Journal of Biochemistry - current issue

Simple Primary Structure, Complex Turnover Regulation and Multiple Roles of Hyaluronan

Itano, N. — 2008-07-31

Hyaluronan is a major macromolecular polysaccharide component of the extra-cellular matrix that confers structural frameworks for cells. Despite its relatively simple chemical composition, hyaluronan mediates many other important functional aspects including signalling activity during embryonic morphogenesis, cellular regeneration and wound healing. Abnormalities in hyaluronan metabolism have been implicated in many diseases, such as inflammatory disorders, cardiovascular diseases and cancer. To date, it has become increasingly clear that hyaluronan production in vertebrates is tightly regulated by three hyaluronan synthases and that hyaluronan catabolism is regulated by an enzymatic degradation reaction involving several hyaluronidases. Together, these discoveries have provided key insights into the physiological roles of hyaluronan and a deeper understanding of the mechanisms underlying altered hyaluronan turnover in diseases. The central aim of this review article is therefore to highlight the multiple roles of hyaluronan in physiological and pathological states via its complex turnover regulation.

Concept, Strategy and Realization of Lectin-based Glycan Profiling

Hirabayashi, J. — 2008-07-31

Lectins are a diverse group of carbohydrate-binding proteins. Each lectin has its own specificity profile. It is believed that lectins exist in all living organisms that produce glycans. From a practical viewpoint, lectins have been used extensively in biochemical fields including proteomics due to their usefulness as detection and enrichment tools for specific glycans. Nevertheless, they have often been underestimated as probes, especially compared with antibodies, because of their low affinity and broad specificity. However, together with the concept of glycomics, such properties of lectins are now considered to be suitable for the task of ‘profiling’ in order to cover a wider range of ligands. Recently there has been rapid movement in the field of proteomics aimed at the investigation of glycan-related biomarkers. This is partly because of limitations of the present approach of simply following changes in protein-level expression, without paying sufficient attention to the fact and effects of glycosylation. The trend is reflected in the frequent use of lectins in the contexts of glycoprotein enrichment and glycan profiling. However, there are many aspects to be considered in using lectins, which differ considerably from antibodies. In this article, the author, as a developer of two unique methodologies, frontal affinity chromatography (FAC) and the lectin microarray, describes critical points concerning the use of lectins, together with the concept, strategy and means to achieve advances in these emerging glycan profiling technologies.

Elongation and Desaturation of Fatty Acids are Critical in Growth, Lipid Metabolism and Ontogeny of Caenorhabditis elegans

Horikawa, M., Nomura, T., Hashimoto, T., Sakamoto, K. — 2008-07-31

Recently, it was reported that a deficit in the mouse stearoyl-CoA desaturase 1 gene decreases biosynthesis and accumulation of fatty acid and revitalizes the β-oxidation of fatty acid. To examine the physiological role of fatty acid desaturase (FAT) and elongase (ELO)-gene transduction in ontogeny, fatty acid accumulation and individual lifespan, we performed bacteria-mediated RNA interference (RNAi) in the nematode Caenorhabditis elegans. Suppression of the expression of FAT-2 gene mRNA caused a drastic decrease in the amount of body fat and defects in egg-hatching. The amount of body fat was markedly decreased, and body size reduced, by down regulation of FAT-6 and FAT-7, whereas lifespan was drastically reduced. RNAi of the FAT-2 gene caused a remarkable increase of the β-oxidation-related gene expression and the DAF-16 transcriptional activity, whereas, ELO-2 RNAi caused a remarkable decrease in fatty acid biosynthesis-related gene expression. Additionally, RNAi of FAT-6 decreased the mRNA levels of the genes involved in fatty acid synthesis, and FAT-7 RNAi increased the mRNA levels of β-oxidation system genes. These results indicated that the elongation and desaturation of fatty acids are integral to various phenomena such as ontogeny and lifespan and play important roles in fatty acid accumulation and consumption.

