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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Covalent Label Transfer Between Peroxisomal Importomer Components Reveals Export-Driven Import Interactions [Cell Biology]

November 13th, 2015 by

Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal Malate Dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes, owing to high degrees of protease susceptibility or resistance, respectively. Here, we present a means for analysis of in vitro import through a covalent biotin label transfer, and employ this method to the import of PEX5C. Label transfer demonstrates that PEX5C construct is monomeric in the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labelling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest, and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import.

The SUMO Ligase PIAS3 Primes ATR for Checkpoint Activation [Signal Transduction]

November 12th, 2015 by Wu, C.-S., Zou, L.

The maintenance of genomic stability relies on the concerted action of DNA repair and DNA damage signaling pathways. The PIAS (protein inhibitor of activated STAT) family of SUMO (small ubiquitin-like modifier) ligases has been implicated in DNA repair, but whether it plays role in DNA damage signaling is still unclear. Here, we show that the PIAS3 SUMO ligase is important for activation of the ATR (ataxia telangiectasia and Rad3 related)-regulated DNA damage signaling pathway. PIAS3 is the only member of the PIAS family that is indispensable for ATR activation. In response to different types of DNA damage and replication stress, PIAS3 plays multiple roles in ATR activation. In cells treated with camptothecin (CPT), PIAS3 contributes to formation of DNA double-stranded breaks. In UV (ultraviolet light) or HU (hydroxyurea) treated cells, PIAS3 is required for efficient ATR autophosphorylation, one of the earliest events during ATR activation. Although PIAS3 is dispensable for ATRIP (ATR-interacting protein) SUMOylation and the ATR-ATRIP interaction, it is required for maintaining the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Together, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling.

In vitro characterization of the type I toxin-antitoxin system bsrE/SR5 from Bacillus subtilis [Gene Regulation]

November 12th, 2015 by Meissner, C., Jahn, N., Brantl, S.

BsrE/SR5 is a new type I toxin/antitoxin system located on the prophage-like region P6 of the B. subtilis chromosome. The bsrE gene encoding a 30 amino acid hydrophobic toxin and the antitoxin gene sr5 overlap at their 3` ends by 112 bp. Overexpression of bsrE causes cell lysis on agar plates. The presence of sr5 ensures a growth advantage of B. subtilis cells. Here, we present a detailed in vitro analysis of bsrE/SR5. The secondary structures of SR5, bsrE mRNA and the SR5/bsrE RNA complex were determined. Apparent binding rate constants (kapp) of wild-type and mutated SR5 species with wild-type bsrE mRNA were calculated and SR5 regions required for efficient inhibition of bsrE mRNA narrowed down. In vivo studies confirmed the in vitro data but indicated that a so far unknown RNA binding protein might exist in B. subtilis that can promote antitoxin/toxin RNA interaction. Using time course experiments, the binding pathway of SR5 and bsrE RNA was elucidated. A comparison with the previously well-characterized type I TA system from the B. subtilis chromosome, bsrG/SR4, reveals similarities, but also significant differences.

Complement component C5a primes retinal pigment epithelial cells for inflammasome activation by lipofuscin-mediated photooxidative damage [Immunology]

November 12th, 2015 by Brandstetter, C., Holz, F. G., Krohne, T. U.

Complement activation, oxidative damage, and activation of the NLRP3 inflammasome have been implicated in retinal pigment epithelium (RPE) pathology in age-related macular degeneration (AMD). Following priming of RPE cells, the NLRP3 inflammasome can be activated by various stimuli such as lipofuscin-mediated photooxidative damage to lysosomal membranes. We investigated whether products of complement activation are capable of providing the priming signal for the inflammasome in RPE cells. Incubation of primary human RPE cells and ARPE-19 cells with complement-competent human serum resulted in upregulation of C5a receptor, but not C3a receptor. Furthermore, it induced expression of pro-IL-1β and enabled IL-1β secretion in response to lipofuscin phototoxicity, thus indicating inflammasome priming by human serum. Complement heat-inactivation, C5 depletion, and C5a receptor inhibition suppressed the priming effect of human serum whereas recombinant C5a likewise induced priming. Conditioned media of inflammasome-activated RPE cells provided an additional priming effect that was mediated by the IL-1 receptor. These results indicate that complement activation product C5a represents a priming signal for RPE cells that allows for subsequent inflammasome activation by stimuli such as lipofuscin-mediated photooxidative damage. This molecular pathway provides a functional link between key factors of AMD pathogenesis including lipofuscin accumulation, photooxidative damage, complement activation, and RPE degeneration and may provide novel therapeutic targets in this disease.
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