Optimization of recombinant human nerve growth factor production in the psychrophilic Pseudoalteromonas haloplanktis

The optimization of production strategy is a very useful tool to attain high level of recombinant protein at a low cost. A promising biotechnological application of psychrophilic bacteria is their use as non-conventional host for the recombinant production of useful proteins. The lowering of the expression temperature can in fact facilitate the correct folding of heterologous proteins that accumulate in insoluble form as inclusion bodies when produced in Escherichia coli. An example of such "difficult" proteins is the human nerve growth factor (hNGF). The gene encoding the mature form of hNGF was expressed in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 at 4 degrees C. Western blotting experiments demonstrated that the protein was produced in soluble form and translocated in the periplasmic space. Furthermore, an analytical gel filtration chromatography confirmed that the recombinant protein was largely in dimeric form. For a more efficient recombinant rhNGF production, the influence of cultivation operational strategies and growth conditions (medium composition, temperature, specific growth rate) on biomass yield and recombinant protein production was investigated in batch and chemostat cultivations. The highest product yield of soluble rhNGF (7.5mg(NGF)g(dryweight)(-1)) has been achieved in batch culture at 4 degrees C on Schatz medium with addition of tryptone and vitamins.     

Superoxide Radical-Mediated Alteration of Synaptosome Membrane Structure and High-Affinity γ-[14C]Aminobutyric Acid Uptake

Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+-ATPase) than the low-affinity system.     

CD1a and MHC class I follow a similar endocytic recycling pathway

CD1 proteins are a family of major histocompatibility complex (MHC) class I-like antigen-presenting molecules that present lipids to T cells. The cytoplasmic tails (CTs) of all human CD1 isoforms, with the exception of CD1a, contain tyrosine-based sorting motifs, responsible for the internalization of proteins by the clathrin-mediated pathway. The role of the CD1a CT, which does not possess any sorting motifs, as well as its mode of internalization are not known. We investigated the internalization and recycling pathways followed by CD1a and the role of its CT. We found that CD1a can be internalized by a clathrin- and dynamin-independent pathway and that it follows a Rab22a- and ADP ribosylation factor (ARF)6-dependent recycling pathway, similar to other cargo internalized independent of clathrin. We also found that the CD1a CT is S-acylated. However, this posttranslational modification does not determine the rate of internalization or recycling of the protein or its localization to detergent-resistant membrane microdomains (DRMs) where we found CD1a to be enriched. We also show that plasma membrane DRMs are essential for efficient CD1a-mediated antigen presentation. These findings place CD1a closer to MHC class I in its trafficking and potential antigen-loading compartments among CD1 isoforms. Furthermore, we identify CD1a as a new marker for the clathrin- and dynamin-independent and DRM-dependent pathway of internalization as well as the Rab22a- and ARF6-dependent recycling pathway.     

The prediction of the circular dichroism of the benzene chromophore: TDDFT calculations and sector rules

The CD spectra of the series PhCH(Me)R, with R = Et (1), nPr (2), iPr (3), and tBu (4), are reported (1-3 for the first time) at room temperature in the 185-280 nm range and at 183 K. These purely hydrocarbon compounds represent the simplest chiral systems containing the phenyl chromophore and exhibit Cotton effects exclusively allied with the benzene transitions. The bands in 1La and 1Lb regions were checked against the available sector rules, with discordant outcomes. Time-dependent density-functional theory calculations, with various functionals and basis sets tested, correctly reproduced the prominent CD bands observed for 1-4.     

Synthesis and biological evaluation of alpha-galactosylceramide (KRN7000) and isoglobotrihexosylceramide (iGb3)

Glycoceramides can activate NKT cells by binding with CD1d to produce IFN-gamma, IL-4, and other cytokines. An efficient synthetic pathway for alpha-galactosylceramide (KRN7000) was established by coupling a protected galactose donor to a properly protected ceramide. During the investigation, it was discovered that when the ceramide was protected with benzyl groups, only beta-galactosylceramide was produced from the glycosylation reaction. In contrast, the ceramide with benzoyl protecting groups produced alpha-galactosylceramide. Isoglobotrihexosylceramide (iGb3) was prepared by glycosylation of Galalpha1-3Galbeta1-4Glc donor with 2-azido-sphingosine in high yield. Biological assays on the synthetic KRN7000 and iGb3 were performed using human and murine iNKT cell clones or hybridomas.     

