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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.40, #3)

Human Alzheimer’s disease synaptic O-GlcNAc site mapping and iTRAQ expression proteomics with ion trap mass spectrometry by Yuliya V. Skorobogatko; John Deuso; Jared Adolf-Bergfoyle; Matthew G. Nowak; Yuesong Gong; Carol Frances Lippa; Keith Vosseller (pp. 765-779).

Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer’s disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.

Keywords: O-GlcNAc; Alzheimer’s disease; Proteomics; iTraq; Post-synaptic density

Substrate and product analogues as human O-GlcNAc transferase inhibitors by Helge C. Dorfmueller; Vladimir S. Borodkin; David E. Blair; Shalini Pathak; Iva Navratilova; Daan M. F. van Aalten (pp. 781-792).

Protein glycosylation on serine/threonine residues with N-acetylglucosamine (O-GlcNAc) is a dynamic, inducible and abundant post-translational modification. It is thought to regulate many cellular processes and there are examples of interplay between O-GlcNAc and protein phosphorylation. In metazoa, a single, highly conserved and essential gene encodes the O-GlcNAc transferase (OGT) that transfers GlcNAc onto substrate proteins using UDP–GlcNAc as the sugar donor. Specific inhibitors of human OGT would be useful tools to probe the role of this post-translational modification in regulating processes in the living cell. Here, we describe the synthesis of novel UDP–GlcNAc/UDP analogues and evaluate their inhibitory properties and structural binding modes in vitro alongside alloxan, a previously reported weak OGT inhibitor. While the novel analogues are not active on living cells, they inhibit the enzyme in the micromolar range and together with the structural data provide useful templates for further optimisation.

Keywords: O-GlcNAc; Post-translational modification; Inhibitor; Signalling; Crystal structure

The dynamic stress-induced “O-GlcNAc-ome” highlights functions for O-GlcNAc in regulating DNA damage/repair and other cellular pathways by Natasha E. Zachara; Henrik Molina; Ker Yi Wong; Akhilesh Pandey; Gerald W. Hart (pp. 793-808).

The modification of nuclear, mitochondrial, and cytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic and essential post-translational modification of metazoans. Numerous forms of cellular injury lead to elevated levels of O-GlcNAc in both in vivo and in vitro models, and elevation of O-GlcNAc levels before, or immediately after, the induction of cellular injury is protective in models of heat stress, oxidative stress, endoplasmic reticulum (ER) stress, hypoxia, ischemia reperfusion injury, and trauma hemorrhage. Together, these data suggest that O-GlcNAc is a regulator of the cellular stress response. However, the molecular mechanism(s) by which O-GlcNAc regulates protein function leading to enhanced cell survival have not been identified. In order to determine how O-GlcNAc modulates stress tolerance in these models we have used stable isotope labeling with amino acids in cell culture to determine the identity of proteins that undergo O-GlcNAcylation in response to heat shock. Numerous proteins with diverse functions were identified, including NF-90, RuvB-like 1 (Tip49α), RuvB-like 2 (Tip49β), and several COPII vesicle transport proteins. Many of these proteins bind double-stranded DNA-dependent protein kinase (PK), or double-stranded DNA breaks, suggesting a role for O-GlcNAc in regulating DNA damage signaling or repair. Supporting this hypothesis, we have shown that DNA-PK is O-GlcNAc modified in response to numerous forms of cellular stress.

Keywords: O-GlcNAc; Cellular stress; Glycosylation; Signal transduction; Chaperone; Cell stress

O-GlcNAcylation of tubulin inhibits its polymerization by Suena Ji; Jeong Gu Kang; Sang Yoon Park; JooHun Lee; Young J. Oh; Jin Won Cho (pp. 809-818).

The attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) to proteins is an abundant and reversible modification that involves many cellular processes including transcription, translation, cell proliferation, apoptosis, and signal transduction. Here, we found that the O-GlcNAc modification pattern was altered during all-trans retinoic acid (tRA)-induced neurite outgrowth in the MN9D neuronal cell line. We identified several O-GlcNAcylated proteins using mass spectrometric analysis, including α- and β-tubulin. Further analysis of α- and β-tubulin revealed that O-GlcNAcylated peptides mapped between residues 173 and 185 of α-tubulin and between residues 216 and 238 of β-tubulin, respectively. We found that an increase in α-tubulin O-GlcNAcylation reduced heterodimerization and that O-GlcNAcylated tubulin did not polymerize into microtubules. Consequently, when O-GlcNAcase inhibitors were co-incubated with tRA, the extent of neurite outgrowth was decreased by 20% compared to control. Thus, our data indicate that the O-GlcNAcylation of tubulin negatively regulates microtubule formation.

