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BBA - General Subjects (v.1760, #8)

Editorial Board (pp. ii).

Factors mediating lipofection potency of a series of cationic phosphonolipids in human cell lines by Daphne Koumbi; Jean-Claude Clement; Zili Sideratou; Jean-Jacques Yaouanc; Dimitris Loukopoulos; Panagoula Kollia (pp. 1151-1159).

A series of cationic liposomes known as cationic phosphonolipids (CPs) were evaluated as vehicles for in vitro gene transfer in K562 erythroleukemia cells and 5637 epithelial carcinoma cells. For each CP and target cell type examined, detailed analyses were performed to determine optimal transfection conditions (lipid/ DNA (+/−) charge ratio, amount of complexed episomal DNA, liposomal and lipoplex size, complexation medium and duration of complex-cell exposure time). Lipofection conditions were determined to be both cell- and lipid-type specific. Complexation medium critically affected transfection competence. The initial size of the liposome was not always predictive of lipofection potency. The lipid chemical composition had a strong impact upon lipofection efficiency; DOPE inclusion in the liposome formulations was found to affect the levels of transgene expression in a cell-dependent way. Notably, effective transgene expression was characterized by prominent plasmid nuclear incorporation. Human Aγ- and ε-globin transgene nuclear incorporation and expression in 5637 cells post GLB.391-mediated lipofection lends credence to its use as a vehicle of therapeutic transgene delivery.

Keywords: Cationic phosphonolipid; Lipofection condition; Gene transfer; Transgene expression; Nuclear DNA incorporation

Subcellular distribution of Plp-40, a lipoprotein in a serotype A strain of Pasteurella multocida by Margaret N. Nsofor; Phillip E. Ryals; Franklin R. Champlin (pp. 1160-1166).

A 40-kDa lipoprotein (Plp-40) is expressed by serotype A strains of Pasteurella multocida in amounts which correlate with the amount of capsular material present. We hypothesized that Plp-40 is exposed at the outer surface of the outer membrane (OM) of the cell and is associated with the serotype A exopolysaccharide material. The objectives of the present study were to confirm the lipoprotein nature of Plp-40 and to determine its subcellular location. Plp-40 maturation was shown to be sensitive to globomycin, thereby confirming it to be a bacterial lipoprotein. Plp-40 was shown to be present in the OM fractions of P. multocida obtained by both sarkosyl extraction and sucrose density gradient centrifugation, as well as in capsule fractions obtained by either hyaluronidase treatment or warm buffer extraction. [³H]palmitic acid-labeled Plp-40 could be removed from the surface of whole cells by exposure to proteinase K. Autoradiography of¹²⁵I-labeled cell surface proteins exhibited a 40-kDa band that was prominent in capsulated strains and greatly diminished in a noncapsulated strain. These results support the hypothesis that Plp-40 is a lipid-modified OM protein, which is exposed on the outer cell surface and is likely associated with serotype A extracellular polysaccharide.

Keywords: Pasteurella multocida; Outer membrane lipoprotein; Cell envelope; Capsule

Antibodies and Fab fragments protect Cu,Zn-SOD against methylglyoxal-induced inactivation by Rukhsana Jabeen; Amin A. Mohammad; Elizabeth C. Elefano; John R. Petersen; Mohammed Saleemuddin (pp. 1167-1174).