d(CGGTGGT) forms an octameric parallel G-quadruplex via stacking of unusual G(:C):G(:C):G(:C):G(:C) octads

Among non-canonical DNA secondary structures, G-quadruplexes are currently widely studied because of their probable involvement in many pivotal biological roles, and for their potential use in nanotechnology. The overall quadruplex scaffold can exhibit several morphologies through intramolecular or intermolecular organization of G-rich oligodeoxyribonucleic acid strands. In particular, several G-rich strands can form higher order assemblies by multimerization between several G-quadruplex units. Here, we report on the identification of a novel dimerization pathway. Our Nuclear magnetic resonance, circular dichroism, UV, gel electrophoresis and mass spectrometry studies on the DNA sequence dCGGTGGT demonstrate that this sequence forms an octamer when annealed in presence of K(+) or NH(4)(+) ions, through the 5'-5' stacking of two tetramolecular G-quadruplex subunits via unusual G(:C):G(:C):G(:C):G(:C) octads.     

Absolute Configuration of Oplopanone Derivatives From Serphidium stenocephalum: ECD Spectra of Acyclic Ketones With Front‐Octant Contributions

The electronic circular dichroism (ECD) spectra of two sesquiterpenoids ( 1 and 2 ) related to oplopanone, obtained from a methanolic extract of the plant Serphidium stenocephalum (Artemisia stenocephala), were measured and reproduced by means of time‐dependent density functional theory (TDDFT) calculations, establishing their absolute configuration. The application of ketone octant rule for carbonyl n‐π* ECD band to compounds 1 and 2 , which include an acyclic carbonyl group, was critically assessed. The peculiar oplopanone skeleton makes a straightforward application of the octant rule impossible, because of the uncertainty related to the shape of the so‐called third nodal surface separating front and back octants. The various group contributions to the carbonyl n‐π* ECD band were estimated with TDDFT calculations on selected molecular models obtained by consecutive dissections from 1 . Chirality 26:39–43, 2013. © 2013 Wiley Periodicals, Inc.     

Quantitative Chemical Proteomics Identifies Novel Targets of the Anti-cancer Multi-kinase Inhibitor E-3810