15N NMR Relaxation Studies of Y14F Mutant of Ketosteroid Isomerase: The Influence of Mutation on Backbone Mobility

2008-07-31

The backbone dynamics of Y14F mutant of ⁵-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by ¹⁵N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S²) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S² values, whereas the S² values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.

Susceptibilities of Phospholipid Membranes Containing Cholesterol or Ergosterol to Gramicidin and its Derivative Incorporated in Lysophospholipid Micelles

Yoshida, N., Mita, T., Onda, M. — 2008-07-31

Complex formation of gramicidin (GA) and desformylgramicidin (des-GA) with sterols was investigated by measuring the intrinsic Trp fluorescence. In organic solvents, the Trp fluorescence of momeric GA was quenched upon binding either cholesterol or ergosterol, but that of monomeric des-GA was not quenched by adding cholesterol. Both dimeric GA and des-GA bound highly to ergosterol, but not to cholesterol, determined by quenching of Trp fluorescence. Furthermore, GA- and des-GA-loaded lysophosphatidylcholine micelles were incubated with phosphatidylcholine vesicles containing cholesterol or ergosterol. The results showed that both monomeric and dimeric peptides hardly bound to cholesterol incorporated into phospholipid vesicles, but markedly bound to ergosterol incorporated into the bilayer membranes. Interestingly, des-GA bound more specifically to the two sterols than GA. In addition, fluorescence resonance energy transfer analysis showed that des-GA bound more specifically to the two sterol than GA.

Modulation of Glucose Uptake in a Human Choriocarcinoma Cell Line (BeWo) by Dietary Bioactive Compounds and Drugs of Abuse

Araujo, J. R., Goncalves, P., Martel, F. — 2008-07-31

The aim of this work was to investigate the putative modulation of glucose uptake in trophoblast cells by several dietary compounds and by drugs of abuse. For this, the acute (26 min) and chronic (48 h) effect of these substances on the apical uptake of ³H-2-deoxy-d-glucose (³H-DG) by a human choriocarcinoma cell line (BeWo) was determined. ³H-DG apical uptake by BeWo cells was time dependent, displayed saturable kinetics (V_max = 1210 ± 29 nmol mg protein^–1 6 min^–1 and K_m = 13.4 ± 0.5 mM) and was insulin-insensitive and cytochalasin B-sensitive (by up to 60%). Acutely, acetaldehyde (30–100 mM), resveratrol, xanthohumol, epigallocatechin-3-gallate (100 µM), chrysin and quercetin (10–100 µM) decreased ³H-DG apical uptake, whereas rutin, catechin (10–100 µM), epicatechin (100 µM) and ethanol (10 mM) increased it. Quercetin and xanthohumol seem to be non-competitive inhibitors of ³H-DG apical uptake, whereas both epigallocatechin-3-gallate and acetaldehyde decreased both the K_m and V_max values. Chronically, rutin and myricetin increased the apical uptake of ³H-DG both isolated (0.1–1 µM) and in combination (both at 1 µM), whereas theophylline (0.1–1 µM) and amphetamine, 3,4-methylenedioxymethamphetamine (0.25–1 µM) and ⁹-tetrahydrocannabinol (1 nM) decreased it. In conclusion, ³H-DG apical uptake by BeWo cells is differentially modulated by different compounds present in drinks and by drugs of abuse.

Evidence for Mitochondrial Localization of P5, a Member of the Protein Disulphide Isomerase Family

2008-07-31

This report demonstrates for the first time that P5, a member of the protein disulphide isomerase (PDI) family, is present in the mitochondria. Various organelles were screened for proteins bearing the CGHC motif using an affinity column conjugated with the phage antibody 5E, which cross-reacts with PDI family proteins. P5 was found in bovine liver mitochondrial extract and identified by Western blot analysis using anti-P5 antibody and by mass spectrometric analysis. Results of cell fractionation, proteinase sensitivity experiments and immuno-electron microscopy supported the mitochondrial localization of P5 and also indicated the presence of ERp57, another PDI family protein, in mitochondria. Our findings will be useful for the elucidation of the translocation mechanism of PDI family proteins and their roles in mitochondria.