Methyl glyoxal (MG) is a highly reactive α-oxoaldehyde that plays an important role in non-enzymatic glycosylation reactions, formation of Advanced Glycation End products (AGEs) and other complications associated with hyperglycemia and related disorders. Unlike sugars, glycation by MG is predominantly arginine directed, which is particularly more damaging since arginine residues have a high-frequency occurrence in ligand and substrate recognition sites in receptor and enzyme active sites. Using bovine erythrocyte Cu,Zn-superoxide dismutase (SOD) as model enzyme, the potential of anti-enzyme antibodies in imparting protection against MG-induced inactivation was investigated. A concentration- and time-dependent inactivation of SOD was observed when the enzyme was incubated with MG. The enzyme lost over 80% activity on incubation with 5 mM MG for 5 days. More marked inactivation was observed in 24 h when the MG concentration was raised up to 30 mM. The SOD inactivation was accompanied by the formation of high molecular weight aggregates as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI/TOF mass spectrometry). Inclusion of specific anti-SOD antibodies raised in rabbits or monomeric Fab fragments derived thereof offered remarkable protection against MG-induced loss in enzyme activity. The protection, however, decreased with increase in the concentration of MG. SELDI/TOF mass spectrometry also revealed that the antibodies restricted the formation of high molecular weight aggregates. The results emphasize the potential of antibody based therapy in combating glycation and related complications.

Keywords: Methyl glyoxal; Glycation; SOD; Polyclonal antibodies

Modulatory influence of dietary resveratrol during different phases of 1,2-dimethylhydrazine induced mucosal lipid-peroxidation, antioxidant status and aberrant crypt foci development in rat colon carcinogenesis by M. Sengottuvelan; R. Senthilkumar; N. Nalini (pp. 1175-1183).

To shed light on the association of lipid peroxidation and antioxidant status with the development of aberrant crypt foci (ACF), we studied the modulatory influence of resveratrol, supplemented in three dietary regimens (initiation, post-initiation and entire period) on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. Rats were administered DMH (20 mg/kg body weight, s.c.) for 15 weeks and were supplemented with resveratrol (8 mg/kg body weight, p.o. everyday) in three dietary regimens. Intestines and colons were analyzed for the levels of diene conjugates (DC), lipid hydroperoxides (LOOHs) and thiobarbituric acid reactive substances (TBARS). Enzymic antioxidants (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX; glutathione S-transferase, GST; and glutathione reductase, GR) and non-enzymic reserve (reduced glutathione, GSH; ascorbate; and α-tocopherol) were also assessed in the intestine and colon. Unsupplemented DMH exposed rats showed significantly decreased levels/activities of tissue DC, LOOHs, TBARS, SOD, CAT, GSH, GR and significantly elevated ( P<0.05) GPX, GST, α-tocopherol and ascorbate as compared to control rats. Resveratrol supplementation during the entire period of the study resulted in significant ( P<0.01) modulation of lipid peroxidation markers and antioxidants status, which were paralleled with ACF suppression, as compared to DMH-alone treated rats. These results indicate that resveratrol effectively inhibits DMH-induced ACF and colonic tumor development.

Keywords: Abbreviations; ACF; Aberrant crypt foci; CAT; Catalase; DC; Diene conjugates; DMH; 1,2-dimethylhydrazine; GLUTs; Glucose transporter; GPX; Glutathione peroxidase; GR; Glutathione reductase; GSH; Reduced glutathione; GST; Glutathione; S; -transferase; LOOH; Lipid hydroperoxides; PUFA; Polyunsaturated fatty acids; SOD; Superoxide dismutase; TBARS; Thiobarbituric acid reactive substancesAntioxidants; Colon carcinogenesis; Oxidative stress; Resveratrol

Characterization of the interaction between human serum albumin and morin by Meng-Xia Xie; Mei Long; Yuan Liu; Chuan Qin; Ying-Dian Wang (pp. 1184-1191).