Keywords: O-GlcNAc; Tubulin; Microtubule; Neuron; Neurite

Activation of the hexosamine biosynthesis pathway and protein O-GlcNAcylation modulate hypertrophic and cell signaling pathways in cardiomyocytes from diabetic mice by Susan A. Marsh; Louis J. Dell’Italia; John C. Chatham (pp. 819-828).

Patients with diabetes have a much greater risk of developing heart failure than non-diabetic patients, particularly in response to an additional hemodynamic stress such as hypertension or infarction. Previous studies have shown that increased glucose metabolism via the hexosamine biosynthesis pathway (HBP) and associated increase in O-linked-β-N-acetylglucosamine (O-GlcNAc) levels on proteins contributed to the adverse effects of diabetes on the heart. Therefore, in this study we tested the hypothesis that diabetes leads to impaired cardiomyocyte hypertrophic and cell signaling pathways due to increased HBP flux and O-GlcNAc modification on proteins. Cardiomyocytes isolated from type 2 diabetic db/db mice and non-diabetic controls were treated with 1 μM ANG angiotensin II (ANG) and 10 μM phenylephrine (PE) for 24 h. Activation of hypertrophic and cell signaling pathways was determined by assessing protein expression levels of atrial natriuretic peptide (ANP), α-sarcomeric actin, p53, Bax and Bcl-2 and phosphorylation of p38, ERK and Akt. ANG II and PE significantly increased levels of ANP and α-actin and phosphorylation of p38 and ERK in the non-diabetic but not in the diabetic group; phosphorylation of Akt was unchanged irrespective of group or treatment. Constitutive Bcl-2 levels were lower in diabetic hearts, while there was no difference in p53 and Bax. Activation of the HBP and increased protein O-GlcNAcylation in non-diabetic cardiomyocytes exhibited a significantly decreased hypertrophic signaling response to ANG or PE compared to control cells. Inhibition of the HBP partially restored the hypertrophic signaling response of diabetic cardiomyocytes. These results suggest that activation of the HBP and protein O-GlcNAcylation modulates hypertrophic and cell signaling pathways in type 2 diabetes.

Keywords: Hypertrophy; Diabetes; Cardiomyocyte; Cell signaling; O-GlcNAc

Inhibition of a bacterial O-GlcNAcase homologue by lactone and lactam derivatives: structural, kinetic and thermodynamic analyses by Yuan He; Abigail K. Bubb; Keith A. Stubbs; Tracey M. Gloster; Gideon J. Davies (pp. 829-839).

The dynamic, intracellular, O-GlcNAc modification is of continuing interest and one whose study through targeted “chemical genetics” approaches is set to increase. Of particular importance is the inhibition of the O-GlcNAc hydrolase, O-GlcNAcase (OGA), since this provides a route to elevate cellular O-GlcNAc levels, and subsequent phenotypic evaluation. Such a small molecule approach complements other methods and potentially avoids changes in protein–protein interactions that manifest themselves in molecular biological approaches to O-GlcNAc transferase knockout or over-expression. Here we describe the kinetic, thermodynamic and three-dimensional structural analysis of a bacterial OGA analogue from Bacteroides thetaiotaomicron, BtGH84, in complex with a lactone oxime (LOGNAc) and a lactam form of N-acetylglucosamine and compare their binding signatures with that of the more potent inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc). We show that both LOGNAc and the N-acetyl gluconolactam are significantly poorer inhibitors than PUGNAc, which may reflect poorer mimicry of transition state geometry and steric clashes with the enzyme upon binding; drawbacks that the phenyl carbamate adornment of PUGNAc helps mitigate. Implications for the design of future generations of inhibitors are discussed.

Keywords: Carbohydrate; Enzyme; X-ray structure; O-GlcNAc; Diabetes; Neurodegeneration

Increased hexosamine pathway flux and high fat feeding are not additive in inducing insulin resistance: evidence for a shared pathway by Robert C. Cooksey; Donald A. McClain (pp. 841-846).

