Rabah Soliymani

Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

BackgroundConsiderable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose.ResultsGenes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells.ConclusionThe results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings.

Antibacterial activities of temporin A analogs

Temporin A (TA) is a small, basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL‐NH2) found in the skin of the European red frog, Rana temporaria. It has variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin‐sensitive and ‐resistant Staphylococcus aureus as well as vancomycin‐resistant Enterococcus faecium strains. In this investigation the antimicrobial activity and structural characteristics of TA synthetic analogs were studied. For antibacterial activity against S. aureus and enterococcal strains, the hydrophobicity of the N‐terminal amino acid of TA was found to be important as well as a positive charge at amino acid position 7, and bulky hydrophobic side chains at positions 5 and 12. Replacing isoleucine with leucine at amino acid positions 5 and 12 resulted in the greatest enhancement of antibacterial activity. In addition, there was little difference between the activities of TA and its all‐D enantiomer, indicating that the peptide probably exerts its effect on bacteria via non‐chiral interactions with membrane lipids.

Antigenic Differences between AS03 Adjuvanted Influenza A (H1N1) Pandemic Vaccines: Implications for Pandemrix-Associated Narcolepsy Risk

Background Narcolepsy results from immune-mediated destruction of hypocretin secreting neurons in hypothalamus, however the triggers and disease mechanisms are poorly understood. Vaccine-attributable risk of narcolepsy reported so far with the AS03 adjuvanted H1N1 vaccination Pandemrix has been manifold compared to the AS03 adjuvanted Arepanrix, which contained differently produced H1N1 viral antigen preparation. Hence, antigenic differences and antibody response to these vaccines were investigated. Methods and Findings Increased circulating IgG-antibody levels to Pandemrix H1N1 antigen were found in 47 children with Pandemrix-associated narcolepsy when compared to 57 healthy children vaccinated with Pandemrix. H1N1 antigen of Arepanrix inhibited poorly these antibodies indicating antigenic difference between Arepanrix and Pandemrix. High-resolution gel electrophoresis quantitation and mass spectrometry identification analyses revealed higher amounts of structurally altered viral nucleoprotein (NP) in Pandemrix. Increased antibody levels to hemagglutinin (HA) and NP, particularly to detergent treated NP, was seen in narcolepsy. Higher levels of antibodies to NP were found in children with DQB1*06∶02 risk allele and in DQB1*06∶02 transgenic mice immunized with Pandemrix when compared to controls. Conclusions This work identified 1) higher amounts of structurally altered viral NP in Pandemrix than in Arepanrix, 2) detergent-induced antigenic changes of viral NP, that are recognized by antibodies from children with narcolepsy, and 3) increased antibody response to NP in association of DQB1*06∶02 risk allele of narcolepsy. These findings provide a link between Pandemrix and narcolepsy. Although detailed mechanisms of Pandemrix in narcolepsy remain elusive, our results move the focus from adjuvant(s) onto the H1N1 viral proteins.

Identification of a histidine-tyrosine cross-link in the active site of the cbb3-type cytochrome c oxidase from Rhodobacter sphaeroides

The heme-copper oxidases constitute a superfamily of terminal dioxygen-reducing enzymes located in the inner mitochondrial or in the bacterial cell membrane. The presence of a mechanistically important covalent bond between a histidine ligand of the copper ion (CuB) in the active site and a generally conserved tyrosine residue nearby has been shown to exist in the canonical cytochrome c oxidases. However, according to sequence alignment studies, this critical tyrosine is missing from the subfamily of cbb3-type oxidases found in certain bacteria. Recently, homology modeling has suggested that a tyrosine residue located in a different helix might fulfill this role in these enzymes. Here, we show directly by methods of protein chemistry and mass spectrometry that there is indeed a covalent link between this tyrosine and the copper-ligating histidine. The identity of the cross-linked tyrosine was determined by showing that the cross-link is not formed when this residue is replaced by phenylalanine, even though structural integrity is maintained. These results suggest a universal functional importance of the histidine-tyrosine cross-link in the mechanism of O2 reduction by all heme-copper oxidases.

Recognition of Porphyromonas gingivalis Gingipain Epitopes by Natural IgM Binding to Malondialdehyde Modified Low-Density Lipoprotein

Objective Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. Methods and Results Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR−/− mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. Conclusion Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis.

LACTB is a filament-forming protein localized in mitochondria

LACTB is a mammalian active-site serine protein that has evolved from a bacterial penicillin-binding protein. Penicillin-binding proteins are involved in the metabolism of peptidoglycan, the major bacterial cell wall constituent, implying that LACTB has been endowed with novel biochemical properties during eukaryote evolution. Here we demonstrate that LACTB is localized in the mitochondrial intermembrane space, where it is polymerized into stable filaments with a length extending more than a hundred nanometers. We infer that LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization. These findings have implications for our understanding of mitochondrial evolution and function.