The interaction of morin with human serum albumin (HSA) has been investigated by using fluorescence, UV absorption and Fourier transform infrared spectroscopic approaches for the first time. Fluorescence data revealed the presence of a specific binding site on HSA for morin, and the binding affinity was 1.13±0.11×10−⁵ L Mol−¹ in the physiological condition. The intrinsic fluorescence of morin was conspicuously enhanced in the presence of HSA due to excited-state proton transfer. The binding ability of morin to protein decreased with the increase of the buffer pH from 6.4 to 8.4, which signified that the level of protonation of the hydroxyl groups played an important role during the drug–protein binding process. From the UV absorption spectra of morin in various pH medium, the dissociation behaviors of the hydroxyl groups on the drug molecule were assigned. The second derivative UV absorption spectra of morin after interacting with HSA were used to elucidate the binding mode of morin to protein. The obvious red shift of the UV absorption band I of morin upon binding to HSA further confirmed the formation of HSA–morin complex, and this property was also utilized to estimate the binding constant. The interaction between morin and HSA induced an obvious reduction of the protein α-helix and β-sheet structures.

Keywords: Morin; Human serum albumin; Fourier transform infrared spectroscopy; Fluorescence; UV absorption; Secondary structure

Expression, purification, and structural prediction of the Ets transcription factor ERM by Sébastien Mauen; Isabelle Huvent; Vincent Raussens; Dominique Demonte; Jean-Luc Baert; Catherine Tricot; Jean-Marie Ruysschaert; Carine Van Lint; Nicole Moguilevsky; Yvan de Launoit (pp. 1192-1201).

The PEA3 group within the Ets family comprises PEA3, ER81, and ERM, three transcription factors of about 500 residues. These factors are highly conserved in their ETS DNA-binding domain and in their two transcriptional activation domains. They are involved in many developmental processes and regulate cancer development via metastasis, as in the case of some breast tumors.Here, we describe the oversynthesis of human ERM from a baculovirus expression vector in Spodoptera frugiperda (Sf9) cells, and the subsequent purification and structural characterization of this protein. Oversynthesis of ERM was confirmed by measuring band intensities on SDS-PAGE gels and by Western blot analysis. Two-step purification by affinity chromatography led to a highly stable protein. Electromobility shift assays suggested that this purified protein is functional, since it recognizes specific Ets DNA-binding sites. We then used circular dichroism and infrared spectrometry to perform a structural analysis of the purified full-length ERM, and compared the results with those of current structural prediction algorithms. Our study indicates that ERM contains a highly structured ETS-domain and suggests that each of the N- and C-terminal transactivating domains also contains an α-helix. In contrast, the 250-residue central domain seems to have very little structure.

Keywords: Ets; Transcription factor; Erm; Baculovirus; Structure

Purification and biochemical characterization of phospholipase A2 from dromedary pancreas by Abir Ben Bacha; Youssef Gargouri; Sofiane Bezzine; Hafedh Mejdoub (pp. 1202-1209).

Dromedary pancreatic PLA2 (DrPLA2) was purified from delipidated pancreases. Pure protein was obtained after heat and acidic treatment (70 °C; pH 3.0), precipitation by ammonium sulphate and ethanol respectively, followed by sequential column chromatographies on Sephadex G-50, MonoS Sepharose, MonoQ Sepharose and C-8 reverse phase high pressure liquid chromatography. Purified DrPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 13748.55 Da. A specific activity of 600 U/mg for purified DrPLA2 was measured at optimal conditions (pH 8.0 and 37 °C) in the presence of 3 mM NaTDC and 7 mM CaCl₂ using PC as substrate. The sequence of the first fourteen amino-acid residues at the N-terminal extremity of DrPLA2 was determined by automatic Edman degradation. One single sequence was obtained and shows a close similarity with all other known pancreatic secreted phospholipases A2.

Keywords: Abbreviations; DrPLA2; dromedary pancreatic phospholipaseA2; sPLA2; secreted pancreatic phospholipase A2; TPP; turkey pancreatic phospholipase; PAF; 1-; O; -alkyl-2-acetyl-; sn; -glycero-3-phosphocholine; RP-HPLC; reverse phase–high pressure liquid chromatography; AU; absorbance unit; DTT; dithiothreitol; EDTA; ethylene diamine tetraacetic acid; EGTA; ethylene glycol-bis (β-aminoethylether); N,N,N′,N; ′-tetraacetic acid; BSA; bovine serum albumine; NaDC; sodium deoxycholate; NaTDC; sodium taurodeoxycholate; TX-100; triton X-100; PC; phosphatidylcholine; TC; ₄; tributyrin; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresisPancreatic phospholipase; PC; Bile salts; Ca; ²⁺; Sequence