Excess fatty acids and carbohydrates have both been implicated in the pathogenesis of type 2 diabetes, and both can reproduce essential features of the disease including insulin resistance and beta cell failure. It has been proposed that both nutrients may regulate metabolism through a common fuel sensing mechanism, namely hexosamine synthesis. We have previously shown that transgenic overexpression of the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), targeted to muscle and fat, leads to insulin resistance mediated by increased O-linked glycosylation of nuclear and cytosolic proteins. We report here that hexosamine-induced insulin resistance is not additive with that induced by high fat feeding. In control mice fed a high fat diet, glucose disposal rates during euglycemic hyperinsulinemia were decreased by 37% (p < 0.02) compared to mice on a low fat diet. Transgenic mice overexpressing GFA and fed a low fat diet exhibited a 51% decrease in glucose disposal compared to controls on a low fat diet (p < 0.001), but no further decrease was evident in the transgenic mice fed a high fat diet. Decreased glucose disposal rates were mirrored by increases in skeletal muscle levels of the principal end product of the hexosamine pathway, UDP-N-acetyl glucosamine. Serum leptin levels, which are modulated both by feeding and hexosamine flux, also show no additivity in their stimulation by GFA overexpression and high fat feeding. These data are consistent with a shared nutrient sensing pathway for high fat and carbohydrate fluxes and a common pathway by which glucose and lipids induce insulin resistance.

Keywords: Insulin resistance; Hexosamine synthesis; N-Acetylglucosamine; O-Linked glycosylation

Direct evidence of O-GlcNAcylation in the apicomplexan Toxoplasma gondii: a biochemical and bioinformatic study by Yobana Perez-Cervera; Grégoire Harichaux; Jörg Schmidt; Françoise Debierre-Grockiego; Vanessa Dehennaut; Ulrike Bieker; Edwige Meurice; Tony Lefebvre; Ralph T. Schwarz (pp. 847-856).

Toxoplasma gondii and Plasmodium falciparum are apicomplexan parasites responsible for serious diseases in humans. Many studies have focused on the post-translational modifications (PTMs) found in the two protists including phosphorylation, acetylation or SUMOylation but only a few of these are concerned with the nuclear and cytosolic-specific glycosylation O-GlcNAcylation. O-GlcNAcylation is a highly dynamic PTM—regulated by the ON and OFF enzymes: O-GlcNAc transferase and O-GlcNAcase—that can compete with phosphorylation but its function remains unclear. In this work, we directly prove the O-GlcNAcylation in T. gondii using antibodies specifically directed against the modification and we strongly suggest its occurrence in P. falciparum. We found that the inducible 70 kDa-Heat Shock Protein is O-GlcNAcylated, or associated with an O-GlcNAc-partner, in T. gondii. Using anti-OGT antibodies we were able to detect the expression of the glycosyltransferase in T. gondii cultured both in human foreskin fibroblast and in Vero cells and report its putative sequence. For the first time the presence of O-GlcNAcylation is unequivocally shown in T. gondii and suspected in P. falciparum. Since the O-GlcNAcylation is implicated in many biological fundamental processes this study opens a new research track in the knowledge of apicomplexans’ life cycle and pathogenic potential.

Keywords: Toxoplasma gondii ; Plasmodium falciparum ; O-GlcNAc; O-GlcNAcylation; O-GlcNAc transferase; OGT

Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody by Scott A. Yuzwa; Anuj K. Yadav; Yuliya Skorobogatko; Thomas Clark; Keith Vosseller; David J. Vocadlo (pp. 857-868).

The microtubule-associated protein tau is known to be post-translationally modified by the addition of N-acetyl-d-glucosamine monosaccharides to certain serine and threonine residues. These O-GlcNAc modification sites on tau have been challenging to identify due to the inherent complexity of tau from mammalian brains and the fact that the O-GlcNAc modification typically has substoichiometric occupancy. Here, we describe a method for the production of recombinant O-GlcNAc modified tau and, using this tau, we have mapped sites of O-GlcNAc on tau at Thr-123 and Ser-400 using mass spectrometry. We have also detected the presence of a third O-GlcNAc site on either Ser-409, Ser-412, or Ser-413. Using this information we have raised a rabbit polyclonal IgG antibody (3925) that detects tau O-GlcNAc modified at Ser-400. Further, using this antibody we have detected the Ser-400 tau O-GlcNAc modification in rat brain, which confirms the validity of this in vitro mapping approach. The identification of these O-GlcNAc sites on tau and this antibody will enable both in vivo and in vitro experiments designed to understand the possible functional roles of O-GlcNAc on tau.

