Quantitative proteomics reveals the Sox system's role in sulphur and arsenic metabolism of phototroph Halorhodospira halophila.

The metabolic process of purple sulphur bacteria's anoxygenic photosynthesis has been primarily studied in Allochromatium vinosum, a member of the Chromatiaceae family. However, the metabolic processes of purple sulphur bacteria from the Ectothiorhodospiraceae and Halorhodospiraceae families remain unexplored. We have analysed the proteome of Halorhodospira halophila, a member of the Halorhodospiraceae family, which was cultivated with various sulphur compounds. This analysis allowed us to reconstruct the first comprehensive sulphur-oxidative photosynthetic network for this family. Some members of the Ectothiorhodospiraceae family have been shown to use arsenite as a photosynthetic electron donor. Therefore, we analysed the proteome response of Halorhodospira halophila when grown under arsenite and sulphide conditions. Our analyses using ion chromatography-inductively coupled plasma mass spectrometry showed that thioarsenates are chemically formed under these conditions. However, they are more extensively generated and converted in the presence of bacteria, suggesting a biological process. Our quantitative proteomics revealed that the SoxAXYZB system, typically dedicated to thiosulphate oxidation, is overproduced under these growth conditions. Additionally, two electron carriers, cytochrome c551/c5 and HiPIP III, are also overproduced. Electron paramagnetic resonance spectroscopy suggested that these transporters participate in the reduction of the photosynthetic Reaction Centre. These results support the idea of a chemically and biologically formed thioarsenate being oxidized by the Sox system, with cytochrome c551/c5 and HiPIP III directing electrons towards the Reaction Centre.

Posttranslational regulation of photosynthetic activity via the TOR kinase in plants.

Chloroplasts are the powerhouse of the plant cell, and their activity must be matched to plant growth to avoid photooxidative damage. We have identified a posttranslational mechanism linking the eukaryotic target of rapamycin (TOR) kinase that promotes growth and the guanosine tetraphosphate (ppGpp) signaling pathway of prokaryotic origins that regulates chloroplast activity and photosynthesis in particular. We find that RelA SpoT homolog 3 (RSH3), a nuclear-encoded enzyme responsible for ppGpp biosynthesis, interacts directly with the TOR complex via a plant-specific amino-terminal region which is phosphorylated in a TOR-dependent manner. Down-regulating TOR activity causes a rapid increase in ppGpp synthesis in RSH3 overexpressors and reduces photosynthetic capacity in an RSH-dependent manner in wild-type plants. The TOR-RSH3 signaling axis therefore regulates the equilibrium between chloroplast activity and plant growth, setting a precedent for the regulation of organellar function by TOR.

Combining metabolic flux analysis with proteomics to shed light on the metabolic flexibility: the case of Desulfovibrio vulgaris Hildenborough.

Introduction: Desulfovibrio vulgaris Hildenborough is a gram-negative anaerobic bacterium belonging to the sulfate-reducing bacteria that exhibits highly versatile metabolism. By switching from one energy mode to another depending on nutrients availability in the environments" it plays a central role in shaping ecosystems. Despite intensive efforts to study D. vulgaris energy metabolism at the genomic, biochemical and ecological level, bioenergetics in this microorganism remain far from being fully understood. Alternatively, metabolic modeling is a powerful tool to understand bioenergetics. However, all the current models for D. vulgaris appeared to be not easily adaptable to various environmental conditions. Methods: To lift off these limitations, here we constructed a novel transparent and robust metabolic model to explain D. vulgaris bioenergetics by combining whole-cell proteomic analysis with modeling approaches (Flux Balance Analysis). Results: The iDvu71 model showed over 0.95 correlation with experimental data. Further simulations allowed a detailed description of D. vulgaris metabolism in various conditions of growth. Altogether, the simulations run in this study highlighted the sulfate-to-lactate consumption ratio as a pivotal factor in D. vulgaris energy metabolism. Discussion: In particular, the impact on the hydrogen/formate balance and biomass synthesis is discussed. Overall, this study provides a novel insight into D. vulgaris metabolic flexibility.