Purification and characterisation of a protease inhibitor from Streptomyces chromofuscus 34-1 with an antiviral activity by Lidiya Angelova; Michèle Dalgalarrondo; Ignat Minkov; Svetla Danova; Nicolai Kirilov; Julia Serkedjieva; Jean-Marc Chobert; Thomas Haertlé; Iskra Ivanova (pp. 1210-1216).

The aim of this study was to purify and characterise a novel protease inhibitor (PISC-2002) isolated from culture supernatants of Streptomyces chromofuscus. PISC-2002 was purified by anion-exchange chromatography, and RP-HPLC analysis. PISC-2002 had a molecular mass of 11.2 kDa and a high content of hydrophobic amino acids and proline. N-terminal sequence gave two sequences differing by one residue. The main sequence is ASLPAVSALVLTV and the shorter sequence is SLPAVSALVLTV. This shows its homology to Streptomyces subtilisin inhibitor family. Besides its large spectrum of powerful inhibitory activities against various serine proteases, PISC-2002 displayed significant antiviral effect against influenza virus A/Rostock/34 (H7N7).

Keywords: Protease inhibitor; Streptomyces; Antiviral; Influenza

The greatly increased amounts of accumulated versican and decorin with specific post-translational modifications may be closely associated with the malignant phenotype of pancreatic cancer by Spyros S. Skandalis; Dimitris Kletsas; Dora Kyriakopoulou; Michalis Stavropoulos; Dimitrios A. Theocharis (pp. 1217-1225).

Pancreatic carcinoma (PC) is a cancer type with highly malignant growth and dissemination pattern of which the mechanisms are poorly understood. However, the malignant phenotype is closely linked to extracellular matrix (ECM) of which proteoglycans (PGs) and hyaluronan (HA) play a crucial role in the control of tumor progression and metastasis. In this study, we demonstrated that versican and decorin, two different PGs with contradictory roles and functions in the pathobiology of cancer, were the main matrix PGs in PC presenting a great increase 27- and 7-fold, respectively, in comparison to normal pancreas (NP). PC was characterized by the disproportional increase of versican compared to decorin, about 4 to 1, with a concurrent increase of HA, which may be closely associated with the growth and aggressiveness of this carcinoma. Significant specific post-translational modifications were also observed in both versican and decorin regarding the type, hydrodynamic size, sulfation pattern and extent of uronate epimerization of their glycosaminoglycan chains (GAGs). In particular, chondroitin sulphate (CS) was the predominant GAG type in both PC-associated versican and decorin. The CS of PC-decorin was increased 11-fold, compared to NP in which dermatan sulfate (DS) was the predominant GAG type in both PGs. The sulfation pattern of GAG chains was significantly altered in PC, since 6-sulfated disaccharides predominated in both versican and decorin with a marked presence of non-sulfated disaccharides accompanied by lower hydrodynamic sizes of both CS and DS chains compared to NP. In conclusion, all these findings agree with the highly malignant phenotype of this cancer and, thus, more studies need to be addressed on the roles of the post-translational modifications of versican and decorin in the biology of cancer.

Keywords: Pancreatic cancer; Proteoglycan; Glycosaminoglycan; Versican; Decorin; Post-translational modifications

Freezing and desiccation tolerance in the moss Physcomitrella patens: An in situ Fourier transform infrared spectroscopic study by Harriëtte Oldenhof; Willem F. Wolkers; John L. Bowman; Fern Tablin; John H. Crowe (pp. 1226-1234).

