Enzymatic methyl esterification of a deamidated form of mouse epidermal growth factor

The enzyme S-adenosylmethionine:protein carboxyl-O-methyl-transferase, type II (EC 2.1.1.77; PCMT) from eukaryotes methyl esterifies peptides containing isoAsp residues, which can arise from spontaneous deamidation of labile Asn residues. We report here a study on in vitro methyl esterification of mouse EGF by bovine brain PCMT. This peptide contains two Asn in the sequences Asn1-Ser2 and Asn16-Gly17. It is known from the literature that the presence of a small residue on the carboxyl side of asparaginyl makes this residue susceptible to deamidation through the spontaneous formation of a succinimide intermediate. Therefore EGF was incubated under deamidating conditions (pH 9.0, 37 degrees for 48 h) and the extent of deamidation monitored by enzymatically measuring the NH3 produced during the alkali treatment: a release of 0.80 mol NH3/mol EGF was calculated. The alkali-treated EGF, analyzed by anion-exchange chromatography, shows two major components identified as native EGF (nEGF) and its deamidated form (dEGF). When incubated in the presence of purified PCMT neither nEGF nor dEGF showed any methyl accepting capability. Since it is known that the three-dimensional structure of a protein may hinder the methyl esterification of a potential ethyl accepting site, dEGF was unfolded by reducing and alkylating the intrachain disulfide bridges. Only a slight increase in the methyl accepting capability could be observed. Conversely, when EGF was deamidated after its unfolding, the resulting protein was stoichiometrically methylated by PCMT, presumably at level of isoAsp16. Our findings strongly suggest that the three-dimensional structure of a protein is a major specificity determinant for both deamidation and methyl esterification processes.     

Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae

The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.     

Non-repair Pathways for Minimizing Protein Isoaspartyl Damage in the Yeast Saccharomyces cerevisiae

The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (EC 2.1.1.77) can initiate the repair of l-isoaspartyl residues to l-aspartyl residues in most organisms, no gene homolog or enzymatic activity is present in the budding yeast Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how proteins containing isoaspartyl residues are metabolized in this organism. Surprisingly, the level of isoaspartyl residues in yeast proteins (50–300 pmol of isoaspartyl residues/mg of protein extract) is comparable with organisms with protein-l-isoaspartyl (d-aspartyl) O-methyltransferase, suggesting a novel regulatory pathway. Interfering with common protein quality control mechanisms by mutating and inhibiting the proteasomal and autophagic pathways in vivo did not increase isoaspartyl residue levels compared with wild type or uninhibited cells. However, the inhibition of metalloproteases in in vitro aging experiments by EDTA resulted in an ∼3-fold increase in the level of isoaspartyl-containing peptides. Characterization by mass spectrometry of these peptides identified several proteins involved in metabolism as targets of isoaspartyl damage. Further analysis of these peptides revealed that many have an N-terminal isoaspartyl site and originate from proteins with short half-lives. These results suggest that one or more metalloproteases participate in limiting isoaspartyl formation by robust proteolysis.     

Formation of Mutagens by Heating Creatine or Amino Acids with Addition or Fatty Acids

α_s1-Casein was deamidated specifically in the moiety of the glutaminyl side chains by transglutaminase. The deamidation was achieved by reversibly blocking the amino groups in substrate protein. The deamidated product was found out to have 80% (11 residues) of the total deamidated glutaminyl residues. No significant conformational changes were observed even after the modifications. Consequently, the solubility in acidic pH regions, especially at pH 5, was increased. On the other hand, the Ca²⁺-sensitivity largely decreased to the extent that nearly all the deamidated product was soluble at 20 mm CaCl₂. Deamidation by transglutaminase may be useful as a new method to solubilize insoluble proteins without destroying protein structures.     

Metaproteomics provides functional insight into activated sludge wastewater treatment

Background

Through identification of highly expressed proteins from a mixed culture activated sludge system this study provides functional evidence of microbial transformations important for enhanced biological phosphorus removal (EBPR).