Acinetobacter type VI secretion system comprises a non-canonical membrane complex.

A. baumannii can rapidly acquire new resistance mechanisms and persist on abiotic surface, enabling the colonization of asymptomatic human host. In Acinetobacter the type VI secretion system (T6SS) is involved in twitching, surface motility and is used for interbacterial competition allowing the bacteria to uptake DNA. A. baumannii possesses a T6SS that has been well studied for its regulation and specific activity, but little is known concerning its assembly and architecture. The T6SS nanomachine is built from three architectural sub-complexes. Unlike the baseplate (BP) and the tail-tube complex (TTC), which are inherited from bacteriophages, the membrane complex (MC) originates from bacteria. The MC is the most external part of the T6SS and, as such, is subjected to evolution and adaptation. One unanswered question on the MC is how such a gigantesque molecular edifice is inserted and crosses the bacterial cell envelope. The A. baumannii MC lacks an essential component, the TssJ lipoprotein, which anchors the MC to the outer membrane. In this work, we studied how A. baumannii compensates the absence of a TssJ. We have characterized for the first time the A. baumannii's specific T6SS MC, its unique characteristic, its membrane localization, and assembly dynamics. We also defined its composition, demonstrating that its biogenesis employs three Acinetobacter-specific envelope-associated proteins that define an intricate network leading to the assembly of a five-proteins membrane super-complex. Our data suggest that A. baumannii has divided the function of TssJ by (1) co-opting a new protein TsmK that stabilizes the MC and by (2) evolving a new domain in TssM for homo-oligomerization, a prerequisite to build the T6SS channel. We believe that the atypical species-specific features we report in this study will have profound implication in our understanding of the assembly and evolutionary diversity of different T6SSs, that warrants future investigation.

An essential role of the reversible electron-bifurcating hydrogenase Hnd for ethanol oxidation in Solidesulfovibrio fructosivorans.

The tetrameric cytoplasmic FeFe hydrogenase Hnd from Solidesulfovibrio fructosivorans (formely Desulfovibrio fructosovorans) catalyses H2 oxidation and couples the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin by using a flavin-based electron-bifurcating mechanism. Regarding its implication in the bacterial physiology, we previously showed that Hnd, which is non-essential when bacteria grow fermentatively on pyruvate, is involved in ethanol metabolism. Under these conditions, it consumes H2 to produce reducing equivalents for ethanol production as a fermentative product. In this study, the approach implemented was to compare the two S. fructosivorans WT and the hndD deletion mutant strains when grown on ethanol as the sole carbon and energy source. Based on the determination of bacterial growth, metabolite consumption and production, gene expression followed by RT-q-PCR, and Hnd protein level followed by mass spectrometry, our results confirm the role of Hnd hydrogenase in the ethanol metabolism and furthermore uncover for the first time an essential function for a Desulfovibrio hydrogenase. Hnd is unequivocally required for S. fructosivorans growth on ethanol, and we propose that it produces H2 from NADH and reduced ferredoxin generated by an alcohol dehydrogenase and an aldehyde ferredoxin oxidoreductase catalyzing the conversion of ethanol into acetate. The produced H2 could then be recycled and used for sulfate reduction. Hnd is thus a reversible hydrogenase that operates in H2-consumption by an electron-bifurcating mechanism during pyruvate fermentation and in H2-production by an electron-confurcating mechanism when the bacterium uses ethanol as electron donor.

Carbon Fixation in the Chemolithoautotrophic Bacterium Aquifex aeolicus Involves Two Low-Potential Ferredoxins as Partners of the PFOR and OGOR Enzymes.