Keywords: O-GlcNAc; Tau; Antibody; Mass spectrometry; Alzheimer disease

O-GlcNAcylation of the Plum pox virus capsid protein catalyzed by SECRET AGENT: characterization of O-GlcNAc sites by electron transfer dissociation mass spectrometry by Young-Cheon Kim; Namrata D. Udeshi; Jeremy L. Balsbaugh; Jeffrey Shabanowitz; Donald F. Hunt; Neil E. Olszewski (pp. 869-876).

The capsid protein of Plum pox virus (PPV-CP) is modified with O-linked β-N-acetylglucosamine (O-GlcNAc). In Arabidopsis thaliana this modification is made by an O-GlcNAc transferase named SECRET AGENT (SEC). Modification of PPV-CP by SEC is hypothesized to have a direct role in the infection process, because virus titer and rate of spread are reduced in SEC mutants. Previous studies used deletion mapping and site-directed mutagenesis to identify four O-GlcNAc sites on the capsid protein that are modified by Escherichia coli-expressed SEC. The infection process was not affected when two of these sites were mutated suggesting that O-GlcNAcylation of these sites does not have a significant role in the infection process or that a subset of the modifications is sufficient. Since it is possible that the mutational mapping approach missed or incorrectly identified O-GlcNAc sites, the modifications produced by E. coli-expressed SEC were characterized using mass spectrometry. O-GlcNAcylated peptides were enzymatically tagged with galactose, the products were enriched on immobilized Ricinus communis agglutinin I and sequenced by electron transfer dissociation (ETD) mass spectrometry. Five O-GlcNAc sites on PPV-CP were identified. Two of these sites were not identified in by the previous mutational mapping. In addition, one site previously predicted by mutation mapping was not detected, but modification of this site was not supported when the mutation mapping was repeated. This study suggests that mapping modification sites by ETD mass spectrometry is more comprehensive and accurate than mutational mapping.

Keywords: O-GlcNAc; Plum pox virus ; Capsid protein; O-GlcNAc transferase; Arabidopsis thaliana ; SECRET AGENT; Electron transfer dissociation tandem mass spectrometry

The E2F-1 associated retinoblastoma-susceptibility gene product is modified by O-GlcNAc by Lance Wells; Chad Slawson; Gerald W. Hart (pp. 877-883).

The retinoblastoma-susceptibility gene product (pRB) is a classical tumor suppressor. pRB regulates a number of cellular processes including proliferation, differentiation, and apoptosis. One of the essential mechanisms by which pRB, and the related p107 and p130 family members, act is through its interactions with the E2F class of transcription factors. E2F-1 transcription is necessary for entry into S-phase during the cell-cycle. pRB binds E2F-1 and represses transcription via recruitment of a histone deacetylase complex and by preventing co-activator complexes from binding E2F-1. Current dogma suggests that phosphorylation of pRB during mid- to late-G1 leads to release of E2F-1 and E2F-1 dependent transcriptional activation of essential S-phase genes. Here we show that pRB, and the related p107 protein, are modified by O-linked β-N-acetylglucosamine (O-GlcNAc) in an in vitro transcription/translation system. Furthermore, we show in vivo that pRB is more heavily glycosylated in G1 of the cell-cycle when pRB is known to be in an active, hypophosphorylated state. Finally, we demonstrate that E2F-1 associated pRB is modified by O-GlcNAc. These studies suggest that regulation of pRB function(s) may be controlled by dynamic O-GlcNAc modification, as well as phosphorylation.

Keywords: O-GlcNAc; pRB; Cell cycle; E2F

Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis by Sang-Hoon Shin; Dona C. Love; John A. Hanover (pp. 885-893).

O-linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAc addition to numerous cellular proteins including transcription and nuclear pore complexes and plays a key role in cellular signaling. One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform. To understand the basis of this selective cytotoxicity, we constructed a fully functional ecdysone-inducible GFP–OGT. Elevated GFP–OGT expression induced a dramatic increase in intracellular O-GlcNAcylated proteins. Furthermore, enhanced OGT expression efficiently triggered programmed cell death. Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity. Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line. These studies suggest that deregulated activity of the mitochondrially targeted mOGT may play a role in triggering the programmed cell death observed with diseases such as diabetes mellitus and neurodegeneration.

Keywords: O-GlcNAc; O-linked N-acetylglucosamine transferase; Apoptosis

Augmented O-GlcNAc signaling attenuates oxidative stress and calcium overload in cardiomyocytes by Gladys A. Ngoh; Lewis J. Watson; Heberty T. Facundo; Steven P. Jones (pp. 895-911).

O-linked β-N-acetylglucosamine (O-GlcNAc) is an inducible, dynamically cycling and reversible post-translational modification of Ser/Thr residues of nucleocytoplasmic and mitochondrial proteins. We recently discovered that O-GlcNAcylation confers cytoprotection in the heart via attenuating the formation of mitochondrial permeability transition pore (mPTP) and the subsequent loss of mitochondrial membrane potential. Because Ca²⁺ overload and reactive oxygen species (ROS) generation are prominent features of post-ischemic injury and favor mPTP formation, we ascertained whether O-GlcNAcylation mitigates mPTP formation via its effects on Ca²⁺ overload and ROS generation. Subjecting neonatal rat cardiac myocytes (NRCMs, n ≥ 6 per group) to hypoxia, or mice (n ≥ 4 per group) to myocardial ischemia reduced O-GlcNAcylation, which later increased during reoxygenation/reperfusion. NRCMs (n ≥ 4 per group) infected with an adenovirus carrying nothing (control), adenoviral O-GlcNAc transferase (adds O-GlcNAc to proteins, AdOGT), adenoviral O-GlcNAcase (removes O-GlcNAc to proteins, AdOGA), vehicle or PUGNAc (blocks OGA; increases O-GlcNAc levels) were subjected to hypoxia–reoxygenation or H₂O₂, and changes in Ca²⁺ levels (via Fluo-4AM and Rhod-2AM), ROS (via DCF) and mPTP formation (via calcein-MitoTracker Red colocalization) were assessed using time-lapse fluorescence microscopy. Both OGT and OGA overexpression did not significantly (P > 0.05) alter baseline Ca²⁺ or ROS levels. However, AdOGT significantly (P < 0.05) attenuated both hypoxia and oxidative stress-induced Ca²⁺ overload and ROS generation. Additionally, OGA inhibition mitigated both H₂O₂-induced Ca²⁺ overload and ROS generation. Although AdOGA exacerbated both hypoxia and H₂O₂-induced ROS generation, it had no effect on H₂O₂-induced Ca²⁺ overload. We conclude that inhibition of Ca²⁺ overload and ROS generation (inducers of mPTP) might be one mechanism through which O-GlcNAcylation reduces ischemia/hypoxia-mediated mPTP formation.

Keywords: Mitochondria; Heart; Metabolism; Glucose

Synthesis of C5-tetrazole derivatives of 2-amino-adipic acid displaying NMDA glutamate receptor antagonism by Fatimazohra Lenda; Nadine Crouzin; Mélanie Cavalier; Janique Guiramand; Fabien Lanté; Gérard Barbanel; Catherine Cohen-Solal; Jean Martinez; Farhate Guenoun; Frédéric Lamaty; Michel Vignes (pp. 913-922).

Five derivatives of 2-amino-adipic acid bearing a tetrazole-substituted in C5 position were synthesized. These compounds displayed selective antagonism towards N-methyl-d-aspartate (NMDA) receptors compared with AMPA receptors, and they were devoid of any neurotoxicity. Among these five analogues, one exhibited a higher affinity for synaptic NMDA responses than the other four. Therefore, C5 tetrazole-substituted of 2-amino-adipic acid represent an interesting series of new NMDA receptor antagonists. This approach may be considered as a new strategy to develop ligands specifically targeted to synaptic or extra-synaptic NMDA receptors.

Keywords: Glutamate; N-Methyl-d-Aspartate (NMDA); Synaptic transmission; Extrasynaptic receptors

The efficient synthesis of isotopically labeled peptide-derived Amadori products and their characterization by Katarzyna Kapczyńska; Piotr Stefanowicz; Łukasz Jaremko; Mariusz Jaremko; Alicja Kluczyk; Zbigniew Szewczuk (pp. 923-932).

Protein glycation is often a cause of diabetes-associated complications. The isotopically labeled peptide-derived Amadori products may serve as standards for quantitative determination of the glycated proteins. In this paper, we discussed various approaches to the synthesis of Amadori products labeled selectively with stable isotopes ²H, ¹³C and ¹⁸O.

Keywords: Amadori rearrangement; Peptides; Glucose; Fructose; Glycation; ¹³C; ²H; ¹⁸O; Isotopes; Labeling; Regioselective; Non-enzymatic protein modifications; Microwave synthesis; NMR studies

The promoting effect of retinoic acid on proliferation of chicken primordial germ cells by increased expression of cadherin and catenins by Minli Yu; Kun Guan; Caiqiao Zhang (pp. 933-941).

Proliferation and cellular aggregation are both crucial features for survival and self-renewal of primordial germ cells (PGCs). Adhesive proteins play pivotal roles in cell–cell adhesion and signal exchanges under the influence of cytokines, growth factors and bioactive metabolites such as retinoic acid (RA). In this study, proliferation-promoting effect of RA on chicken PGCs was investigated by revealing changes in adhesive proteins E-cadherin and α/β catenins. PGCs were isolated from the genital ridge of 4-day-old chicken embryos and cultured on embryonic fibroblast feeder. RA (10⁻⁷–10⁻⁵ M) increased PGCs aggregation and mRNA expression of E-cadherin and α/β-catenins. Furthermore, E-cadherin and β-catenin protein expression levels were increased by RA treatment. However, RA-elicited effect was significantly attenuated by a PKC inhibitor H₇. In addition, the number and area of PGC colonies were increased by RA treatment at 10⁻⁷–10⁻⁵ M. Again, this increase was reduced by combined treatment of H₇. The proliferating effect of RA on PGCs was further confirmed by increased mRNA expression of cyclins, CCND1 and CCNE1, and cyclin-dependent kinases 6 and 2, which are critical for G1–S progression in cell cycle. Moreover, flow cytometry analysis confirmed that RA-treated PGC populations displayed a significant increase in the proportion of S and G2 phase cells. Likewise, this stimulating action was hindered by combined H₇ treatment. These results indicate that RA, as a bioactive metabolite of vitamin A, may promote PGC proliferation and increase intercellular aggregation of PGCs via E-cadherin and α/β-catenins expression through the PKC signaling pathway.

Keywords: Primordial germ cells; Retinoic acid; Cadherin; Catenin; Proliferation; Chicken

Proteomic analysis of childhood de novo acute myeloid leukemia and myelodysplastic syndrome/AML: correlation to molecular and cytogenetic analyses by Maria Braoudaki; Fotini Tzortzatou-Stathopoulou; Athanasios K. Anagnostopoulos; Chrisa Papathanassiou; Konstantinos Vougas; Kalliopi Karamolegou; George Th. Tsangaris (pp. 943-951).

The aim of this study was to investigate the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML) and to provide additional data regarding the proteomic analysis of AML. The protein profiles obtained were correlated to cytogenetic and molecular analyses. Bone marrow (BM) and peripheral blood (PB) samples were obtained during MDS diagnosis, at MDS transformation to AML, at de novo AML diagnosis and 3 months following treatment. As controls, non-leukemic pediatric patients were studied. Cytogenetic and molecular analyses were carried out by G banding and polymerase chain reaction followed by sequencing, respectively. Differential proteomic analysis was performed by two-dimensional gel electrophoresis and protein identification by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. No significant correlations were noted between protein patterns and cytogenetic or molecular analyses. Certain suppressor genes, metabolic enzymes, immunoglobulins and actin-binding proteins were differentially expressed by BM or PB plasma and cell lysates compared to controls. The obtained data showed that vitamin D and gelsolin played contradicting roles in contributing and restraining leukemogenesis, while MOES, EZRI and AIFM1 could be considered as biomarkers for AML.

Keywords: Childhood AML; MDS; Proteomics; Mass spectrometry; Two-dimensional gel electrophoresis

Rab32 and the remodeling of the imaginal midgut in Helicoverpa armigera by Li Hou; Jin-Xing Wang; Xiao-Fan Zhao (pp. 953-961).

Midgut remodeling is a complex physiological process in holometabolous insects. During midgut remodeling, the larval midgut is decomposed by apoptosis or autophagy during metamorphosis, and the degraded larval midgut is partially absorbed as nutrients by the imaginal midgut for its formation. The molecular mechanism involved in this process is not clear. Here, we found that a Rab protein, which we have named HaRab32, is related to the organogenesis of insect imaginal midgut. Results show that HaRab32 is up-regulated in epidermis and midgut during metamorphosis. Its expression could be up-regulated by 20E. Immunohistochemistry shows Rab32 is distributed in the epithelium of the imaginal midgut during metamorphosis. Knockdown of HaRab32 by RNA interference disturbs the formation of the imaginal midgut. These data imply HaRab32 plays important roles in midgut remodeling by participating in the imaginal midgut formation.

Keywords: Helicoverpa armigera ; Rab32; Midgut remodeling; 20E

iFC²: an integrated web-server for improved prediction of protein structural class, fold type, and secondary structure content by Ke Chen; Wojciech Stach; Leila Homaeian; Lukasz Kurgan (pp. 963-973).

Several descriptors of protein structure at the sequence and residue levels have been recently proposed. They are widely adopted in the analysis and prediction of structural and functional characteristics of proteins. Numerous in silico methods have been developed for sequence-based prediction of these descriptors. However, many of them do not have a public web-server and only a few integrate multiple descriptors to improve the predictions. We introduce iFC² (integrated prediction of fold, class, and content) server that is the first to integrate three modern predictors of sequence-level descriptors. They concern fold type (PFRES), structural class (SCEC), and secondary structure content (PSSC-core). The server exploits relations between the three descriptors to implement a cross-evaluation procedure that improves over the predictions of the individual methods. The iFC² annotates fold and class predictions as potentially correct/incorrect. When tested on datasets with low-similarity chains, for the fold prediction iFC² labels 82% of the PFRES predictions as correct and the accuracy of these predictions equals 72%. The accuracy of the remaining 28% of the PFRES predictions equals 38%. Similarly, our server assigns correct labels for over 79% of SCEC predictions, which are shown to be 98% accurate, while the remaining SCEC predictions are only 15% accurate. These results are shown to be competitive when contrasted against recent relevant web-servers. Predictions on CASP8 targets show that the content predicted by iFC² is competitive when compared with the content computed from the tertiary structures predicted by three best-performing methods in CASP8. The iFC² server is available at http://biomine.ece.ualberta.ca/1D/1D.html .

Keywords: Protein structure classification; Secondary structure; Structural class; Fold type; SCOP

PROlocalizer: integrated web service for protein subcellular localization prediction by Kirsti Laurila; Mauno Vihinen (pp. 975-980).

Subcellular localization is an important protein property, which is related to function, interactions and other features. As experimental determination of the localization can be tedious, especially for large numbers of proteins, a number of prediction tools have been developed. We developed the PROlocalizer service that integrates 11 individual methods to predict altogether 12 localizations for animal proteins. The method allows the submission of a number of proteins and mutations and generates a detailed informative document of the prediction and obtained results. PROlocalizer is available at http://bioinf.uta.fi/PROlocalizer/ .

Keywords: Protein localization prediction; Cell compartments; Mutations; Disease-causing mutations; Prediction method

Biophysical characterization of recombinant HIV-1 subtype C virus infectivity factor by Daniela Gallerano; Siva Charan Devanaboyina; Ines Swoboda; Birgit Linhart; Irene Mittermann; Walter Keller; Rudolf Valenta (pp. 981-989).

HIV-1 virus infectivity factor (Vif) is one of the four accessory proteins that are characteristic of primate lentiviruses and critically required for the infection of host cells. Vif plays a key role in replication and transmission of the virus in non-permissive cells, such as primary T cells and macrophages. Using co-precipitation and co-fractionation techniques, evidence has been provided that Vif interacts with a variety of host proteins, such as the cytidine deaminases APOBEC3G and 3F, the Cullin5/EloBC ubiquitin–ligase complex, Fyn and Hck tyrosine kinases, as well as with viral components, such as the immature Gag precursor and viral RNA. We report on the expression, purification and molecular characterization of a folded recombinant subtype C Vif. Vif was expressed in E. coli with a C-terminal hexahistidine tag and purified by nickel affinity chromatography. We obtained approximately 5 mg protein per liter of bacterial culture, with a purity >95%. The expected molecular mass of 23.7 kDa was confirmed by mass spectrometry. Although dynamic light scattering and small angle X-ray scattering measurements revealed the presence of high molecular weight aggregates in the protein preparation, circular dichroism analysis showed that the protein contains mainly folded β-sheet elements and exhibits remarkable thermal stability (T _m > 95°C). Recombinant Vif may be used as a tool to study its biological functions and tertiary structure, as well as for the development of diagnostic, therapeutic and preventive strategies for HIV-1 infections.

Keywords: HIV; Virus infectivity factor (Vif); E.coli expression; Circular dichroism; Thermal stability

Accurate prediction of the burial status of transmembrane residues of α-helix membrane protein by incorporating the structural and physicochemical features by Chengqi Wang; Shuyan Li; Lili Xi; Huanxiang Liu; Xiaojun Yao (pp. 991-1002).

Predicting the burial status (the residue exposure to the lipid bilayer or buried within the protein core) of transmembrane (TM) residues of α-helix membrane protein (αHMP) is of great importance for genome-wide annotation and for experimental researchers to elucidate diverse physiological processes. In this work, we developed a new computational model that can be used for predicting the burial status of TM residues of αHMP. By incorporating physicochemical scales and conservation index, an efficient prediction model using least squares support vector machine (LS-SVM) was developed. The model was developed from 43 protein chains and its prediction ability was evaluated by an independent test set of other non-redundant ten protein chains. The prediction accuracy of our method was much better than the results of the reported works. Our results demonstrate that the LS-SVM prediction model incorporating structural and physicochemical features derived from sequence information could greatly improve the prediction accuracy.

Keywords: Burial status of transmembrane residues; Recursive feature elimination (RFE); Least squares support vector machine (LS-SVM)

ESM-1 silencing decreased cell survival, migration, and invasion and modulated cell cycle progression in hepatocellular carcinoma by Yun Hee Kang; Na Young Ji; Chung Il Lee; Hee Gu Lee; Jae Wha Kim; Young IL Yeom; Dae Ghon Kim; Seung Kew Yoon; Jong Wan Kim; Pil Je Park; Eun Young Song (pp. 1003-1013).

Endothelial cell-specific molecule-1 (ESM-1) is a secretory proteoglycan comprising a mature polypeptide of 165 amino acids and a single dermatan sulfate. The aim of this study was to evaluate endothelial cell-specific molecule-1 (ESM-1) as a hepatocellular carcinoma (HCC) marker and to analyze the effect of ESM-1 gene silencing in hepatocellular carcinoma cells. RT-PCR and Western Blot analysis revealed overexpression of ESM-1 in human HCC liver tissue and in serum from patients with HCC. Sandwich ELISA assay was used for quantitative analysis of ESM-1 in serum. Levels of ESM-1 were significantly elevated in the serum of patients with HCC (n = 40) as compared to serum from patients with hepatitis (AH, n = 40; CH, n = 39) or liver cirrhosis (n = 40) or from healthy subjects (n = 40). The accuracy of ESM-1 for HCC was higher than that of α-fetoprotein (AFP) according to ROC curve analysis. Expression of ESM-1 siRNA decreased cell survival through the inhibition of NF-κB pathway and induced cell cycle arrest by PTEN induction resulting in the inhibition of cyclin D1 in SK-Hep1 cells. Furthermore, ESM-1 silencing inhibited cell migration and invasion of SK-Hep1 cells. This study demonstrates that ESM-1 as a potential tumor marker is overexpressed in most tissues and serum in the presence of HCC and is involved with cell survival, cell cycle progression, migration, and invasion of hepatocellular carcinoma cells. Based on our results, we suggest that ESM-1 or a combination of ESM-1 and AFP is useful markers for diagnosis of HCC and ESM-1 may be useful therapeutic target of hepatocellular carcinoma.

Keywords: ESM-1; Hepatocellular carcinoma; Diagnostic marker; Cell cycle; Cell migration

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.40, #3)

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.40, #3)