Redundant pathways of sunscreen biosynthesis in a cyanobacterium

Route of the sun block: according to empirical evidence, sun-screening mycosporine-like amino acids (MAAs) in Eukarya originate from the shikimic acid pathway, whereas in cyanobacteria, biosynthesis of the MAA shinorine reportedly occurs through the pentose phosphate pathway. However, gene deletion shows that the cyanobacterium Anabaena variabilis ATCC 29143 does not biosynthesise shinorine exclusively by this route.     

Thiazolopyrimidine Inhibitors of 2‐Methylerythritol 2,4‐Cyclodiphosphate Synthase (IspF) from Mycobacterium tuberculosis and Plasmodium falciparum

A library of 40 000 compounds was screened for inhibitors of 2‐methylerythritol 2,4‐cyclodiphosphate synthase (IspF) protein from Arabidopsis thaliana using a photometric assay. A thiazolopyrimidine derivative resulting from the high‐throughput screen was found to inhibit the IspF proteins of Mycobacterium tuberculosis, Plasmodium falciparum, and A. thaliana with IC₅₀ values in the micromolar range. Synthetic efforts afforded derivatives that inhibit IspF protein from M. tuberculosis and P. falciparum with IC₅₀ values in the low micromolar range. Several compounds act as weak inhibitors in the P. falciparum red blood cell assay.     

O‐Methyltransferase Is Shared between the Pentose Phosphate and Shikimate Pathways and Is Essential for Mycosporine‐Like Amino Acid Biosynthesis in Anabaena variabilis ATCC 29413

The parent core structure of mycosporine‐like amino acids (MAAs) is 4‐deoxygadusol, which, in cyanobacteria, is derived from conversion of the pentose phosphate pathway intermediate sedoheptulose 7‐phosphate by the enzymes 2‐epi‐5‐epivaliolone synthase (EVS) and O‐methyltransferase (OMT). Yet, deletion of the EVS gene from Anabaena variabilis ATCC 29413 was shown to have little effect on MAA production, thus suggesting that its biosynthesis is not exclusive to the pentose phosphate pathway. Herein, we report how, using pathway‐specific inhibitors, we demonstrated unequivocally that MAA biosynthesis occurs also via the shikimate pathway. In addition, complete in‐frame gene deletion of the OMT gene from A. variabilis ATCC 29413 reveals that, although biochemically distinct, the pentose phosphate and shikimate pathways are inextricably linked to MAA biosynthesis in this cyanobacterium. Furthermore, proteomic data reveal that the shikimate pathway is the predominate route for UV‐induced MAA biosynthesis.     

pH‐responsive poly(ethylene glycol)‐poly(ϵ‐caprolactone)‐poly(glutamic acid) polymersome as an efficient doxorubicin carrier for cancer therapy

The synthesis, characterization and potential application in the doxorubicin (Dox) delivery system of a biodegradable polypeptide‐based block copolymer, poly(ethylene glycol)₂₀₀₀‐poly(?‐caprolactone)₆₀₀₀‐poly(glutamic acid)₁₀₀₀ (PEG₂₀₀₀‐PCL₆₀₀₀‐PGA₁₀₀₀), was investigated. The copolymer was synthesized via ring‐opening polymerization and characterized by ¹H NMR and Fourier transform IR. The synthesized copolymer could self‐assemble into aggregates and the critical aggregation concentration was 0.23 mg mL^?1. Transmission electron microscopy indicated that spherical polymersomes formed with a desirable size about 180 nm. Therefore Dox was encapsulated into these polymersomes, and then we investigated its applications in a drug delivery system. These Dox‐loaded polymersomes (PolyDox) were characterized by dynamic light scattering, zeta potential and pH responsiveness measurements. In vitro drug release indicated that the release rate of drug from PolyDox was pH‐responsive and significantly decreased. The drug pharmacokinetic parameters were improved in comparison to the group treated with free Dox, which proved the prolonged Dox release from PolyDox. A WST‐1 assay indicated a low toxicity and good compatibility of copolymer to cells within 48 h. The results also showed that PolyDox appeared to induce a higher anti‐tumor effect. Cell uptake results indicated that PolyDox displayed higher cellular uptake in A549 cells. Endocytosis inhibition results demonstrated that the internalization of PolyDox was mostly mediated by the fluid‐phase endocytosis pathway. © 2017 Society of Chemical Industry     

Studies addressing the importance of charge in the binding of fosmidomycin-like molecules to deoxyxylulosephosphate reductoisomerase

Fosmidomycin and its homologue FR900098 are inhibitors of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, which is part of the mevalonate-independent isoprenoid biosynthetic pathway. Replacement of the phosphonate moiety by uncharged sulfone or sulfonamide partial structures resulted in complete loss of activity. Dropping one of the two negative charges resulted in a marked decrease in activity. Through occupation of a hydrophobic binding site, some activity could be regained, leading to compounds with micromolar activity against cultured malaria parasites.     