Aquifex aeolicus is a microaerophilic hydrogen- and sulfur -oxidizing bacterium that assimilates CO2 via the reverse tricarboxylic acid cycle (rTCA). Key enzymes of this pathway are pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OGOR), which are responsible, respectively, for the reductive carboxylation of acetyl-CoA to pyruvate and of succinyl-CoA to 2-oxoglutarate, two energetically unfavorable reactions that require a strong reduction potential. We have confirmed, by biochemistry and proteomics, that A. aeolicus possesses a pentameric version of these enzyme complexes ((αßγδε)2) and that they are highly abundant in the cell. In addition, we have purified and characterized, from the soluble fraction of A. aeolicus, two low redox potential and oxygen-stable [4Fe-4S] ferredoxins (Fd6 and Fd7, E0 = -440 and -460 mV, respectively) and shown that they can physically interact and exchange electrons with both PFOR and OGOR, suggesting that they could be the physiological electron donors of the system in vivo. Shotgun proteomics indicated that all the enzymes assumed to be involved in the rTCA cycle are produced in the A. aeolicus cells. A number of additional enzymes, previously suggested to be part of a putative partial Wood-Ljungdahl pathway used for the synthesis of serine and glycine from CO2 were identified by mass spectrometry, but their abundance in the cell seems to be much lower than that of the rTCA cycle. Their possible involvement in carbon assimilation is discussed.

PrkA is an ATP-dependent protease that regulates sporulation in Bacillus subtilis.

In Bacillus subtilis, sporulation is a sequential and highly regulated process. Phosphorylation events by histidine kinases are key points in the phosphorelay that initiates sporulation, but serine/threonine protein kinases also play important auxiliary roles in this regulation. PrkA has been proposed to be a serine protein kinase expressed during the initiation of sporulation and involved in this differentiation process. Additionally, the role of PrkA in sporulation has been previously proposed to be mediated via the transition phase regulator ScoC, which in turn regulates the transcriptional factor σK and its regulon. However, the kinase activity of PrkA has not been clearly demonstrated, and neither its autophosphorylation nor phosphorylated substrates have been unambiguously established in B. subtilis. We demonstrated here that PrkA regulation of ScoC is likely indirect. Following bioinformatic homology searches, we revealed sequence similarities of PrkA with the ATPases associated with diverse cellular activities ATP-dependent Lon protease family. Here, we showed that PrkA is indeed able to hydrolyze α-casein, an exogenous substrate of Lon proteases, in an ATP-dependent manner. We also showed that this ATP-dependent protease activity is essential for PrkA function in sporulation since mutation in the Walker A motif leads to a sporulation defect. Furthermore, we found that PrkA protease activity is tightly regulated by phosphorylation events involving one of the Ser/Thr protein kinases of B. subtilis, PrkC. Taken together, our results clarify the key role of PrkA in the complex process of B. subtilis sporulation.

Characterization of the Achromobacter xylosoxidans Type VI Secretion System and Its Implication in Cystic Fibrosis.

Bacteria of the genus Achromobacter are environmental germs, with an unknown reservoir. It can become opportunistic pathogens in immunocompromised patients, causing bacteremia, meningitis, pneumonia, or peritonitis. In recent years, Achromobacter xylosoxidans has emerged with increasing incidence in patients with cystic fibrosis (CF). Recent studies showed that A. xylosoxidans is involved in the degradation of the respiratory function of patients with CF. The respiratory ecosystem of patients with CF is colonized by bacterial species that constantly fight for space and access to nutrients. The type VI secretion system (T6SS) empowers this constant bacterial antagonism, and it is used as a virulence factor in several pathogenic bacteria. This study aimed to investigate the prevalence of the T6SS genes in A. xylosoxidans isolated in patients with CF. We also evaluated clinical and molecular characteristics of T6SS-positive A. xylosoxidans strains. We showed that A. xylosoxidans possesses a T6SS gene cluster and that some environmental and clinical isolates assemble a functional T6SS nanomachine. A. xylosoxidans T6SS is used to target competing bacteria, including other CF-specific pathogens. Finally, we demonstrated the importance of the T6SS in the internalization of A. xylosoxidans in lung epithelial cells and that the T6SS protein Hcp is detected in the sputum of patients with CF. Altogether, these results suggest for the first time a role of T6SS in CF-lung colonization by A. xylosoxidans and opens promising perspective to target this virulence determinant as innovative theranostic options for CF management.

Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants.

The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin-Benson-Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.

The electron-bifurcating FeFe-hydrogenase Hnd is involved in ethanol metabolism in Desulfovibrio fructosovorans grown on pyruvate.

Desulfovibrio fructosovorans, a sulfate-reducing bacterium, possesses six gene clusters encoding six hydrogenases catalyzing the reversible oxidation of H2 into protons and electrons. Among them, Hnd is an electron-bifurcating hydrogenase, coupling the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin with electrons derived from H2 . It was previously hypothesized that its biological function involves the production of NADPH necessary for biosynthetic purposes. However, it was subsequently demonstrated that Hnd is instead a NAD+ -reducing enzyme, thus its specific function has yet to be established. To understand the physiological role of Hnd in D. fructosovorans, we compared the hnd deletion mutant with the wild-type strain grown on pyruvate. Growth, metabolite production and consumption, and gene expression were compared under three different growth conditions. Our results indicate that hnd is strongly regulated at the transcriptional level and that its deletion has a drastic effect on the expression of genes for two enzymes, an aldehyde ferredoxin oxidoreductase and an alcohol dehydrogenase. We demonstrated here that Hnd is involved in ethanol metabolism when bacteria grow fermentatively and proposed that Hnd might oxidize part of the H2 produced during fermentation generating both NADH and reduced ferredoxin for ethanol production via its electron bifurcation mechanism.

Identification and characterization of a noncanonical menaquinone-linked formate dehydrogenase.

The molybdenum/tungsten-bis-pyranopterin guanine dinucleotide family of formate dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting because of their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates used by partner subunits interacting with the catalytic hub. Here, we unveiled two noncanonical FDHs in Bacillus subtilis, which are organized into two-subunit complexes with unique features, ForCE1 and ForCE2. We show that the formate oxidoreductase catalytic subunit interacts with an unprecedented partner subunit, formate oxidoreductase essential subunit, and that its amino acid sequence within the active site deviates from the consensus residues typically associated with FDH activity, as a histidine residue is naturally substituted with a glutamine. The formate oxidoreductase essential subunit mediates the utilization of menaquinone as an electron acceptor as shown by the formate:menadione oxidoreductase activity of both enzymes, their copurification with menaquinone, and the distinctive detection of a protein-bound neutral menasemiquinone radical by multifrequency electron paramagnetic resonance (EPR) experiments on the purified enzymes. Moreover, EPR characterization of both FDHs reveals the presence of several [Fe-S] clusters with distinct relaxation properties and a weakly anisotropic Mo(V) EPR signature, consistent with the characteristic molybdenum/bis-pyranopterin guanine dinucleotide cofactor of this enzyme family. Altogether, this work enlarges our knowledge of the FDH family by identifying a noncanonical FDH, which differs in terms of architecture, amino acid conservation around the molybdenum cofactor, and reactivity.

Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts.

In the chloroplast, Calvin-Benson-Bassham enzymes are active in the reducing environment created in the light by electrons from the photosystems. In the dark, these enzymes are inhibited, mainly caused by oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent, measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges is simultaneous. In oxidized CP12, the C23-C31 pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C66-C75 pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment: a cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66-C75 pair than for the C23-C31 pair. The redox mid-potentials for the two cysteine pairs differ and are similar to those found for glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase, consistent with the regulatory role of CP12.

A new type of flexible CP12 protein in the marine diatom Thalassiosira pseudonana.