In situ Fourier transform infrared spectroscopy (FTIR) was used in order to obtain more insights in the underlying protective mechanisms upon freezing and drying of ABA-treated tissues of the moss Physcomitrella patens. The effects of different treatments on the membrane phase behaviour, glassy state, and overall protein secondary structure were studied. We found that growth on ABA resulted in the accumulation of sucrose: up to 22% of the tissue on a dry weight basis, compared to only 3.7% in non-ABA-treated tissues. Sucrose functions as a protectant during freezing and drying, but accumulation of sucrose alone is not sufficient for survival. ABA-treated tissue survives a freeze–thaw cycle down to −80 °C only after addition of an additional cryoprotectant (DMSO). Survival correlates with preservation of membrane phase behaviour. We found that ABA-treated P. patens can survive slow but not rapid drying down to water contents as low as 0.02 g H₂O per g DW. Rapidly and slowly dried ABA-treated tissues were found to have similar sugar compositions and glass transition temperatures. The average strength of hydrogen bonding in the cytoplasmic glassy matrix, however, was found to be increased upon slow drying. In addition, slowly dried tissues were found to have a higher relative proportion of α-helical structures compared to rapidly dried tissues.

Keywords: Abbreviations; DMSO; dimethyl sulfoxide; FTIR; Fourier transform infrared spectroscopy; LEA; late embryogenesis abundant; T; _g; glass transition temperature; T; _m; membrane phase transition temperature; WTC; wavenumber temperature coefficientAbscisic acid; Desiccation tolerance; FTIR spectroscopy; Glassy behaviour; LEA-proteins; Membrane phase behaviour; Sucrose

Differential O-glycosylation in cortical and medullary thymocytes by Georgina Alvarez; Ricardo Lascurain; Pedro Hernández-Cruz; Daniel Tetaert; Pierre Degand; Patricia Gorocica; Blanca Espinosa; Edgar Zenteno; Raúl Chávez (pp. 1235-1240).

Differentiation of T lymphocytes is characterized by variable expression of CD8/CD4 co-receptor molecules and changes in the glycosylation pattern. In this work, O-glycosylation was analyzed in microsomes from murine thymocytes purified with the PNA and Amaranthus leucocarpus (ALL) lectins, specific for the T antigen (Galβ1,3GalNAc1,0 Ser/Thr) in cortical and medullary thymocytes, respectively. Three peptides were used as acceptors for UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyl-transferase (GalNAc transferase); the peptide motif TTSAPTTS was the best glycosylated one. Cortical ALL−PNA+ thymocytes showed two-fold higher GalNAc transferase activity than ALL+PNA− thymocytes; however, capillary electrophoresis showed a higher proportion of di- versus mono-glycosylated peptides for ALL+PNA− than for ALL−PNA+. We compared the GalNAc transferase activity of thymocytes from dexamethasone-treated mice versus control mice. GalNAc transferase activity was six-fold higher in thymocytes from control mice than from dexamethasone-treated mice; the rate of di-glycosylated peptides for dexamethosone-resistant ALL+ was two-fold higher than for ALL− thymocytes. Our results confirm an upregulated biosynthesis of O-glycosidically linked glycans on T cell surface glycoproteins, and suggest that the modification of GalNAc transferase activity plays a relevant role during the maturation process of thymic cells.

Keywords: O-glycosylation; Thymocytes; Lectins; Amaranthus lectin; Peanut agglutinin; Apoptosis

Identification of N-terminal acetylation of recombinant human prothymosin α in Escherichia coli by Jun Wu; Shaohong Chang; Xing Gong; Dianxin Liu; Qingjun Ma (pp. 1241-1247).