Inactivation of the Ketoreductase gilU Gene of the Gilvocarcin Biosynthetic Gene Cluster Yields New Analogues with Partly Improved Biological Activity

Four new analogues of the gilvocarcin‐type aryl‐C‐glycoside antitumor compounds, namely 4′‐hydroxy gilvocarcin V (4′‐OH‐GV), 4′‐hydroxy gilvocarcin M, 4′‐hydroxy gilvocarcin E and 12‐demethyl‐defucogilvocarcin V, were produced through inactivation of the gilU gene. The 4′‐OH‐analogues showed improved activity against lung cancer cell lines as compared to their parent compounds without 4′‐OH group (gilvocarcins V and E). The structures of the sugar‐containing new mutant products indicate that the enzyme GilU acts as an unusual ketoreductase involved in the biosynthesis of the C‐glycosidically linked deoxysugar moiety of the gilvocarcins. The structures of the new gilvocarcins indicate substrate flexibility of the post‐polyketide synthase modifying enzymes, particularly the C‐glycosyltransferase and the enzyme responsible for the sugar ring contraction. The results also shed light into biosynthetic sequence of events in the late steps of biosynthetic pathway of gilvocarcin V.     

Monitoring of the Synthesis of Hyperbranched Poly(urea‐urethane)s by Real‐Time Attenuated Total Reflection (ATR)‐FT‐IR Spectroscopy

Summary: The reaction of 2,4‐TDI and DEA, as an A₂ + B^*B₂ polymerization system towards hyperbranched HPUs was followed using in situ ATR‐FT‐IR spectroscopy. The decrease in intensity of the NCO absorption band of the reactive isocyanate group of 2,4‐TDI along with the formation and growth of the new characteristic bands of urethane and urea groups were detected. The reactivity difference of both NH and OH groups towards the NCO group at low temperatures was proven. The rate of the reaction was found to be affected by changing the temperature, the rate of addition of the B^*B₂ monomer and the type of solvent. Moreover, the increase of the carbonyl vibration and the amide II bands of urea was very obvious during the addition of the stopper DEA. Thus, it was possible to verify the individual reaction steps of this complex polyreaction and to correlate these with the structural development of the resulting macromolecules.

Characteristic vibration bands of urethane and urea groups in the IR spectra (1 780–1 480 cm^?1) during the polymerization reaction.     

Brefeldin A Limits N-Hexanoylsphingosine-Induced Accumulation of Natural Ceramide via the Salvage Pathway by Enhancing Glucosylation

Cells actively metabolize exogenously administered N-hexanoylsphingosine (C6-Cer) to natural (i.e. long-chain) ceramide (LC-Cer) via the sphingosine (Sph) salvage pathway, namely via C6-Cer deacylation and Sph reacylation with a long-chain fatty acid. Based on the observation that the mycotoxin brefeldin A (BFA), a Golgi complex disassembler, impairs C6-Cer-evoked LC-Cer accumulation, it has been hypothesized that the integrity of the above-mentioned organelle might be necessary for C6-Cer processing via the salvage pathway and that BFA might block the phenomenon at the step short-chain ceramide deacylation. The present study shows that BFA indeed attenuates C6-Cer-evoked LC-Cer accumulation in human neurotumor CHP-100 cells: evidence is however provided that the phenomenon is not due to impaired synthesis of LC-Cer, but to its enhanced conversion to glucosylceramide. The possibility is discussed that this outcome might be a consequence of the BFA well-established property to induce the merging of the cis-Golgi region with endoplasmic reticulum, namely the compartments in which glucosylceramide synthase and ceramide synthases have been reported to reside.     

