Zn2+-dependent activation of the Trk signaling pathway induces phosphorylation of the brain-enriched tyrosine phosphatase STEP: molecular basis for Zn2+-induced ERK MAPK activation [Neurobiology]

November 16th, 2015 by Poddar, R., Rajagopal, S., Shuttleworth, C. W., Paul, S.

Excessive release of Zn2+ in the brain is implicated in the progression of acute brain injuries. Although several signaling cascades have been reported to be involved in Zn2+-induced neurotoxicity, a potential contribution of tyrosine phosphatases in this process has not been well explored. Here we show that exposure to high concentrations of Zn2+ led to a progressive increase in phosphorylation of the striatal-enriched phosphatase (STEP), a component of the excitotoxic-signaling pathway that plays a role in neuroprotection. Zn2+ mediated phosphorylation of STEP61 at multiple sites (hyperphosphorylation) was induced by the up-regulation of brain-derived neurotropic factor (BDNF), tropomyosin receptor kinase (Trk) signaling and activation of cAMP-dependent PKA (protein kinase A). Mutational studies further showed that differential phosphorylation of STEP61 at the PKA sites, ser160 and ser221 regulates the affinity of STEP61 towards its substrates. Consistent with these findings we also show that BDNF/Trk/PKA mediated signaling is required for Zn2+-induced phosphorylation of extracellular regulated kinase 2 (ERK2), a substrate of STEP that is involved in Zn2+-dependent neurotoxicity. The strong correlation between the temporal profile of STEP61 hyperphosphorylation and ERK2 phosphorylation indicates that loss of function of STEP61 through phosphorylation is necessary for maintaining sustained ERK2 phosphorylation. This interpretation is further supported by the findings that deletion of the STEP gene led to a rapid and sustained increase in ERK2 phosphorylation within minutes of exposure to Zn2+. The study provides further insight into the mechanisms of regulation of STEP61 and also offers a molecular basis for the Zn2+-induced sustained activation of ERK2.
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Structural plasticity of the protein plug that traps newly packaged genomes in podoviridae virions [Microbiology]

November 16th, 2015 by

Bacterial viruses of the P22-like family encode a specialized tail needle essential for genome stabilization after DNA-packaging and implicated in Gram-negative cell envelope penetration. The atomic structure of P22 tail needle (gp26) crystallized at acidic pH reveals a slender fiber containing an N-terminal trimer-of-hairpins tip. Though the length and composition of tail needles vary significantly in Podoviridae, unexpectedly, the amino acid sequence of the N-terminal tip is exceptionally conserved in more than two hundred genomes of P22-like phages and prophages. In this paper, we used X-ray crystallography and EM to investigate the neutral pH structure of three tail needles from bacteriophage P22, HK620 and Sf6. In all cases, we found the N-terminal tip is poorly structured, in stark contrast to the compact trimer-of-hairpins seen in gp26 crystallized at acidic pH. Hydrogen/deuterium exchange mass spectrometry, limited proteolysis, circular dichroism spectroscopy and gel filtration chromatography revealed that the N-terminal tip is highly dynamic in solution and unlikely to adopt a stable trimeric conformation at physiological pH. This is supported by the cryo-EM reconstruction of P22 mature virion tail, where the density of gp26 N-terminal tip is incompatible with a trimer-of-hairpins. We propose the tail needle N-terminal tip exists in two conformations: a pre-ejection extended conformation, which seals the portal vertex after genome-packaging and a post-ejection trimer-of-hairpins that form upon its release from the virion. The conformational plasticity of the tail needle N-terminal tip is built in the amino acid sequence, explaining its extraordinary conservation in nature.

Ankyrin-G inhibits endocytosis of cadherin dimers [Membrane Biology]

November 16th, 2015 by Cadwell, C. M., Jenkins, P. M., Bennett, V., Kowalczyk, A. P.

Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Thus, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. Yet, the mechanisms that control junctional plasticity are not fully understood. VE-cadherin is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of the cadherin and stabilizes it at the plasma membrane. Here, we report that induced cis-dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis-dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions.

Downregulation of miRs 203, 887, 3619 and 182 prevent vimentin-triggered, phospholipase D (PLD)-mediated cancer cell invasion [Signal Transduction]

November 15th, 2015 by Fite, K., Gomez-Cambronero, J.