Microglial Cell Death Induced by Glycated Bovine Serum Albumin: Nitric Oxide Involvement

2008-07-31

Nonenzymatic glycation results in the formation of advanced glycation end products (AGEs) through a nonenzymatic multistep reaction of reducing sugars with proteins. AGEs have been suspected to be involved in the pathogenesis of several chronic clinical neurodegenerative complications including Alzheimer's disease, which is characterized with the activation of microglial cells in neuritic plaques. To find out the consequence of this activation on microglial cells, we treated the cultured microglial cells with different glycation levels of Bovine Serum Albumin (BSA) which were prepared in vitro. Extent of glycation of protein has been characterized during 16 weeks of incubation with glucose. Treatment of microglial cells with various levels of glycated albumin induced nitric oxide (NO) production and consequently cell death. We also tried to find out the mode of death in AGE-activated microglial cells. Altogether, our results suggest that AGE treatment causes microglia to undergo NO-mediated apoptotic and necrotic cell death in short term and long term, respectively. NO production is a consequence of iNOS expression in a JNK dependent RAGE signalling after activation of RAGE by AGE–BSA.

Characterization and Secondary Structure Analysis of Endostatin Covalently Modified by Polyethylene Glycol and Low Molecular Weight Heparin

Tan, H., Yang, S., Feng, Y., Liu, C., Cao, J., Mu, G., Wang, F. — 2008-07-31

Endostatin (ES), as an angiogenesis inhibitor, has been approved by the State Food and Drug Administration (SFDA) in China for the treatment of patients with non-small-cell lung cancer. However, as a protein drug, there are a lot of obstacles on its clinical application, such as need of high dose to maintain its efficacy, expensive and poor stability, etc and limits its clinical use. In order to overcome these shortcomings, we chemically modified ES by polyethylene glycol and low molecular weight heparin (LMWH), respectively. The changes of the secondary structure of the modified products were studied by Fourier transform infrared spectroscopy and Circular dichroism spectra to obtain better ES derivatives. Our study demonstrated that the modified products have a better heat tolerance than ES towards. The result of secondary structure analysis suggests the percentage of β-turn in whole protein is an important factor on the activity and heat stability and ES modified by LMWH can maintain higher activity and its secondary structure.

NMR Studies on the Equilibrium Unfolding of Ketosteroid Isomerase by Urea

2008-07-31

Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of ¹H-¹⁵N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the β5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the β6-strand. The results thus suggest that the interface region primarily around the β5- and β6-strands could play an important role as the starting positions in the unfolding process of KSI.

Protein Methyltransferase Activities in Commercial In vitro Translation Systems

Denman, R. B. — 2008-07-31

Protein arginine methylation is a well-known post-translational modification that has been shown to occur in rabbit reticulocyte in vitro translation lysates (RRL); however, it is not known whether this is a general feature of in vitro-produced proteins from other eukaryotic cell-free translation systems, particularly insect-derived lysates (ICL). Because methylation can affect protein localization, RNA binding and protein–protein interactions this may be of great importance as in vitro-produced proteins are often used in assays of protein function. Here, I report the presence of base-stable and base-labile methyltransferase activities in RRL, ICL and wheat germ in vitro extracts (WGE). Indeed, the presence of CARM1 in RRL and ICL and a class II protein arginine methyltransferase activity (PRMT5/7) is documented in all three systems. Additionally, the lysine methyltransferase that modifies eukaryotic elongation factor 1A (eEF-1A) was detected in ICL and WGE. Importantly, using a defined set of substrates under identical conditions I show that all three in vitro systems contain different complements of the various methyltransferases. These data suggest that three systems can be used in a complementary fashion to investigate the effect(s) of post-translational modification on protein function.