BACKGROUND: CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background. METHODS: A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12. RESULTS: Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions. CONCLUSIONS: These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle. Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta. While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism. Video Abstract.

ppGpp influences protein protection, growth and photosynthesis in Phaeodactylum tricornutum.

Chloroplasts retain elements of a bacterial stress response pathway that is mediated by the signalling nucleotides guanosine penta- and tetraphosphate ((p)ppGpp). In the model flowering plant Arabidopsis, ppGpp acts as a potent regulator of plastid gene expression and influences photosynthesis, plant growth and development. However, little is known about ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here, we studied the function of ppGpp in the diatom Phaeodactylum tricornutum using transgenic lines containing an inducible system for ppGpp accumulation. We used these lines to investigate the effects of ppGpp on growth, photosynthesis, lipid metabolism and protein expression. We demonstrate that ppGpp accumulation reduces photosynthetic capacity and promotes a quiescent-like state with reduced proliferation and ageing. Strikingly, using nontargeted proteomics, we discovered that ppGpp accumulation also leads to the coordinated upregulation of a protein protection response in multiple cellular compartments. Our findings highlight the importance of ppGpp as a fundamental regulator of chloroplast function across different domains of life, and lead to new questions about the molecular mechanisms and roles of (p)ppGpp signalling in photosynthetic eukaryotes.

Zinc Binds to RRM2 Peptide of TDP-43.

Transactive response DNA and RNA binding protein 43 kDa (TDP-43) is a highly conserved heterogeneous nuclear ribonucleoprotein (hnRNP), which is involved in several steps of protein production including transcription and splicing. Its aggregates are frequently observed in motor neurons from amyotrophic lateral sclerosis patients and in the most common variant of frontotemporal lobar degeneration. Recently it was shown that TDP-43 is able to bind Zn2+ by its RRM domain. In this work, we have investigated Zn2+ binding to a short peptide 256-264 from C-terminus of RRM2 domain using isothermal titration calorimetry, electrospray ionization mass spectrometry, QM/MM simulations, and NMR spectroscopy. We have found that this peptide is able to bind zinc ions with a Ka equal to 1.6 × 105 M-1. Our findings suggest the existence of a zinc binding site in the C-terminal region of RRM2 domain. Together with the existing structure of the RRM2 domain of TDP-43 we propose a model of its complex with Zn2+ which illustrates how zinc might regulate DNA/RNA binding.

Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices.

Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ.

Interaction between Silver Nanoparticles and Two Dehydrogenases: Role of Thiol Groups.

Widely used silver nanoparticles (AgNPs) are readily accessible to biological fluids and then surrounded by proteins. However, interactions between AgNPs and proteins are poorly understood. Two dehydrogenases, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and malate dehydrogenase (MDH), are chosen to investigate these interactions. Ag bound to thiol groups of these enzymes significantly decreases the number of free thiols available. Dose-dependent inhibition of enzyme activities is observed in both AgNPs and Ag+ treatments. Based on the concentration required to inhibit 50% activity, GAPDH and MDH are 24-30 fold more sensitive to Ag+ than to AgNPs suggesting that the measured 4.2% Ag+ containing AgNPs can be responsible for the enzymes inhibition. GAPDH, with a thiol group in its active site, is more sensitive to Ag than MDH, displaying many thiol groups but none in its active site, suggesting that thiol groups at the active site strongly determines the sensitivity of enzymes toward AgNPs. In contrast, the dramatic changes of circular dichroism spectra show that the global secondary structure of MDH under AgNPs treatment is more altered than that of GAPDH. In summary, this study shows that the thiol groups and their location on these dehydrogenases are crucial for the AgNPs effects.

Identification of a new natural gastric lipase inhibitor from star anise.