Breast cancer is a leading cause of morbidity and mortality among women. Metastasis is initiated after epithelial-mesenchymal-transition (EMT). We have found a connection between EMT markers and the expression of 4 microRNAs (miRs), mediated by the signaling enzyme phospholipase D (PLD). Low aggressive MCF-7 and BT-474 breast cancer cells have low endogenous PLD enzymatic activity and cell invasion, concomitantly with high expression of miR-203, 887 and 3619 (that decrease PLD2 translation and a luciferase reporter) and miR-182 (targeting PLD1) that are therefore tumor-suppresor like miRs. The combination miR-887+miR-3619 abolished >90% PLD enzymatic activity. Conversely, post-EMT MDA-MB-231 and BT-549 cells, have low miR expression, high levels of PLD1/2 and high aggressiveness. The latter was reversed by ectopically transfecting the miRs, which was negated by silencing miRs with specific siRNAs. We uncovered that the molecular mechanism is that E-cadherin triggers expression of the miRs in pre-EMT cells, whereas Vimentin dampens expression of the miRs in post-EMT, invasive cells. This novel work identifies for the first time a set of miRs that are activated by a major pre-EMT marker and deactivated by a post-EMT marker, boosting the transition from low invasion to high invasion, as mediated by the key phospholipid metabolism enzyme PLD.
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Structure and Properties of Non-Processive, Salt-Requiring, Acidophilic Pectin Methylesterases from Aspergillus niger Provides Insights into the Key Determinants of Processivity Control [Enzymology]

November 15th, 2015 by

Many pectin methylesterases (PMEs) are expressed in plants to modify plant cell-wall pectins for various physiological roles. These pectins are also attacked by PMEs from phytopathogens and phytophagous insects. The de-methylesterification by PMEs of the O6-methylester groups of the homogalacturonan (HG) component of pectin, exposing galacturonic acids, can occur processively or non-processively, respectively describing sequential versus single de-methylesterification events occurring before enzyme-substrate dissociation. The high-resolution X-ray structures of a PME from Aspergillus niger in deglycosylated and Asn-linked N-acetylglucosamine-stub forms reveal a 10⅔-turn parallel beta helix (similar to but with less extensive loops than bacterial, plant and insect PMEs). Capillary electrophoresis shows that this PME is non-processive, halophilic and acidophilic. Molecular-dynamics simulations and electrostatic-potential calculations reveal very different behavior and properties compared to processive PMEs. Specifically, uncorrelated rotations are observed about the glycosidic bonds of a partially de-methylesterified decasaccharide model substrate, in sharp contrast to the correlated rotations of processive PMEs, and the substrate-binding groove is negatively not positively charged.
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Genome-wide Screening of Regulators of Catalase Expression: Role of a Transcription Complex and Histone and tRNA Modification Complexes on Adaptation to Stress [Signal Transduction]

November 13th, 2015 by Garcia, P., Encinar del Dedo, J., Ayte, J., Hidalgo, E.

In response to environmental cues, the MAP kinase Sty1-driven signaling cascade activates hundreds of genes to induce a robust anti-stress cellular response in fission yeast. Thus, upon stress imposition Sty1 transiently accumulates in the nucleus where it up-regulates transcription through the Atf1 transcription factor. Several regulators of transcription and translation have been identified as important to mount an integral response to oxidative stress, such as the SAGA or Elongator complexes, respectively. With the aim of identifying new regulators of this massive gene expression program, we have used a GFP-based protein reporter and screened a fission yeast deletion collection using flow cytometry. We find that the levels of catalase fused to GFP, both before and after a threat of peroxides, are altered in hundreds of strains lacking components of chromatin modifiers, transcription complexes and modulators of translation. Thus, the transcription elongation complex Paf1, the histone methylase Set1-COMPASS and the translation-related Trm112 dimers are all involved in full expression of Ctt1-GFP and in wild-type tolerance to peroxides.
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Activation of exogenous fatty acids to acyl-acyl carrier protein cannot bypass FabI inhibition in Neisseria [Microbiology]

November 13th, 2015 by Yao, J., Bruhn, D. F., Frank, M. W., Lee, R. E., Rock, C. O.

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine if Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype and minimal inhibition of DNA, RNA, and protein synthesis showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria are supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria.

Developmental Stage-dependent Regulation of Prolyl 3-Hydroxylation in Tendon Type I Collagen [Glycobiology and Extracellular Matrices]

November 13th, 2015 by Taga, Y., Kusubata, M., Ogawa-Goto, K., Hattori, S.

