A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells

Whereas bacterial expression systems are widely used for production of uniformly or selectively ¹⁵N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively ¹⁵N-labeled proteins in insect cells. The quantities of ¹⁵N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the ¹⁵N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.     

Efficient expression of isotopically labeled peptides for high resolution NMR studies: application to the Cdc42/Rac binding domains of virulent kinases in Candida albicans

The production of bioactive peptides and small protein fragments is commonly achieved via solid-phase chemical synthesis. However, such techniques become unviable and prohibitively expensive when the peptides are large (e.g., >30 amino acids) or when isotope labeling is required for NMR studies. Expression and purification of large quantities of unfolded peptides in E. coli have also proved to be difficult even when the desired peptides are carried by fusion proteins such as GST. We have developed a peptide expression system that utilizes a novel fusion protein (SFC120) which is highly expressed and directs the peptides to inclusion bodies, thereby minimizing in-cell proteolysis whilst maintaining high yields of peptide expression. The expressed peptides can be liberated from the carrier protein by CNBr cleavage at engineered methionine sites or through proteolysis by specific proteases for peptides containing methionine residues. In the present systems, we use CNBr, due to the absence of methionine residues in the target peptides, although other cleavage sites can be easily inserted. We report the production of six unfolded protein fragments of different composition and lengths (19 to 48 residues) derived from the virulent effector kinases, Cla4 and Ste20 of Candida albicans. All six peptides were produced with high yields of purified material (30–40 mg/l in LB, 15–20 mg/l in M9 medium), pointing to the general applicability of this expression system for peptide production. The enrichment of these peptides with ¹⁵N, ¹⁵N/¹³C and even ¹⁵N/¹³C/²H isotopes is presented allowing speedy assignment of poorly-resolved resonances of flexible peptides.     

Differently labeled peptide ligands for rapid investigation of receptor expression on a new human glioblastoma cell line

The neuropeptides vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and substance P (SP) as well as insulin and insulin-like growth factor 1 (IGF-1) were labeled with biotin, fluorescent dyes, and radioactivity to characterize the expression of peptide receptor of a novel cancer cell line, established from a human glioblastoma multiforme. Thus, not only binding sites could be detected but advantages and disadvantages of the different labels could be compared, too. With all three markers, the presence or absence of the receptors could be answered rapidly and sensitively. The glioblastoma cells express receptors for VIP (IC₅₀ = 9 nM ± 30%), insulin (K_d = 0.66 nM ± 14%, B_max = 0.028 nM ± 13%), and IGF-1 (K_d = 21 nM ± 25%, B_max = 1.65 nM ± 24%), but there are no binding sites for NPY and SP. As especially VIP and IGF-1 receptors are expressed in huge amounts, these receptors might be an interesting target for tumor diagnostics and therapy.     

Conformational changes in truncated p47phox proteins monitored by fluorescent labeling

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the reduction of oxygen to O^– ₂ at the expense of NADPH. During activation, the cytosolic oxidase components p47^phox and p67^phox, each containing two Src homology 3 (SH3) domains, migrate to the plasma membrane. p47^phox and p67^phox associate with cytochrome b₅₅₈, a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid, as an activating agent. Activators of the oxidase in vitro cause exposure of the SH3 domains of p47^phox, which has probably been masked by the C-terminal region of this protein in a resting state. We show here that the fluorescence exhibited by the covalently labeled N,N-di-methyl-N(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine (IANBD) was increased when N-terminal-truncated p47^phox-(SH3)₂-C was treated with anionic amphiphiles. This finding was similar to the results obtained with the full-length p47^phox. However, the fluorescence of C-terminal-truncated p47^phox-N-(SH3)₂ and that of both C-terminal and N-terminal truncated p47^phox-(SH3)₂ were not altered by the activators. These results indicate that the C-terminal region of p47^phox is a primary target of the conformational change during the activation of NADPH oxidase.     

Interactions of a didomain fragment of the Drosophila Sex-lethal protein with single-stranded uridine-rich oligoribonucleotides derived from the transformer and Sex-lethal messenger RNA precursors: NMR with residue-selective [5-2H]uridine substitutions

Proteins that contain two or more copies of the RNA-binding domain [ribonucleoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to be involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, residue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the present study, we analyzed the interaction of a Drosophila Sex-lethal (Sxl) protein fragment, consisting of two RBDs (RBD1–RBD2), with two distinct target RNAs derived from the tra and Sxl mRNA precursors with guanosine and adenosine, respectively, in a position near the 5-terminus of a uridine stretch. First, we prepared a [5-²H]uridine phosphoramidite, and synthesized a series of ²H-labeled RNAs, in which all of the uridine residues except one were replaced by [5-²H]uridine in the target sequence, GU₈C. By observing the H5-H6 TOCSY cross peaks of the series of ²H-labeled RNAs complexed with the Sxl RBD1–RBD2, all of the base H5-H6 proton resonances of the target RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other target RNAs, GU₂GU₈, AU₈, and UAU₈, were assigned by comparison with those of GU₈C. We found that the uridine residue prior to the G or A residue is essential for proper interaction with the protein, and that the interaction is tighter for A than for G. Moreover, the H1 resonance assignments were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the unusual C2-endo ribose conformation in the complex.     