Methodology/Principal Findings

A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P. The microbial communities were dominated by the uncultured polyphosphate-accumulating organism “Candidatus Accumulibacter phosphatis”. When EBPR failed, the sludge was dominated by tetrad-forming α-Proteobacteria. Representative and reproducible 2D gel protein separations were obtained for all sludge samples. 638 protein spots were matched across gels generated from the phosphate removing sludges. 111 of these were excised and 46 proteins were identified using recently available sludge metagenomic sequences. Many of these closely match proteins from “Candidatus Accumulibacter phosphatis” and could be directly linked to the EBPR process. They included enzymes involved in energy generation, polyhydroxyalkanoate synthesis, glycolysis, gluconeogenesis, glycogen synthesis, glyoxylate/TCA cycle, fatty acid β oxidation, fatty acid synthesis and phosphate transport. Several proteins involved in cellular stress response were detected.

Conclusions/Significance

Importantly, this study provides direct evidence linking the metabolic activities of “Accumulibacter” to the chemical transformations observed in EBPR. Finally, the results are discussed in relation to current EBPR metabolic models.     

Enzymatic attributes of an l-isoaspartyl methyltransferase from Candida utilis and its role in cell survival

BackgroundsSpontaneous deamidation and isoaspartate (IsoAsp) formation contributes to aging and reduced longevity in cells. A protein-l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) is responsible for minimizing IsoAsp moieties in most organisms.MethodsPCMT was purified in its native form from yeast Candida utilis. The role of the native PCMT in cell survival and protein repair was investigated by manipulating intracellular PCMT levels with Oxidized Adenosine (AdOx) and Lithium Chloride (LiCl). Proteomic Identification of possible cellular targets was carried out using 2-dimensional gel electrophoresis, followed by on-Blot methylation and mass spectrometric analysis.ResultsThe 25.4 kDa native PCMT from C. utilis was found to have a K_m of 3.5 µM for AdoMet and 33.36 µM for IsoAsp containing Delta Sleep Inducing Peptide (DSIP) at pH 7.0. Native PCMT comprises of 232 amino acids which is coded by a 698 bp long nucleotide sequence. Phylogenetic comparison revealed the PCMT to be related more closely with the prokaryotic homologs. Increase in PCMT levels in vivo correlated with increased cell survival under physiological stresses. PCMT expression was seen to be linked with increased intracellular reactive oxygen species (ROS) concentration. Proteomic identification of possible cellular substrates revealed that PCMT interacts with proteins mainly involved with cellular housekeeping. PCMT effected both functional and structural repair in aged proteins in vitro.General significanceIdentification of PCMT in unicellular eukaryotes like C. utilis promises to make investigations into its control machinery easier owing to the familiarity and flexibility of the system.     

Repair of Isoaspartate Formation Modulates the Interaction of Deamidated 4E-BP2 with mTORC1 in Brain

In eukaryotes, a rate-limiting step of translation initiation is recognition of the mRNA 5′ m⁷GpppN cap structure by the eukaryotic initiation factor 4F (eIF4F), a heterotrimeric complex consisting of the cap-binding protein, eIF4E, along with eIF4G, and eIF4A. The eIF4E-binding proteins (4E-BPs) repress translation by disrupting eIF4F formation, thereby preventing ribosome recruitment to the mRNA. Of the three 4E-BPs, 4E-BP2 is the predominant paralog expressed in the mammalian brain and plays an important role in synaptic plasticity and learning and memory. 4E-BP2 undergoes asparagine deamidation, solely in the brain, during early postnatal development. Deamidation spontaneously converts asparagines into a mixture of aspartates or isoaspartates, the latter of which may be destabilizing to proteins. The enzyme protein l-isoaspartyl methyltransferase (PIMT) prevents isoaspartate accumulation by catalyzing the conversion of isoaspartates to aspartates. PIMT exhibits high activity in the brain, relative to other tissues. We report here that 4E-BP2 is a substrate for PIMT. In vitro deamidated 4E-BP2 accrues isoapartyl residues and is methylated by recombinant PIMT. Using an antibody that recognizes 4E-BP2, which harbors isoaspartates at the deamidation sites, Asn⁹⁹ and Asn¹⁰², we demonstrate that 4E-BP2 in PIMT−/− brain lysates contains isoaspartate residues. Further, we show that 4E-BP2 containing isoaspartates lacks the augmented association with raptor that is a feature of deamidated 4E-BP2.     

Control of Cellular Bcl-xL Levels by Deamidation-Regulated Degradation

The cellular concentration of Bcl-x_L is among the most important determinants of treatment response and overall prognosis in a broad range of tumors as well as an important determinant of the cellular response to several forms of tissue injury. We and others have previously shown that human Bcl-x_L undergoes deamidation at two asparaginyl residues and that DNA-damaging antineoplastic agents as well as other stimuli can increase the rate of deamidation. Deamidation results in the replacement of asparginyl residues with aspartyl or isoaspartyl residues. Thus deamidation, like phosphorylation, introduces a negative charge into proteins. Here we show that the level of human Bcl-x_L is constantly modulated by deamidation because deamidation, like phosphorylation in other proteins, activates a conditional PEST sequence to target Bcl-x_L for degradation. Additionally, we show that degradation of deamidated Bcl-x_L is mediated at least in part by calpain. Notably, we present sequence and biochemical data that suggest that deamidation has been conserved from the simplest extant metazoans through the human form of Bcl-x_L, underscoring its importance in Bcl-x_L regulation. Our findings strongly suggest that deamidation-regulated Bcl-x_L degradation is an important component of the cellular rheostat that determines susceptibility to DNA-damaging agents and other death stimuli.     

A tethered bilayer assembled on top of immobilized calmodulin to mimic cellular compartmentalization

Background

Biomimetic membrane models tethered on solid supports are important tools for membrane protein biochemistry and biotechnology. The supported membrane systems described up to now are composed of a lipid bilayer tethered or not to a surface separating two compartments: a ”trans” side, one to a few nanometer thick, located between the supporting surface and the membrane; and a “cis” side, above the synthetic membrane, exposed to the bulk medium. We describe here a novel biomimetic design composed of a tethered bilayer membrane that is assembled over a surface derivatized with a specific intracellular protein marker. This multilayered biomimetic assembly exhibits the fundamental characteristics of an authentic biological membrane in creating a continuous yet fluid phospholipidic barrier between two distinct compartments: a “cis” side corresponding to the extracellular milieu and a “trans” side marked by a key cytosolic signaling protein, calmodulin.

Methodology/Principal Findings

We established and validated the experimental conditions to construct a multilayered structure consisting in a planar tethered bilayer assembled over a surface derivatized with calmodulin. We demonstrated the following: (i) the grafted calmodulin molecules (in trans side) were fully functional in binding and activating a calmodulin-dependent enzyme, the adenylate cyclase from Bordetella pertussis; and (ii) the assembled bilayer formed a continuous, protein-impermeable boundary that fully separated the underlying calmodulin (trans side) from the above medium (cis side).

Conclusions

The simplicity and robustness of the tethered bilayer structure described here should facilitate the elaboration of biomimetic membrane models incorporating membrane embedded proteins and key cytoplasmic constituents. Such biomimetic structures will also be an attractive tool to study translocation across biological membranes of proteins or other macromolecules.     

Analysis of autophagy genes in microalgae: chlorella as a potential model to study mechanism of autophagy

Background

Microalgae, with the ability to mitigate CO₂ emission and produce carbohydrates and lipids, are considered one of the most promising resources for producing bioenergy. Recently, we discovered that autophagy plays a critical role in the metabolism of photosynthetic system and lipids production. So far, more than 30-autophagy related (ATG) genes in all subtypes of autophagy have been identified. However, compared with yeast and mammals, in silico and experimental research of autophagy pathways in microalgae remained limited and fragmentary.

Principal Findings

In this article, we performed a genome-wide analysis of ATG genes in 7 microalgae species and explored their distributions, domain structures and evolution. Eighteen “core autophagy machinery” proteins, four mammalian-specific ATG proteins and more than 30 additional proteins (including “receptor-adaptor” complexes) in all subtypes of autophagy were analyzed. Data revealed that receptor proteins in cytoplasm-to-vacuole targeting and mitophagy seem to be absent in microalgae. However, most of the “core autophagy machinery” and mammalian-specific proteins are conserved among microalgae, except for the ATG9-cycling system in Chlamydomonas reinhardtii and the second ubiquitin-like protein conjugation complex in several algal species. The catalytic and binding residues in ATG3, ATG5, ATG7, ATG8, ATG10 and ATG12 are also conserved and the phylogenetic tree of ATG8 coincides well with the phylogenies. Chlorella contains the entire set of the core autophagy machinery. In addition, RT-PCR analysis verified that all crucial ATG genes tested are expressed during autophagy in both Chlorella and Chlamydomonas reinhardtii. Finally, we discovered that addition of 3-Methyladenine (a PI3K specific inhibitor) could suppress the formation of autophagic vacuoles in Chlorella.