A Novel Approach of Targeted Ablation of Mammary Carcinoma Cells Through Luteinizing Hormone Receptors using Hecate-CGβ Conjugate

Recent studies have shown that human and animal mammary gland carcinoma cell line express luteinizing hormone receptors (LHRs). We have examined the cytotoxic effect of Hecate-CGβ conjugate, that is, fusion of a lytic peptide (Hecate) and a 15-amino acid fragment of the CGβ-chain in vitro. To test the hypothesis that the Hecate-CGβ conjugate selectively abolishes cells possessing LHR, estrogen dependent and independent human breast cancer cell lines (MCF-7; MDA-MB-231) and a mouse Leydig tumor cell line (BLT-1) were treated in vitro with Hecate-CGβ conjugate and Hecate alone. Cytotoxic effects of the Hecate-CGβ conjugate and the Hecate alone was measured by lactate dehydrogenase (LDH) release immediately after treatment. We observed that the Hecate-CGβ conjugate selectively, in dose-dependent manner destroys cells possessing LHR in lower concentrations of preparate comparing to the Hecate alone and that the cytotoxic effect is strongly correlated with the number of LHR. Using Western blot analysis we characterized the LHR on membranes of MDA-MB-231, MCF-7 and BLT-1 tumor cell lines. In addition, we showed the evaluation of inhibition potential of the Hecate-CGβ conjugate to LHR. At a concentration of 33 µM the conjugate inhibited (50%; IC50) the binding of CG to LHR.We suggest further development of this novel approach for the treatment of breast cancer by the Hecate-CGβ for in vivo trials.

Binding of Hepcidin to Plasma Proteins

To the Editor: The hepcidin hormone of 25 amino acid residues is a key regulator of iron homeostasis (1). Hepcidin has been shown to bind in vitro to α2-macroglobulin (α2M)1 and albumin in human plasma (2). It is not known, however, to what extent hepcidin is bound to proteins in vivo. For purposes of clinical interpretation, it is critical to know whether protein-bound or free hepcidin is being quantified. To characterize the binding of hepcidin to plasma proteins, we used gel filtration to fractionate 0.5 mL serum samples from both healthy individuals and patients undergoing hematopoietic stem cell transplantation. We similarly fractionated mixtures of hepcidin and α2M or albumin [1 g/L in 10 mmol/L potassium phosphate buffer, pH 7.4, containing 150 mmol/L NaCl (PBS)]. We used a Superdex™ 200 10/300 GL column (GE Healthcare Biosciences) equilibrated with PBS at a flow rate 0.5 mL/min. The column was calibrated with synthetic hepcidin (Peptide Institute), human albumin, and α2M. We collected 0.5-mL fractions and measured hepcidin in the fractions by HPLC–tandem mass spectrometry (LC-MS/MS) as previously described (3). We observed a single peak, corresponding to …

Imaging mass spectrometry: a new tool for kidney disease investigations

Matrix-assisted laser desorption ionization (MALDI)-profiling and imaging mass spectrometry are promising technologies for measuring hundreds of different molecules directly on tissues. For instance, small molecules, drugs and their metabolites, endogenous lipids, carbohydrates and complex peptides/proteins can be measured at the same time without significant disruption of sample integrity. In this review, the potential of MALDI-profiling/imaging technologies in disease proteomics, drug action and studies of cellular processes in the context of kidney tissue is described. Spatial and sequence information obtained in tissue MALDI-profiling/imaging studies can be correlated with other mass spectrometry-based techniques, auxiliary imaging technologies and routine (immuno) histochemical staining.

Proteomic analysis of the palmitoyl protein thioesterase 1 interactome in SH-SY5Y human neuroblastoma cells

UNLABELLED Neuronal ceroid lipofuscinoses (NCL) are a group of inherited progressive childhood disorders, characterized by early accumulation of autofluorescent storage material in lysosomes of neurons or other cells. Clinical symptoms of NCL include: progressive loss of vision, mental and motor deterioration, epileptic seizures and premature death. CLN1 disease (MIM#256730) is caused by mutations in the CLN1 gene, which encodes palmitoyl protein thioesterase 1 (PPT1). In this study, we utilised single step affinity purification coupled to mass spectrometry (AP-MS) to unravel the in vivo substrates of human PPT1 in the brain neuronal cells. Protein complexes were isolated from human PPT1 expressing SH-SY5Y stable cells, subjected to filter-aided sample preparation (FASP) and analysed on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. A total of 23 PPT1 interacting partners (IP) were identified from label free quantitation of the MS data by SAINT platform. Three of the identified PPT1 IP, namely CRMP1, DBH, and MAP1B are predicted to be palmitoylated. Our proteomic analysis confirmed previously suggested roles of PPT1 in axon guidance and lipid metabolism, yet implicates the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway. BIOLOGICAL SIGNIFICANCE The significance of this work lies in the unravelling of putative in vivo substrates of human CLN1 or PPT1 in brain neuronal cells. Moreover, the PPT1 IP implicate the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway.