis in an amyloid beta (Aβ) toxin induced rat model of AD like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampal derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation, and reduced Aβ toxin mediated toxicity and neurodegeneration, leading to behavioral recovery in rat AD model. ETH inhibited Aβ mediated suppression of neurogenic and Akt-Wnt/β-catenin pathway gene's expression in the hippocampus. ETH activated the PI3K/Akt and Wnt/β-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K/Akt and Wnt/β-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1 and GSK-3β. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K/Akt and Wnt/β-catenin signaling.
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ATM-dependent phosphorylation of the Fanconi anemia protein PALB2 promotes the DNA Damage Response [DNA and Chromosomes]

September 29th, 2015 by Guo, Y., Feng, W., Sy, S. M. H., Huen, M. S. Y.

The Fanconi anemia protein PALB2, also known as FANCN, protects genome integrity by regulating DNA repair and cell cycle checkpoints. Exactly how PALB2 functions may be temporally coupled with detection and signaling of DNA damage is not known. Intriguingly, we found that PALB2 is transformed into a hyper-phosphorylated state in response to ionizing radiation (IR). IR treatment specifically triggered PALB2 phosphorylation at Ser157 and Ser376 in manners that required the master DNA Damage Response (DDR) kinase Ataxia telangiectasia mutated (ATM), revealing potential mechanistic links between PALB2 and the ATM-dependent DDRs. Consistently, dysregulated PALB2 phosphorylation resulted in sustained activation of DDRs. Full-blown PALB2 phosphorylation also required the breast and ovarian susceptible gene product BRCA1, highlighting important roles of the BRCA1-PALB2 interaction in orchestrating cellular responses to genotoxic stress. In summary, our phosphorylation analysis of tumour suppressor protein PALB2 uncovers new layers of regulatory mechanisms in the maintenance of genome stability and tumor suppression.

Structural mechanism of the interaction of Alzheimer’s disease A{beta} fibrils with the NSAID sulindac sulfide [Molecular Biophysics]

September 28th, 2015 by

Alzheimer's disease is the most severe neurodegenerative disease worldwide. In the past years, a plethora of small molecules interfering with amyloid-β (Aβ) aggregation have been suggested. However, their mode of interaction with amyloid fibers is not understood. Non-steroidal anti-inflammatory drugs (NSAIDs) are known γ-secretase modulators (GSMs). It has been suggested that NSAIDs are pleiotrophic and can interact with more than one pathomechanism. We present here a magic angle spinning (MAS) solid-state NMR study that shows that the NSAID sulindac sulfide interacts specifically with Alzheimer's disease Aβ fibrils. We find that sulindac sulfide does not induce drastic architectural changes in the fibrillar structure, but intercalates between the two β-strands of the amyloid fibril and binds to hydrophobic cavities, which are found consistently in all analyzed structures. The characteristic D23-K28 salt bridge is not affected upon interacting with sulindac sulfide. The primary binding site is located in the vicinity of residue G33, a residue involved in M35 oxidation. The results presented here could be useful in the search for pharmacologically active molecules which can potentially be employed as lead structures to guide the design of small molecules for the treatment of Alzheimer's disease.

Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector [Membrane Biology]

September 28th, 2015 by Couoh-Cardel, S., Milgrom, E., Wilkens, S.

The membrane sector (Vo) of the proton pumping vacuolar ATPase (V-ATPase; V1Vo-ATPase) from Saccharomyces cerevisiae was purified to homogeneity and its structure was characterized by electron microscopy (EM) of single molecules and two-dimensional (2-D) crystals. Projection images of negatively stained Vo 2-D crystals showed a ring like structure with a large asymmetric mass at the periphery of the ring. A cryo EM reconstruction of Vo from single particle images showed subunits a and d in close contact on the cytoplasmic side of the proton channel. A comparison of 3-D reconstructions of free Vo and Vo as part of holo V1Vo revealed that the cytoplasmic N-terminal domain of subunit a (aNT) must undergo a large conformational change upon enzyme disassembly or (re)assembly from Vo, V1 and subunit C. Isothermal titration calorimetry using recombinant subunit d and aNT revealed that the two proteins bind each other with a Kd of ~ 5 μM. Treatment of purified Vo sector with lyso 1-palmitoyl phosphatidylglycerol (LPPG) resulted in selective release of subunit d, allowing purification of a VoΔd complex. Passive proton translocation assays revealed that both Vo and VoΔd are impermeable to protons. We speculate that the structural change in subunit a upon release of V1 from Vo during reversible enzyme dissociation plays a role in blocking passive proton translocation across free Vo and that the interaction between aNT and d seen in free Vo functions to stabilize the Vo sector for efficient reassembly of V1Vo.

Retinal attachment instability is diversified among mammalian melanopsins [Molecular Biophysics]

September 28th, 2015 by

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics, by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extra-cellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.

A Foerster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers [Protein Structure and Folding]

September 28th, 2015 by Booth, E. A., Vane, E. W., Dovala, D., Thorner, J.