The identification and isolation of bioactive compounds are of great interest in the drug delivery field, despite being a difficult task. We describe here an innovative strategy for the identification of a new gastric lipase inhibitor from star anise for the treatment of obesity. After plant screening assays for gastric lipase inhibition, star anise was selected and investigated by bioactivity guided fractionation. MALDI-TOF mass spectrometry and peptide mass fingerprinting allowed the detection of an inhibitor covalently bound to the catalytic serine of gastric lipase. A mass-directed screening approach using UPLC-HRMS and accurate mass determination searching identified the flavonoid myricitrin-5-methyl ether (M5ME) as a lipase inhibitor. The inhibitory activity was rationalized based on molecular docking, showing that M5ME is susceptible to nucleophilic attack by gastric lipase. Overall, our data suggest that M5ME may be considered as a potential candidate for future application as a gastric lipase inhibitor for the treatment of obesity.

The Pkn22 Kinase of Nostoc PCC 7120 Is Required for Cell Differentiation via the Phosphorylation of HetR on a Residue Highly Conserved in Genomes of Heterocyst-Forming Cyanobacteria.

Hanks-type kinases encoding genes are present in most cyanobacterial genomes. Despite their widespread pattern of conservation, little is known so far about their role because their substrates and the conditions triggering their activation are poorly known. Here we report that under diazotrophic conditions, normal heterocyst differentiation and growth of the filamentous cyanobacterium Nostoc PCC 7120 require the presence of the Pkn22 kinase, which is induced under combined nitrogen starvation conditions. By analyzing the phenotype of pkn22 mutant overexpressing genes belonging to the regulatory cascade initiating the development program, an epistatic relationship was found to exist between this kinase and the master regulator of differentiation, HetR. The results obtained using a bacterial two hybrid approach indicated that Pkn22 and HetR interact, and the use of a genetic screen inducing the loss of this interaction showed that residues of HetR which are essential for this interaction to occur are also crucial to HetR activity both in vitro and in vivo. Mass spectrometry showed that HetR co-produced with the Pkn22 kinase in Escherichia coli is phosphorylated on Serine 130 residue. Phosphoablative substitution of this residue impaired the ability of the strain to undergo cell differentiation, while its phosphomimetic substitution increased the number of heterocysts formed. The Serine 130 residue is part of a highly conserved sequence in filamentous cyanobacterial strains differentiating heterocysts. Heterologous complementation assays showed that the presence of this domain is necessary for heterocyst induction. We propose that the phosphorylation of HetR might have been acquired to control heterocyst differentiation.

Characterization of pepsin from rabbit gastric extract, its action on ß-casein and the effects of lipids on proteolysis.

Rabbit gastric extract (RGE) is a source of gastric enzymes for in vitro digestion studies. While its gastric lipase activity has been characterized and compared to other lipases, its pepsin activity has not been studied. We measured pepsin activity in RGE using both hemoglobin and azocoll as substrates, and identified the protein separated by SDS-PAGE as a type II-4 mature pepsin of 328 amino acid residues using Edman sequencing, LC-MS/MS analysis and intact mass measurement. As a proof-of-concept that RGE was suitable for in vitro digestion of both proteins and lipids, it was used for studying the proteolysis of ß-casein under conditions mimicking the early stages of intragastric digestion. ß-Casein was displayed either in solution or at the surface of a ß-casein-stabilized rapeseed oil emulsion to investigate the impact of lipids and lipolysis on proteolysis. Proteolysis of ß-casein was quantified based on the kinetics of ß-casein disappearance, the identification of various peptides generated upon digestion and their variation with time. The results obtained with RGE were highly similar to those obtained with equivalent amounts of porcine pepsin used as a reference standard. Digestion of ß-casein was slower when it was displayed at the oil-water interface and some degradation peptides were transiently observed at higher levels and for a longer time than with ß-casein in solution, or accumulated upon digestion. N-terminal sequencing of the main isolated peptides revealed a sequential action of pepsin starting from the hydrophobic C-terminal end of ß-casein, which was impaired by the interaction of ß-casein with lipids.