3-Hydroxyproline (3-Hyp) unique to collagen is a fairly rare post-translational modification. Recent studies have suggested a function of prolyl 3-hydroxylation in fibril assembly and its relationships with certain disorders, including recessive osteogenesis imperfecta and high myopia. However, no direct evidence for the physiological and pathological roles of 3-Hyp has been presented. In this study, we first estimated the overall alterations in prolyl hydroxylation in collagens purified from skin, bone, and tail tendon of 0.5-18-month-old rats by LC-MS analysis with stable isotope-labeled collagen, which was recently developed as an internal standard for highly accurate collagen analyses. 3-Hyp was found to significantly increase in tendon collagen until 3 months after birth and then remain constant, while increased prolyl 3-hydroxylation was not observed in skin and bone collagens. Site-specific analysis further revealed that 3-Hyp was increased in tendon type I collagen in a specific sequence region, including a previously known modification site at Pro707 and newly identified sites at Pro716 and Pro719, at the early ages. The site-specific alterations in prolyl 3-hydroxylation with aging were also observed in bovine Achilles tendon. We postulate that the significant increases in 3-Hyp at the consecutive modification sites are correlated with tissue development in tendon. The present findings suggest that prolyl 3-hydroxylation incrementally regulates collagen fibril diameter in tendon.

Identification of critical paraoxonase 1 residues involved in high density lipoprotein interaction [Protein Structure and Folding]

November 13th, 2015 by

Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated protein with atherosclerosis-protective and systemic anti-oxidant functions. We recently showed that PON1, myeloperoxidase (MPO) and HDL bind to one another in vivo forming a functional ternary complex (Huang, Y. et al J. Clin. Invest. 2013 123(9):3815-28). However, specific residues on PON1 involved in the HDL-PON1 interaction remain unclear. Unambiguous identification of protein residues involved in docking interactions to lipid surfaces poses considerable methodological challenges. Here we describe a new strategy that uses a novel synthetic photoactivatable and click chemistry taggable phospholipid probe, which when incorporated into HDL, was used to identify amino acid residues on PON1 that directly interact with the lipoprotein phospholipid surface. Several specific PON1 residues (Leu9, Tyr185 and Tyr293) were identified through covalent cross-links with the lipid probes using affinity isolation coupled to liquid chromatography with on-line tandem mass spectrometry. Based upon the crystal structure for PON1, the identified residues are all localized in relatively close proximity on the surface of PON1, defining a domain that binds to the HDL lipid surface. Site-specific mutagenesis of the identified PON1 residues (Leu9, Tyr185 and Tyr293), coupled with functional studies, reveals their importance in PON1 binding to HDL, and both PON1 catalytic activity and stability. Specifically, the residues identified on PON1 provide important structural insights into PON1-HDL interaction. More generally, the new photoactivatable and affinity tagged lipid probe developed herein should prove to be a valuable tool for identifying contact sites supporting protein interactions with lipid interfaces such as found on cell membranes or lipoproteins.

Human Type IV P-type ATPases that Work as Plasma Membrane Phospholipid Flippases, and Their Regulation by Caspase and Calcium [Membrane Biology]

November 13th, 2015 by Segawa, K., Kurata, S., Nagata, S.

In plasma membranes, flippases translocate aminophospholipids such as phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) from the extracellular to the cytoplasmic leaflet. Mammalian ATP11C, a type IV P-type ATPase (P4-ATPase), acts as a flippase at the plasma membrane. Here, by expressing 12 human P4-ATPases in ATP11C-deficient cells, we determined that ATP8A2 and ATP11A can also act as plasma membrane flippases. As with ATP11C, ATP8A2 and ATP11A localized to the plasma membrane in a CDC50A-dependent manner. ATP11A was cleaved by caspases during apoptosis, and a caspase-resistant ATP11A blocked apoptotic PtdSer exposure. In contrast, ATP8A2 was not cleaved by caspase, and cells expressing ATP8A2 did not expose PtdSer during apoptosis. Similarly, high Ca2+ concentrations inhibited the ATP11A and ATP11C PtdSer-flippase activity, but ATP8A2′s flippase activity was relatively resistant to Ca2+. ATP11A and ATP11C were ubiquitously expressed in human and mouse adult tissues. In contrast, ATP8A2 was expressed in specific tissues, such as the brain and testis. Thus, ATP8A2 may play a specific role in translocating PtdSer in these tissues.
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