Purification and Kinetic Characterization of 6-Phosphofructo-1-kinase from the Liver of Gilthead Sea Bream (Sparus Aurata)

Mediavilla, D., Meton, I., Baanante, I. V. — 2008-07-31

6-Phosphofructo-1-kinase (PFK) was purified to homogeneity from liver of gilthead sea bream (Sparus aurata) and kinetic properties of the enzyme were determined. The native enzyme had an apparent molecular mass of 510 kDa and was composed of 86 kDa subunits, suggesting homohexameric structure. At pH 7, S. aurata liver PFK (PFKL) showed sigmoidal kinetics for fructose-6-phosphate (fru-6-P) and hyperbolic kinetics for ATP. Fructose-2,6-bisphosphate (fru-2,6-P₂) converted saturation curves for fru-6-P to hyperbolic and activated PFKL synergistically with AMP. Fru-2,6-P₂ counteracted the inhibition caused by ATP, ADP and citrate. Compared to the S. aurata muscle isozyme, PFKL had lower affinity for fru-6-P, higher cooperativity, hyperbolic kinetics in relation to ATP, increased susceptibility to inhibition by ATP, and was less affected by AMP, ADP and inhibition by 3-phosphoglycerate, phosphoenolpyruvate, 6-phosphogluconate or phosphocreatine. The effect of starvation-refeeding on PFKL expression was studied at the levels of enzyme activity and protein content in the liver of S. aurata. Our findings indicate that short-term recovery of PFKL activity after refeeding previously starved fish, may result from allosteric regulation by fru-2,6-P₂, whereas combination of activation by fru-2,6-P₂ and increase in protein content may determine the long-term recovery of the enzyme activity.

Molecular Characterization, Heterologous Expression and Kinetic Analysis of Recombinant Plasmodium falciparum Thymidylate Kinase

Kandeel, M., Kitade, Y. — 2008-07-31

The gene encoding for thymidylate kinase from Plasmodium falciparum was obtained by PCR and expressed in Escherichia coli and the enzyme was investigated as a possible new drug target. The enzyme is a homodimer exhibiting maximal kinase activity over a wide pH range of 7–9 and is characterized by marked stability. Compared with the human enzyme, the recombinant P. falciparum TMP kinase showed a broader spectrum of substrate specificity. The enzyme not only phosphorylates dTMP and dUMP but can also tolerate the bulkier purines dGMP, GMP and dIMP. Initial velocity studies showed that the K_m values for TMP and dGMP are 22 and 30 µM, respectively. The turnover number k_cat(TMP) was found to be 3.4 s^–1, a value indicating the higher catalytic efficiency of the plasmodium enzyme. From the present study, we suggest that the design of appropriate inhibitors especially purine based compounds could have a selective inhibitory effect on the parasite enzyme.

Nicotine Suppresses Tunicamycin-Induced, But Not Thapsigargin-Induced, Expression of GRP78 during ER Stress-Mediated Apoptosis in PC12 Cells

2008-07-31

We previously reported that nicotine protected against tunicamycin (Tm)-induced ER stress-mediated apoptosis, but not thapsigargin (Tg)-induced apoptosis in PC12 cells. In the present study, we report that the expression of glucose-regulated protein 78 (GRP78) was suppressed by nicotine in Tm-treated PC12 cells. Interestingly, the GRP78 expression was not changed by nicotine in Tg-treated cells. Moreover, nicotine reduced the activation of caspase-12 in Tm-treated cells, but not in Tg-treated cells. These results suggest that nicotine prevented Tm-induced ER stress-mediated apoptosis by attenuating an early stage of Tm-induced ER stress. It was possible that the suppression of GRP78 expression by nicotine was achieved through the suppression of the Ire1-XBP1 and/or ATF6 pathways. We observed that nicotine suppressed the Tm-induced, but not Tg-induced, splicing of XBP1 mRNA, and also suppressed the Tm-induced, but not Tg-induced, production of cleaved ATF6 in PC12 cells. These results indicate that the suppression of Ire1-XBP1 and ATF6 pathways contributes to the suppression of GRP78 expression by nicotine in Tm-treated PC12 cells, suggesting that nicotine suppresses a common step upstream of both the Ire1-XBP1 and ATF6 pathways which are required for the expression of GRP78 during Tm-induced ER stress.