Synthesis and characterization of cytidine derivatives that inhibit the kinase IspE of the non-mevalonate pathway for isoprenoid biosynthesis

The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are attractive targets for the development of novel drugs against malaria and tuberculosis. This pathway is used exclusively by the corresponding pathogens, but not by humans. A series of water-soluble, cytidine-based inhibitors that were originally designed for the fourth enzyme in the pathway, IspD, were shown to inhibit the subsequent enzyme, the kinase IspE (from Escherichia coli). The binding mode of the inhibitors was verified by co-crystal structure analysis, using Aquifex aeolicus IspE. The crystal structures represent the first reported example of a co-crystal structure of IspE with a synthetic ligand and confirmed that ligand binding affinity originates mainly from the interactions of the nucleobase moiety in the cytidine binding pocket of the enzyme. In contrast, the appended benzimidazole moieties of the ligands adopt various orientations in the active site and establish only poor intermolecular contacts with the protein. Defined binding sites for sulfate ions and glycerol molecules, components in the crystallization buffer, near the well-conserved ATP-binding Gly-rich loop of IspE were observed. The crystal structures of A. aeolicus IspE nicely complement the one from E. coli IspE for use in structure-based design, namely by providing invaluable structural information for the design of inhibitors targeting IspE from Mycobacterium tuberculosis and Plasmodium falciparum. Similar to the enzymes from these pathogens, A. aeolicus IspE directs the OH group of a tyrosine residue into a pocket in the active site. In the E. coli enzyme, on the other hand, this pocket is lined by phenylalanine and has a more pronounced hydrophobic character.     

Synthesis of sustainable integrated biorefinery via reaction pathway synthesis: Economic,incremental enviromental burden and energy assessment with multiobjective optimization

With the increasing attention toward sustainable development, biomass has been identified as one of the most promising sources of renewable energy. To convert biomass into value‐added products and energy, an integrated processing facility, known as an integrated biorefinery is needed. To date, various biomass conversion systems such as gasification, pyrolysis, anaerobic digestion and fermentation are well established. Due to a large number of technologies available, systematic synthesis of a sustainable integrated biorefinery which simultaneously considers economic performance, environmental impact, and energy requirement is a challenging task. To address this issue, multiobjective optimization approaches are used in this work to synthesize a sustainable integrated biorefinery. In addition, a novel approach (incremental environmental burden) to assess the environmental impact for an integrated biorefinery is presented. To illustrate the proposed approach, a palm‐based biomass case study is solved. © 2014 American Institute of Chemical Engineers AIChE J, 61: 132–146, 2015     

Rab21 Protein Is Degraded by Both the Ubiquitin-Proteasome Pathway and the Autophagy-Lysosome Pathway

Rab21 is a GTPase protein that is functional in intracellular trafficking and involved in the pathologies of many diseases, such as Alzheimer’s disease (AD), glioma, cancer, etc. Our previous work has reported its interaction with the catalytic subunit of gamma-secretase, PS1, and it regulates the activity of PS1 via transferring it from the early endosome to the late endosome/lysosome. However, it is still unknown how Rab21 protein itself is regulated. This work revealed that Rab21 protein, either endogenously or exogenously, can be degraded by the ubiquitin-proteasome pathway and the autophagy-lysosome pathway. It is further observed that the ubiquitinated Rab21 is increased, but the total protein is unchanged in AD model mice. We further observed that overexpression of Rab21 leads to increased expression of a series of genes involved in the autophagy-lysosome pathway. We speculated that even though the ubiquitinated Rab21 is increased due to the impaired proteasome function in the AD model, the autophagy-lysosome pathway functions in parallel to degrade Rab21 to keep its protein level in homeostasis. In conclusion, understanding the characters of Rab21 protein itself help explore its potential as a target for therapeutic strategy in diseases.     

Octacosanol Enhances the Proliferation and Migration of Human Umbilical Vein Endothelial Cells via Activation of the PI3K/Akt and MAPK/Erk Pathways

Atherosclerosis is characterized by endothelial dysfunction, lipid deposition, fibro‐proliferative reactions and inflammation. Octacosanol is a high‐molecular‐weight primary aliphatic alcohol. As the main component of a cholesterol‐lowering drug, octacosanol could inhibit lipids accumulation and cholesterol metabolism. To explore the indication of octacosanol on endothelial protection, we evaluated its effects on the proliferation and migration of human umbilical vein endothelial cells (HUVEC). Cell viability assay using methyl thiazolyl tetrazolium and 5‐ethynyl‐2′‐deoxyuridine revealed that 3.125 μg/ml octacosanol promoted the proliferation of HUVEC. A cell migration assay indicated that 0.781 and 3.125 μg/ml octacosanol increased the migration of HUVEC. Moreover, the phosphorylation levels of Akt and Erk1/2 were significantly elevated under exposure to octacosanol. Blocking the activation of Akt and Erk with their potent inhibitors LY294002 and PD98059, respectively, markedly attenuated the octacosanol‐induced proliferation and migration of HUVEC. These findings demonstrated for the first time that octacosanol enhanced the proliferation and migration of HUVEC and mediated these effects through activation of the PI3K/Akt and MAPK/Erk1/2 signaling pathways.     

Interaction of a Tat substrate and a Tat signal peptide with thylakoid lipids at the air-water interface

The Tat machinery enables folded proteins to be translocated across biological membranes. In vitro studies have shown that Tat substrates can interact with membranes prior to translocation. In this study we investigated the initial states of this interaction with thylakoid lipid monolayers at the air-water interface by using monolayer techniques combined with infrared reflection-absorption spectroscopy (IRRAS). We used enhanced green fluorescent protein (EGFP) as a model substrate and the signal peptide SP16 from the 16 kDa protein of the spinach oxygen-evolving complex (OEC16). We found that the signal peptide is essential for the interaction of the model substrate with lipid monolayers. IRRA spectroscopy showed an increased amount of α-helical secondary structure elements for the chimeric model substrate i16/EGFP (SP16 fused to EGFP) compared with EGFP; this can be attributed to the signal peptide.     

Collateral Glucose-Utlizing Pathwaya in Diabetic Polyneuropathy

Diabetic polyneuropathy (DPN) is the most common neuropathy manifested in diabetes. Symptoms include allodynia, pain, paralysis, and ulcer formation. There is currently no established radical treatment, although new mechanisms of DPN are being vigorously explored. A pathophysiological feature of DPN is abnormal glucose metabolism induced by chronic hyperglycemia in the peripheral nerves. Particularly, activation of collateral glucose-utilizing pathways such as the polyol pathway, protein kinase C, advanced glycation end-product formation, hexosamine biosynthetic pathway, pentose phosphate pathway, and anaerobic glycolytic pathway are reported to contribute to the onset and progression of DPN. Inhibitors of aldose reductase, a rate-limiting enzyme involved in the polyol pathway, are the only compounds clinically permitted for DPN treatment in Japan, although their efficacies are limited. This may indicate that multiple pathways can contribute to the pathophysiology of DPN. Comprehensive metabolic analysis may help to elucidate global changes in the collateral glucose-utilizing pathways during the development of DPN, and highlight therapeutic targets in these pathways.     

Role of Glycolysis/Gluconeogenesis and HIF-1 Signaling Pathways in Rats with Dental Fluorosis Integrated Proteomics and Metabolomics Analysis

Fluoride is widely distributed, and excessive intake will lead to dental fluorosis. In this study, six offspring rats administrated 100 mg/L sodium fluoride were defined as the dental fluorosis group, and eight offspring rats who received pure water were defined as the control group. Differentially expressed proteins and metabolites extracted from peripheral blood were identified using the liquid chromatography tandem mass spectrometry and gas chromatography mass spectrometry, with the judgment criteria of fold change >1.2 or <0.83 and p < 0.05. A coexpression enrichment analysis using OmicsBean was conducted on the identified proteins and metabolites, and a false discovery rate (FDR) < 0.05 was considered significant. Human Protein Atlas was used to determine the subcellular distribution of hub proteins. The Gene Cards was used to verify results. A total of 123 up-regulated and 46 down-regulated proteins, and 12 up-regulated and 2 down-regulated metabolites were identified. The significant coexpression pathways were the HIF-1 (FDR = 1.86 × 10⁻³) and glycolysis/gluconeogenesis (FDR = 1.14 × 10⁻¹⁰). The results of validation analysis showed the proteins related to fluorine were mainly enriched in the cytoplasm and extrinsic component of the cytoplasmic side of the plasma membrane. The HIF-1 pathway (FDR = 1.01 × 10⁻⁷) was also identified. Therefore, the HIF-1 and glycolysis/gluconeogenesis pathways were significantly correlated with dental fluorosis.