Deconvoluting the biology and druggability of protein lipidation using chemical proteomics

Lipids are indispensable cellular building blocks, and their post-translational attachment to proteins makes them important regulators of many biological processes. Dysfunction of protein lipidation is also implicated in many pathological states, yet its systematic analysis presents significant challenges. Thanks to innovations in chemical proteomics, lipidation can now be readily studied by metabolic tagging using functionalized lipid analogs, enabling global profiling of lipidated substrates using mass spectrometry. This has spearheaded the first deconvolution of their full scope in a range of contexts, from cells to pathogens and multicellular organisms. Protein N-myristoylation, S-acylation, and S-prenylation are the most well-studied lipid post-translational modifications because of their extensive contribution to the regulation of diverse cellular processes. In this review, we focus on recent advances in the study of these post-translational modifications, with an emphasis on how novel mass spectrometry methods have elucidated their roles in fundamental biological processes.     

Stability of hydroxylamino- and amino-intermediates from reduction of 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene

Hydroxylamines, produced as intermediates in the reductive metabolism of 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene between nitroaromatic parent compounds and corresponding amines, were unstable in aqueous solution in the presence of O₂. Reactions of hydroxylamines to compounds other than amines may be the major cause of poor mass balance observations in bioremediation systems where only aminated products are monitored. Results demonstrate the formation of azoxy compounds as products of abiotic aryl-hydroxylamine reactions.     

Down-modulation of nuclear localisation and pro-fibrogenic effect of 4-hydroxy-2,3-nonenal by thiol- and carbonyl-reagents

Among the oxidative breakdown products of ω-6 unsaturated fatty acids, the aldehyde 4-hydroxy-2,3-nonenal (HNE) is receiving increasing attention for its potential pathophysiological implication, which at least partly lies on the demonstrated ability to modulate gene expression of a number of genes. Here we show that a marked down-modulation of HNE nuclear localisation in cells of a macrophage line (J774-A1) can be afforded by treatment with sulfydryl and carbonyl reagents without significantly interfering with cell viability. As regards the addition of thiol-group reagents to the cell suspension, N-ethylmaleimide (NEM) led to a sustained decrease of HNE nuclear localisation, while 4-(chloromercuri)-benzene-sulfonic acid (PCMBS) gave a similar but more transient effect. Hydroxylamine (HYD), a carbonyl-group reagent, was also able to inhibit HNE nuclear localisation. The actual efficacy of the inhibitors used was then tested on the HNE-induced stimulation of transforming growth factor β1 (TGFβ1) production by J774-A1 cells. Indeed, the thiol reagents NEM and PCMBS, both markedly down-modulating HNE nuclear localisation, were able to inhibit HNE-induced increase of TGFβ1 protein synthesis. The carbonyl reagent HYD was less effective on this respect, producing strong but incomplete protection against HNE-induced TGFβ1 increase. Taken together, the results indicate that sulfydryl groups are involved in the process of HNE cellular internalisation, while both sulfydryl and carbonyl groups are involved in the process of HNE nuclear translocation, and consequently in the modulation of gene expression by the aldehyde. Further, an actual demonstration is provided that HNE-induced effect on gene regulation can be efficiently counteracted by suitable interference with HNE biochemistry.     

Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii: (II) Involvement of the PGR5–PGRL1 pathway under anaerobic conditions

In oxygenic photosynthesis, cyclic electron flow around photosystem I denotes the recycling of electrons from stromal electron carriers (reduced nicotinamide adenine dinucleotide phosphate, NADPH, ferredoxin) towards the plastoquinone pool. Whether or not cyclic electron flow operates similarly in Chlamydomonas and plants has been a matter of debate. Here we would like to emphasize that despite the regulatory or metabolic differences that may exist between green algae and plants, the general mechanism of cyclic electron flow seems conserved across species. The most accurate way to describe cyclic electron flow remains to be a redox equilibration model, while the supramolecular reorganization of the thylakoid membrane (state transitions) has little impact on the maximal rate of cyclic electron flow. The maximum capacity of the cyclic pathways is shown to be around 60 electrons transferred per photosystem per second, which is in Chlamydomonas cells treated with 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and placed under anoxic conditions. Part I of this work (aerobic conditions) was published in a previous issue of BBA-Bioenergetics (vol. 1797, pp. 44–51) (Alric et al., 2010).     