Novel drugs are designed against specific molecular targets, but almost unavoidably they bind non-targets, which can cause additional biological effects that may result in increased activity or, more frequently, undesired toxicity. Chemical proteomics is an ideal approach for the systematic identification of drug targets and off-targets, allowing unbiased screening of candidate interactors in their natural context (tissue or cell extracts).E-3810 is a novel multi-kinase inhibitor currently in clinical trials for its anti-angiogenic and anti-tumor activity. In biochemical assays, E-3810 targets primarily vascular endothelial growth factor and fibroblast growth factor receptors. Interestingly, E-3810 appears to inhibit the growth of tumor cells with low to undetectable levels of these proteins in vitro, suggesting that additional relevant targets exist. We applied chemical proteomics to screen for E-3810 targets by immobilizing the drug on a resin and exploiting stable isotope labeling by amino acids in cell culture to design experiments that allowed the detection of novel interactors and the quantification of their dissociation constant (K_{d imm}) for the immobilized drug. In addition to the known target FGFR2 and PDGFRα, which has been described as a secondary E-3810 target based on in vitro assays, we identified six novel candidate kinase targets (DDR2, YES, LYN, CARDIAK, EPHA2, and CSBP). These kinases were validated in a biochemical assay and—in the case of the cell-surface receptor DDR2, for which activating mutations have been recently discovered in lung cancer—cellular assays.Taken together, the success of our strategy—which integrates large-scale target identification and quality-controlled target affinity measurements using quantitative mass spectrometry—in identifying novel E-3810 targets further supports the use of chemical proteomics to dissect the mechanism of action of novel drugs.The “target deconvolution” process, namely, the identification and characterization of proteins bound by a drug of interest (1), is a crucial step in drug development that allows definition of the compound selectivity and the early detection of potential side effects. Target deconvolution can be achieved by means of systematic in vitro biochemical assays measuring the ability of the drug to interact with candidate binders and, if they are enzymes, interfere with their activity. An alternative approach is chemical proteomics (chemoproteomics), which combines affinity chromatography and proteomic techniques (2, 3). Up-to-date chemical proteomics essentially consists of three main steps: (i) drug immobilization on a solid phase; (ii) drug affinity chromatography to capture drug targets in complex protein mixtures, such as cell or tissue lysates; and (iii) mass spectrometry (MS)-based¹ identification of the proteins retained by the immobilized drug (4–6).In chemical proteomics, the affinity chromatography step is typically performed under mild conditions, to allow the identification of all possible natural binders. The drawback of using mild, non-denaturing conditions is the significant number of proteins nonspecifically binding to the solid phase, which, once identified via MS, can be difficult to discern from genuine drug targets. The relatively high number of such nonspecific binders has limited the widespread use of this strategy.More recently, the development and implementation of quantitative strategies in proteomics based on the use of differentially stable isotopes to label proteomes from distinct functional states, together with significant technological and instrumental developments in the MS field concerning sensitivity and throughput, have largely allowed this limitation to be overcome. One of the most popular labeling techniques is stable isotope labeling by amino acids in cell culture (SILAC) (7). In SILAC, dividing cells are cultured in media supplemented with amino acids containing stable isotopic variants of carbon (¹²C/¹³C), nitrogen (¹⁴N/¹⁵N), or hydrogen (¹H/²H), which are incorporated into newly synthesized proteins during cell division. When extensive labeling (>98%) of cells is achieved upon the appropriate number of replications, light and heavy cells are differentially treated (e.g. exposed to drug versus vehicle), mixed in equal proportion, and subjected to proteomics analysis by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Peptides from the two functional states can be distinguished by their specific delta mass values, and their intensity ratio in MS spectra is directly proportional to the relative abundance of the corresponding proteins in the initial protein extract. Robust analysis of SILAC data is possible with dedicated software, such as MaxQuant (8). The application of SILAC strategies to interactomic studies is an efficient means of discerning specific from background binders (9). When applied to chemical proteomics, quantitative proteomics is crucial, as it offers quality filters to discern genuine drug interactors from proteins binding to the solid phase, with the use of different experimental setups (4, 5).In this study, we successfully coupled SILAC with chemical proteomics to carry out an unbiased screening of protein interactors of the anti-cancer drug E-3810, currently in Phase II clinical trials. E-3810 is a novel multi-kinase inhibitor, a class of targeted drug that comprises different molecules currently used in clinical practice (e.g. imatinib, dasatinib, sunitinib, sorafenib) (10). E-3810 exhibits both anti-tumor and anti-angiogenic properties (11). In preclinical studies, E-3810 showed broad anti-tumor activity in vivo, when used as monotherapy in a variety of human xenografts, or in conjunction with conventional chemotherapy (11, 12).Cellular vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs) are the principal targets of E-3810, as previously demonstrated by in vitro kinase assays, which showed that E-3810 inhibited VEGFR-1, -2, and -3 and FGFR-1 and -2 in the nanomolar range (11). Studies performed on several kinase inhibitors demonstrated that these molecules can elicit pleiotropic effects not easily explained by the sole inhibition of their known targets (13, 14). These effects are in most cases due to an inhibitory activity of the drug on additional kinase targets not tested in vitro that may lead to synergistic anti-cancer effects or undesirable toxicity. This could also be the case for E-3810, which was shown to inhibit in vitro additional kinase targets with high affinity, and which is able to inhibit the growth of tumor cells expressing low to undetectable levels of VEGFRs/FGFRs, suggesting that its spectrum of target inhibition has not been fully explored (11).We thus established a SILAC-based chemical proteomic platform composed of a set of affinity chromatography experiments using E-3810 immobilized on agarose resin and incubated with SILAC-labeled extract from the ovarian cancer cell line A2780. We identified proteins interacting with the resin via MS and took advantage of SILAC-based protein quantitation to discern genuine from background binders and derive quantitative information about the specific interactions. Our findings demonstrate that additional targets of E-3810 exist and that these targets may contribute to the anticancer effect of E-3810.