Functional modification of protein through N-terminal acetylation is common in eukaryotes but rare in prokaryotes. Prothymosin α is an essential protein in immune stimulation and apoptosis regulation. The protein is N-terminal acetylated in eukaryotes, but similar modification has never been found in recombinant protein produced in prokaryotes. In this study, two mass components of recombinant human prothymosin α expressed in Escherichia coli were identified and separated by RP-HPLC. Mass spectrometry of the two components showed that one of them had a 42 Da mass increment as compared with the theoretical mass of human prothymosin α, which suggested a modification of acetylation. The mass of another one was equal to that of the theoretical one. Peptides mass spectrometry of the modified component showed that the 42-Da mass increment occurred in the N-terminal peptide domain, and MS/MS peptide sequencing of the N-terminal peptide found that the acetylated modification occurred at the N-terminal serine residue. So, part of the recombinant human prothymosin α produced by E. coli was N-terminal acetylated. This finding adds a new clue for the mechanism of acetylated modification in prokaryotes, and also suggested a new method for production of N-terminal modificated prothymosin α and thymosin α1.

Keywords: N-terminal acetylation; prothymosin α; Escherichia coli; prokaryote

Multiple ligand-binding properties of the lipocalin member chicken α₁-acid glycoprotein studied by circular dichroism and electronic absorption spectroscopy: The essential role of the conserved tryptophan residue by Ferenc Zsila; Hisami Matsunaga; Zsolt Bikádi; Jun Haginaka (pp. 1248-1273).

Multiple ligand-binding properties of the 30-kDa chicken α₁-acid glycoprotein (cAGP), a member of the lipocalin protein family, were investigated for the first time by using circular dichroism (CD) and UV/Vis absorption spectroscopy methods. By measuring induced CD (ICD) spectra, high-affinity binding ( K_a≈10⁵–10⁶ M⁻¹) of several drugs, dyes and natural compounds to cAGP was demonstrated including antimalarial agents (quinacrine, primaquine), phenotiazines (chlorpromazine, methylene blue), propranolol, non-steroidal antiinflammatory drugs (ketoprofen, diclofenac), tamoxifen, diazepam, tacrine, dicoumarol, cationic dyes (auramine O, thioflavine T, ethidium bromide), benzo[a]pyrene,l-thyroxine, bile pigments (bilirubin, biliverdin), alkaloids (piperine, aristolochic acid), saturated and unsaturated fatty acids. Analysis of the extrinsic CD spectra with the study of the covalently modified protein and CD displacement experiments revealed that a single Trp26 residue of cAGP conserved in the whole lipocalin family is part of the binding site, and it is essentially involved in the ligand-binding process via π–π stacking interaction resulting in the appearance of strong induced CD bands due to the non-degenerate intermolecular exciton coupling between the π–π* transitions of the stacked indole ring–ligand chromophore. The finding that cAGP is able to accommodate a broad spectrum of ligands belonging to different chemical classes suggests that its core β-barrel cavity is unusually wide containing overlapping sub-sites. Significance of these new data in understanding of the ligand-binding properties of other lipocalins, especially that of human AGP, and potential practical applications are briefly discussed. Overall, cAGP serves as a simple, ultimate model to extend our knowledge on ligand-binding properties of lipocalins and to study the role of tryptophan residues in molecular recognition processes.

Keywords: Abbreviations; AAc; aristolochic acid; ArO; auramine O; BLG; bovine β-lactoglobulin; BP; benzo[a]pyrene; BR; bilirubin; BV; biliverdin; cAGP; chicken α; ₁; -acid glycoprotein; CD; circular dichroism; CE; cotton effect; CHLP; chlorpromazine; cPA; cis; -parinaric acid; DC; dicoumarol; DCF; diclofenac; DZ; diazepam; EBr; ethidium bromide; FWHM; full width at half maximum; hAGP; human α; ₁; -acid glycoprotein; ICD; induced circular dichroism; KPF; ketoprofen; L; /; P; ligand/protein molar ratio; mdeg; millidegree; MB; methylene blue; NSAID; non-steroidal antiinflammatory drug; OMCHI; ovomucoid from chicken; PA; palmitic acid; PP; piperine; PRQ; primaquine; PRP; propranolol; QR; quinacrine; T; ₄; l; -thyroxin; ThT; thioflavin T; TMX; tamoxifen; UV/Vis; ultraviolet–visibleAromatic stacking; β-Barrel; Chicken α; ₁; -acid glycoprotein; Ligand binding; Lipocalin; Induced circular dichroism; Non-degenerate exciton coupling; Tryptophan