Mass spectrometric study of Photosystem II heterogeneity in oxygen and nitrogen production: Effects of magnesium and of phosphorylation of pea thylakoids

Photosystem II (PS II) is capable of the oxidation of both water and hydroxylamine with the production of O₂- and N₂-production, respectively. The resulting changes in the partial pressure of the respective gases can be measured by an appropriate mass spectrometric set-up. Analysis of single turn-over flash saturation curves of O₂- and N₂-production has been performed to determine the relative optical cross sections of the competent PS II units and absolute amounts of their fractions in pea thylakoids. We studied the changes of these parameters upon Mg²⁺-induced transition of thylakoid membrane from unstacked to stacked configuration and upon protein phosphorylation of the stacked samples. The results showed a 2.5-fold increase of effective antenna size of PS II units competent in either O₂- or N₂-production after addition of 10 mM MgCl₂ to cation-depleted thylakoids, which indicates a potential capability of both - and -units to carry out these alternative reactions. However, we observed a significant difference in the amounts of PS II units competent in O₂- or N₂-production, with a ratio of 1:4 in unstacked thylakoids, and reciprocal alterations in stacked ones. This represents an increase by about 20% and a 2-fold decrease of O₂- and N₂-evolving units, respectively, yielding a ratio of 1:1.5, which implies a heterogeneity of PS II with respect to these reactions, the capabilities of - and -units being distinct. The phosphorylation of stacked thylakoids did not essentially influence the antenna size of O₂- and N₂-evolving PS II units but caused opposite and reciprocal changes in their amounts, approximately 30% decrease and increase, respectively, to a ratio of 1:3. The relationship of the structure-function heterogeneity in PS II with implications for current models of photosynthetic regulation mechanisms is discussed.     

羟胺切割Neurturin融合蛋白表达载体的构建、表达产物纯化及生物活性

 Neurturin (NTN)是新近发现的一种神经营养因子 ,是 GDNF家族的成员之一 .将 5′端引入了羟胺切割位点的 h NTN基因克隆到硫氧还蛋白融合表达载体 p Thio His A,在宿主菌 BL2 1中获得了稳定、高效表达 ,表达产物以包涵体形式存在 .在变性条件下经羟胺切割、柱层析纯化后复性 ,获得纯度达 90 %以上的 rh NTN.经鸡胚背根神经节 (DRG)培养法测定具有生物学活性 .     

Non-photochemical reduction of thylakoid photosynthetic redox carriers in vitro: Relevance to cyclic electron flow around photosystem I?

Non-photochemical (dark) increases in chlorophyll a fluorescence yield associated with non-photochemical reduction of redox carriers (F_npr) have been attributed to the reduction of plastoquinone (PQ) related to cyclic electron flow (CEF) around photosystem I. In vivo, this rise in fluorescence is associated with activity of the chloroplast plastoquinone reductase (plastid NAD(P)H:plastoquinone oxidoreductase) complex. In contrast, this signal measured in isolated thylakoids has been attributed to the activity of the protein gradient regulation-5 (PGR5)/PGR5-like (PGRL1)-associated CEF pathway. Here, we report a systematic experimentation on the origin of F_npr in isolated thylakoids. Addition of NADPH and ferredoxin to isolated spinach thylakoids resulted in the reduction of the PQ pool, but neither its kinetics nor its inhibitor sensitivities matched those of F_npr. Notably, F_npr was more rapid than PQ reduction, and completely insensitive to inhibitors of the PSII Q_B site and oxygen evolving complex as well as inhibitors of the cytochrome b₆f complex. We thus conclude that F_npr in isolated thylakoids is not a result of redox equilibrium with bulk PQ. Redox titrations and fluorescence emission spectra imply that F_npr is dependent on the reduction of a low potential redox component (E_m about − 340 mV) within photosystem II (PSII), and is likely related to earlier observations of low potential variants of Q_A within a subpopulation of PSII that is directly reducible by ferredoxin. The implications of these results for our understanding of CEF and other photosynthetic processes are discussed.     

A simple method for the purification of an antimicrobial peptide in recombinant Escherichia coli

A magainin derivative, designated MSI-344, was produced in Escherichia coli as fusion protein, by utilizing a truncated amidophsphoribosyltransferase of E. coli as a fusion partner. Bacterial cells transformed with the gene encoding the fusion protein were grown to a high cell density and induced with isopropyl-1-thio-b-D-galatoside (IPTG) to initiate product expression. The fusion protein was accumulated into cytoplasmic inclusion body and recombinant MSI-344 was released from the fusion partner by hydroxylamine treatment. Following cleavage of the fusion protein with hydroxylamine, the released MSI-344 was purified to homogeneity by cationic exchange chromatography. The final purity was at least 95% by reversed-phase high performance liquid chromatography (RP-HPLC). Purified recombinant MSI-344 was found to be indistinguishable from the synthetic peptide determined by amino acid sequences and antimicrobial activity assay.     

Characteristics of nitrogenous substrate conversion by anammox enrichment

The characteristics of nitrogenous substrates conversion by anammox enrichment were investigated using batch experiments. The anammox enrichment was proved able to convert hydroxylamine to hydrazine, as well as convert hydrazine to ammonia anaerobically, with the average conversion rates of 0.207 and 0.031 mmol gVSS⁻¹ h⁻¹. It could convert hydroxylamine and nitrite simultaneously, with ammonia as an intermediate product. The maximum conversion rates of hydroxylamine and nitrite were 0.535 and 0.145 mmol gVSS⁻¹ h⁻¹, respectively. Their conversion rates were enhanced by 26.7% and 120.7%, respectively, by raising the ratio of hydroxylamine to nitrite from 1:1 to 2:1. The characteristics of nitrogenous substrate conversion by anammox enrichment could be explained using the speculative anammox pathway based on van de Graaf model.