Protein Tyrosine Phosphatase Receptor Type Z Dephosphorylates TrkA Receptors and Attenuates NGF-dependent Neurite Outgrowth of PC12 Cells

Shintani, T., Noda, M. — 2008-07-31

Protein tyrosine phosphatase receptor type Z (Ptprz/Ptp / RPTPβ) is a receptor-like protein tyrosine phosphatase (RPTP) which is predominantly expressed in the central nervous system. Tropomyosin-related kinases (Trks) are single-pass transmembrane molecules that are highly expressed in the developing nervous system. Upon the ligand binding of neurotrophins, Trk receptors are activated through autophosphorylation of tyrosine residues; however, the PTPs responsible for the negative regulation of Trk receptors have not been fully elucidated. Here, we identified Ptprz as a specific PTP that efficiently dephosphorylates TrkA as a substrate. Co-expression of Ptprz with Trk receptors in 293T cells showed that Ptprz suppresses the ligand-independent tyrosine phosphorylation of TrkA, but not of TrkB or TrkC, and that Ptprz attenuates TrkA activation induced by nerve growth factor (NGF). Co-expression analyses with TrkA mutants revealed that Ptprz dephosphorylates phosphotyrosine residues in the activation loop of the kinase domain, which are requisite for activation of the TrkA receptor. Consistent with these findings, forced expression of Ptprz in PC12D cells markedly inhibited neurite extension induced by a low dose of NGF. In addition, an increment in the tyrosine phosphorylation of TrkA was observed in the brain of Ptprz-deficient mice. Ptprz thus appears to be one of the PTPs which regulate the activation and signalling of TrkA receptors.

An O6-methylguanine-DNA Methyltransferase-like Protein from Thermus thermophilus Interacts with a Nucleotide Excision Repair Protein

Morita, R., Nakagawa, N., Kuramitsu, S., Masui, R. — 2008-07-31

The major damage to DNA caused by alkylating agents involves the formation of O⁶-methylguanine (O⁶-meG). Almost all species possess O⁶-methylguanine-DNA-methyltransferase (Ogt) to repair such damage. Ogt repairs O⁶-meG lesions in DNA by stoichiometric transfer of the methyl group to a cysteine residue in its active site (PCHR). Thermus thermophilus HB8 has an Ogt homologue, TTHA1564, but in this case an alanine residue replaces cysteine in the putative active site. To reveal the possible function of TTHA1564 in processing O⁶-meG-containing DNA, we characterized the biochemical properties of TTHA1564. No methyltransferase activity for synthetic O⁶-meG-containing DNA could be detected, indicating TTHA1564 is an alkyltransferase-like protein. Nevertheless, gel shift assays showed that TTHA1564 can bind to DNA containing O⁶-meG with higher affinity (9-fold) than normal (unmethylated) DNA. Experiments using a fluorescent oligonucleotide suggested that TTHA1564 recognizes O⁶-meG in DNA using the same mechanism as other Ogts. We then investigated whether TTHA1564 functions as a damage sensor. Pull-down assays identified 20 proteins, including a nucleotide excision repair protein UvrA, which interacts with TTHA1564. Interaction of TTHA1564 with UvrA was confirmed using a surface plasmon resonance assay. These results suggest the possible involvement of TTHA1564 in DNA repair pathways.