Insights into ATP Synthase Structure and Function Using Affinity and Site-Specific Spin Labeling

A variety of different approaches has been used during the last couple of decades to investigatestructure and function relationships within the catalytic portion of the F₀F₁-ATP synthase andof its interactions with the proton-translocator F₀. In our group, we employ ESR spectroscopywith the use of stable organic radicals, so-called spin labels, as reporter groups. The radicalsare either attached to substrates/ligands or specifically inserted into the protein structure bysite-specific spin labeling. Both approaches bear intrinsic advantages for their special usesand result in the specific information that is available through ESR, e.g., structural changesdue to binding of effector molecules (e.g., Mg²⁺ ions), conformational transitions duringcatalytic turnover, distance information on radicals bound at 20 Å or less, and information onthe binding characteristics of labeled substrates. This review summarizes the results of a varietyof different approaches we have used during the last years to study, with the help of ESRspectroscopy, the structure of the nucleotide binding sites of F₁-ATPases of different originsas well as interactions with F₀ subunits.     

Assessing the impacts of biochar-blended urea on nitrogen use efficiency and soil retention in wheat production

Improving nitrogen (N) use efficiency (NUE) in crop plants is important to reduce the negative impact of excessive N on the environment. Although biochar-blended fertilizer had been increasingly tested in crop production, the fate of fertilized N in soil and plant had not been elucidated in field conditions. In this study, a novel biochar-blended urea (BU) was prepared by pelleting maize straw biochar, bentonite, sepiolite, carboxymethylcellulose sodium, and chitosan with urea (commercial urea without biochar [CU]). N fertilization in a winter wheat field was treated with BU and CU at both 265 kg N ha^?1 (HL) and 186 kg N ha^?1 (LN). Within a treatment plot, a microplot was fertilized with ¹⁵N-labeled urea at a relevant N level. We investigated the influence of fertilizer management on biomass, grain yield, bioaccumulation of nutrient, soil properties, ¹⁵N isotopic abundance, and greenhouse gas emissions. Microscopic and spectroscopic analysis showed that micro/nanonetwork of biochar could bind N to form a loss control agglomerated particle, and organo-mineral coatings on BU may protect N from quick release. Compared with CU, BU significantly increased grain yield by 13% and 38%, and grain N allocation by 19% and 55%, respectively, at HN and LN level. The total recovery of urea ¹⁵N in wheat plant (¹⁵N based NUE) was 32.8% under CU regardless of N rates but increased to 41.7% (HN rate) and 56.3% (LN rate) under BU. Whereas, the soil proportion (soil residual ¹⁵N) was 20.1% and 13.4% under CU but 32.5% and 18.8% under BU, in 0-20cm topsoil, respectively, at HN and LN rate. Compared with the CU, BU had no effect on CO₂ and CH₄ emissions but significantly reduced the total N₂O emission by 23%–28%. These important findings suggested that BU can be beneficial to uplift plant NUE to reduce reactive N loading but boost crop production.     

N-azidoacetylmannosamine-mediated chemical tagging of gangliosides

Peracetylated N-alpha-azidoacetylmannosamine (Ac(4)ManNAz) is metabolized by cells to CMP-azidosialic acid. It has been demonstrated previously that in this way azidosialic acid-containing glycoproteins are formed that can be labeled on the cell surface by a modified Staudinger ligation. Here, we first demonstrate that the same procedure also results in the formation of azidosialic acid-containing gangliosides. Deoxymannojirimycin, an inhibitor of N-glycan processing in proteins, decreases the total cell surface labeling in Jurkat cells by approximately 25%. Inhibition of ganglioside biosynthesis with N-[5-(adamantan-1-yl-methoxy)-pentyl]1-deoxynojirimycin reduces cell surface labeling by approximately 75%. In conclusion, exposure of cells to Ac(4)ManNAz allows in vivo chemical tagging of gangliosides.     

Use of 5′-[<Emphasis Type="Italic">p</Emphasis>-(Fluorosulfonyl)benzoyl] Guanosine as an Affinity Probe for the Guanine Nucleotide-Binding Site of Transducin

Transducin (T) mediates vision in retinal rods by transmitting light signals detected by rhodopsin to a cGMP phosphodiesterase. The flow of information relies on a subunit association/dissociation cycle of T regulated by a guanine nucleotide exchange/hydrolysis reaction. 5′-[p-(Fluorosulfonyl)benzoyl] guanosine (FSBG) was synthesized and examined here as an affinity label for the guanine nucleotide binding site of T. Although the relative binding affinity of FSBG to T was much lower than for GTP and β,γ-imido-guanosine 5′-triphosphate (GMPPNP), the incorporation of FSBG to T inhibited its light-dependent [³H] GMPPNP binding activity in a concentration dependent manner. Additionally, GDP, GTP and GTP analogs hindered the binding of [³H] FSBG to T. These results demonstrated that FSBG could be used to specifically modify the active site of T. In addition, FSBG was not capable of dissociating T from T:photoactivated rhodopsin complexes, suggesting that in this case FSBG is acting as a GDP analog.     

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis

BackgroundDuring pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence.MethodsWe examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis.ResultsPancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin.ConclusionOur findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.