Prior studies in both budding yeast (Saccharomyces cerevisiae) and in human cells have established that septin protomers assemble into linear hetero-octameric rods with two-fold rotational symmetry. In mitotically-growing yeast cells, five septin subunits are expressed (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1) and assemble into two types of rods that differ only in their terminal subunit: Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Shs1. EM analysis has shown that, under low-salt conditions, the Cdc11-capped rods polymerize end-to-end to form long paired filaments, whereas Shs1-capped rods form arcs, spirals and rings. To develop a facile method to study septin polymerization in vitro, we exploited our previous work where we generated septin complexes in which all endogenous cysteine (Cys) residues were eliminated by site-directed mutagenesis, except an introduced E294C mutation in Cdc11 in these experiments. Mixing samples of a preparation of such single-Cys containing Cdc11-capped rods that have been separately derivatized with organic dyes that serve as donor and acceptor, respectively, for FRET provided a spectroscopic method to monitor filament assembly mediated by Cdc11-Cdc11 interaction and to measure its affinity under specified conditions. Modifications of this same FRET scheme also allow us to assess whether Shs1-capped rods are capable of end-to-end association either with themselves or with Cdc11-capped rods. This FRET approach also was used to follow the binding to septin filaments of a septin-interacting protein, the type II myosin-binding protein Bni5.
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Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain [Molecular Bases of Disease]

September 28th, 2015 by

Functional and deep sequencing studies have combined to demonstrate the involvement of APOBEC3B in cancer mutagenesis. APOBEC3B is a single-stranded DNA cytosine deaminase that functions normally as a nuclear-localized restriction factor of DNA-based pathogens. However, it is overexpressed in cancer cells and elicits an intrinsic preference for 5'-TC motifs in single-stranded DNA, which is the most frequently mutated dinucleotide in breast, head/neck, lung, bladder, cervical, and several other tumor types. In many cases, APOBEC3B mutagenesis accounts for the majority of both dispersed and clustered (kataegis) cytosine mutations. Here, we report the first structures of the APOBEC3B catalytic domain in multiple crystal forms. These structures reveal a tightly closed active site conformation and suggest that substrate accessibility is regulated by adjacent flexible loops. Residues important for catalysis are identified by mutation analyses and the results provide insights into the mechanism of target site selection. We also report a nucleotide (dCMP) bound crystal structure that informs a multi-step model for binding single-stranded DNA. Overall, these high-resolution crystal structures provide a framework for further mechanistic studies and the development of novel anti-cancer drugs to inhibit this enzyme, dampen tumor evolution, and minimize adverse outcomes such as drug resistance and metastasis.

The Relationship Between Glycan-Binding and Direct Membrane Interactions in Vibrio cholerae Cytolysin, a Channel-Forming Toxin [Membrane Biology]

September 28th, 2015 by

Bacterial pore-forming toxins (PFTs) are structurally diverse pathogen-secreted proteins that form cell-damaging channels in the membranes of host cells. Most PFTs are released as water-soluble monomers that first oligomerize on the membrane before inserting a transmembrane channel. To modulate specificity and increase potency, many PFTs recognize specific cell-surface receptors that increase the local toxin concentration on cell membranes thereby facilitating channel formation. Vibrio cholerae cytolysin (VCC) is a toxin secreted by the human pathogen responsible for pandemic cholera disease and acts as a defensive agent against the host immune system. While it has been shown that VCC utilizes specific glycan receptors on the cell surface, additional direct contacts with the membrane must also play a role in toxin binding. To better understand the nature of these interactions, we conducted a systematic investigation of the membrane-binding surface of VCC to identify additional membrane interactions important in cell targeting. Through cell-based assays on several human-derived cell-lines we show that VCC is unlikely to utilize high-affinity protein receptors like structurally similar toxins from Staphylococcus aureus. Next, we identified a number of specific amino-acid residues that greatly diminish the VCC potency against cells and investigated the interplay between glycan-binding and these direct lipid contacts. Finally, we used model membranes to parse the importance of these key residues in lipid and cholesterol binding. Our study provides a complete functional map of the VCC membrane-binding surface and insights into the integration of sugar, lipid, and cholesterol binding-interactions.
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Rotenone-induced Impairment of Mitochondrial Electron Transport Chain Confers a Selective Priming Signal for NLRP3 Inflammasome Activation [Immunology]

September 28th, 2015 by Won, J.-H., Park, S., Hong, S., Son, S., Yu, J.-W.

Mitochondrial dysfunction is considered crucial for NLRP3 inflammasome activation partly through its release of mitochondrial toxic products such as mitochondrial ROS (mROS) and mitochondrial DNA (mtDNA). While previous studies have shown that classical NLRP3-activating stimulations lead to mROS generation and mtDNA release, it remains poorly understood whether and how mitochondrial damage-derived factors may contribute to NLRP3 inflammasome activation. Here, we demonstrate that impairment of the mitochondrial electron transport chain by rotenone licenses NLRP3 inflammasome activation only upon costimulation with ATP, but not with nigericin or alum. Rotenone-induced priming of NLRP3 in the presence of ATP triggered the formation of speck-like NLRP3 or ASC aggregates and the association of NLRP3 with ASC, resulting in NLRP3-dependent caspase-1 activation. Mechanistically, rotenone confers a priming signal for NLRP3 inflammasome activation only in the context of aberrant high-grade, but not low-grade, mROS production and mitochondrial hyperpolarization. By contrast, rotenone/ATP-mediated mtDNA release and mitochondrial depolarization are likely to be merely an indication of mitochondrial damage rather than triggering factors for NLRP3 inflammasome activation. Our results provide a molecular insight into the selective contribution made by mitochondrial dysfunction to the NLRP3 inflammasome pathway.
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