A novel fluorescent method employing the FRET-based biosensor “LIBRA�? for the identification of ligands of the inositol 1,4,5-trisphosphate receptors by Akihiro Nezu; Akihiko Tanimura; Takao Morita; Akiko Shitara; Yosuke Tojyo (pp. 1274-1280).

LIBRA is a fluorescent biosensor of inositol 1,4,5-trisphosphate (IP₃) and is composed of the ligand-binding domain of the rat type 3 IP₃ receptor and cyan and yellow fluorescent proteins. We examined the responses of LIBRA and its IP₃-insensitive mutant LIBRA-N to compounds known to inhibit IP₃-induced Ca²⁺ release. Heparin, a competitive antagonist of IP₃ receptors, increased the emission ratio of LIBRA but not that of LIBRA-N. In contrast, 2-aminoethoxydiphenyl borate, a known non-competitive inhibitor of IP₃ receptor, decreased the emission ratios of both LIBRA and LIBRA-N. Thus, the concurrent use of LIBRA-N with LIBRA identifies nonspecific responses. These results indicate that LIBRA and its mutant control can be used to detect specific agonists and antagonists of IP₃ receptors. We also demonstrate the utility of LIBRA and LIBRA-N in discriminating between specific and nonspecific responses in intact cells.

Keywords: Abbreviations; 2APB; 2-aminoethoxydiphenyl borate; Ach; acetylcholine; CCD; charge-coupled device; CFP; cyan fluorescent protein; ECFP; enhanced CFP; EYFP; enhanced YFP; FRET; fluorescence resonance energy transfer; ICM; intracellular-like medium; IP; ₃; inositol 1,4,5-trisphosphate; IP; ₃; R; IP; ₃; receptor; PLC; phospholipase C; YFP; yellow fluorescent proteinInositol 1,4,5-trisphosphate (IP; ₃; ); IP; ₃; -binding domain; Heparin; Fluorescence resonance energy transfer (FRET); LIBRA

Deviation of carbohydrate metabolism by the SIT4 phosphatase in Saccharomyces cerevisiae by Willy Jablonka; Simón Guzmán; Jorge Ramírez; Mónica Montero-Lomelí (pp. 1281-1291).

A prominent phenotype of the yeast sit4 mutant, which lacks the Ser–Thr phosphatase Sit4, is hyper-accumulation of glycogen and the failure to grow on respiratory substrates. We investigated whether these two phenotypes are linked by studying the metabolic response to SIT4 deletion. Although the sit4 mutant failed to grow on respiratory substrates, in the exponential growth, phase respiration was de-repressed; active respiration was confirmed by measuring oxygen consumption and NADH generation. However, the fermentation rate and the internal glucose 6-phosphate and pyruvate levels were reduced, while glycogen content was high. Respiro-fermentative and respiratory substrates such as galactose, glycerol and ethanol were directed toward glycogen synthesis, which indicates that sit4 mutant deviates metabolism to glycogenesis by activating a glycogen futile cycle and depleting cells of Krebs cycle intermediates. An important feature of the sit4 mutant was the lack of growth under anaerobic conditions, suggesting that respiration is necessary to meet the energy requirements of the cell. Addition of aspartic acid, which can restore Krebs cycle intermediates, partially restored growth on ethanol. Our findings suggest that inhibition of Sit4 activity may be essential for redirecting carbohydrate flux to gluconeogenesis and glycogen storage.

Keywords: Ser–Thr phosphatase; SIT4; Carbohydrate metabolism; Glycogen

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BBA - General Subjects (v.1760, #8)