Conclusions

Taken together, Chlorella may represent a potential model organism to investigate autophagy pathways in photosynthetic eukaryotes. The study will not only promote understanding of the general features of autophagic pathways, but also benefit the production of Chlorella-derived biofuel with future commercial applications.     

Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses

Background

Black flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized.

Methodology/Principal Findings

Two Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins are inhibitors of serine proteases, we hypothesized that SV-66 and/or −170 were involved in the anticoagulant activity of black fly saliva. Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation. Mutational analysis suggested that SV-66 is a canonical BPTI-like inhibitor. Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin. Surface plasmon resonance studies indicated that rSV-66 bound with highest affinity to elastase (K_D = 0.4 nM) and to the active site of FXa (K_D = 3.07 nM). We propose the name “Simukunin” for this novel protein.

Conclusions

We conclude that Simukunin preferentially inhibits Factor Xa. The inhibition of elastase and cathepsin G further suggests this protein may modulate inflammation, which could potentially affect pathogen transmission.     

A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris

Background

Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers.

Methodology/Principal Findings

Codon-optimized “Opti-Pgp” and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from T_m ∼40°C to 49°C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids.

Conclusion

The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins.     

Control of process-induced asparaginyl deamidation during manufacture of Erwinia chrysanthemi l-asparaginase

During the manufacture of the chemotherapeutic enzyme Erwinia chrysanthemi l-asparaginase, a small proportion (approximately 5–15%) of acidic variants, including deamidated species, are observed. Although the deamidated forms appear to have similar specific activity and quaternary structure to the unmodified enzyme, monitoring and control of these forms is important from a regulatory perspective. The extent of Asn to Asp deamidation directly correlates with the time taken to thaw the Erwinia cells. Erwinia l-asparaginase is a tetrameric enzyme containing one site, Asn₂₈₁, theoretically very labile to deamidation due to the sequence Asn-Gly. Structurally, this part of the protein sequence is completely buried inside the tetramer, but solvent-exposed upon tetramer dissociation. During the cell thawing and alkaline lysis sequence of the process, lengthening the cell thaw times by up to 24 h allowed tetramer to reassociate, protected Asn₂₈₁ from deamidation and reduced the acidic species content of the l-asparaginase from approximately 17% to 9% as measured by weak cation-exchange (WCX) HPLC. The correlation of cell thaw time with acidic species content was also confirmed using capillary zone electrophoresis (CZE) and peptide mapping. These studies demonstrate that cell thaw time is an important, if unexpected, control variable for l-asparaginase deamidation.     

Needles in the EST haystack: large-scale identification and analysis of excretory-secretory (ES) proteins in parasitic nematodes using expressed sequence tags (ESTs)

Background

Parasitic nematodes of humans, other animals and plants continue to impose a significant public health and economic burden worldwide, due to the diseases they cause. Promising antiparasitic drug and vaccine candidates have been discovered from excreted or secreted (ES) proteins released from the parasite and exposed to the immune system of the host. Mining the entire expressed sequence tag (EST) data available from parasitic nematodes represents an approach to discover such ES targets.

Methods and Findings

In this study, we predicted, using EST2Secretome, a novel, high-throughput, computational workflow system, 4,710 ES proteins from 452,134 ESTs derived from 39 different species of nematodes, parasitic in animals (including humans) or plants. In total, 2,632, 786, and 1,292 ES proteins were predicted for animal-, human-, and plant-parasitic nematodes. Subsequently, we systematically analysed ES proteins using computational methods. Of these 4,710 proteins, 2,490 (52.8%) had orthologues in Caenorhabditis elegans, whereas 621 (13.8%) appeared to be novel, currently having no significant match to any molecule available in public databases. Of the C. elegans homologues, 267 had strong “loss-of-function” phenotypes by RNA interference (RNAi) in this nematode. We could functionally classify 1,948 (41.3%) sequences using the Gene Ontology (GO) terms, establish pathway associations for 573 (12.2%) sequences using Kyoto Encyclopaedia of Genes and Genomes (KEGG), and identify protein interaction partners for 1,774 (37.6%) molecules. We also mapped 758 (16.1%) proteins to protein domains including the nematode-specific protein family “transthyretin-like” and “chromadorea ALT,” considered as vaccine candidates against filariasis in humans.

Conclusions

We report the large-scale analysis of ES proteins inferred from EST data for a range of parasitic nematodes. This set of ES proteins provides an inventory of known and novel members of ES proteins as a foundation for studies focused on understanding the biology of parasitic nematodes and their interactions with their hosts, as well as for the development of novel drugs or vaccines for parasite intervention and control.     

Up-regulation of NDRG2 in senescent lens epithelial cells contributes to age-related cataract in human

Background

Human N-Myc downstream regulated gene2 (NDRG2), a novel gene has been cloned and shown to be related to a number of cellular processes, including proliferation, differentiation, stress, and apoptosis. NDRG2 has also been linked to age-related Alzheimer''s disease. Since the role of this gene in senescence is limited, we have investigated the potential role of NDRG2 in human lens epithelial cells (HLECs), a paradigm implicated in age-related cataract.

Methodology/Principal Findings

Cultured HLECs (SRA01/04) were subjected to prolonged exposure to low dose of H₂O₂ to simulate senescence. After being exposed to 50 µM H₂O₂ for 2 weeks, HLECs senescent-morphological changes appeared, cell viability decreased dramatically, cell proliferation reduced from 37.4% to 16.1%, and senescence-associated β-galactosidase activity increased from 0 to 90.3%. Ndrg2 protein expression was also significantly increased in these senescent cells. To induce overexpression of NDRG2, SRA01/04 cells were infected with the adenoviral vector of NDRG2. In these cells, overexpression of NDRG2 resulted in a fibroblast-like appearance and the cell viability decreased about 20%. In addition, the NDRG2-overexpression cells demonstrated 20% lower viability when exposed to 50–200 µM H₂O₂ for acute oxidative stress. Furthermore, the expression of NDRG2 from age-related cataracts was up-regulated 2-fold at both mRNA and protein levels compared with the clear lenses.

Conclusions/Significance

NDRG2 is up regulated not only in the ageing process of HLECs in vitro but also in the cells from human age-related cortical cataract in vivo. Up-regulation of NDRG2 induces cell morphological changes, reduces cell viability, and especially lowers cellular resistance to oxidative stress. NDRG2-mediated affects in HLECs may associate with age-related cataract formation.     

The concentration of soluble extracellular amyloid-β protein in acute brain slices from CRND8 mice

Background

Many recent studies of the effects of amyloid-β protein (Aβ) on brain tissue from amyloid precursor protein (APP) overexpressing mice have concluded that Aβ oligomers in the extracellular space can profoundly affect synaptic structure and function. As soluble proteins, oliomers of Aβ can diffuse through brain tissue and can presumably exit acute slices, but the rate of loss of Aβ species by diffusion from brain slices and the resulting reduced concentrations of Aβ species in brain slices are unknown.

Methodology/Principal Findings

Here I combine measurements of Aβ_1–42 diffusion and release from acute slices and simple numerical models to measure the concentration of Aβ_1–42 in intact mice (in vivo) and in acute slices from CRND8 mice. The in vivo concentration of diffusible Aβ_1–42 in CRND8 mice was 250 pM at 6 months of age and 425 pM at 12 months of age. The concentration of Aβ_1–42 declined rapidly after slice preparation, reaching a steady-state concentration within one hour. 50 µm from the surface of an acute slice the steady-state concentration of Aβ was 15–30% of the concentration in intact mice. In more superficial regions of the slice, where synaptic physiology is generally studied, the remaining Aβ is less than 15%. Hence the concentration of Aβ_1–42 in acute slices from CRND8 mice is less than 150 pM.

Conclusions/Significance

Aβ affects synaptic plasticity in the picomolar concentration range. Some of the effects of Aβ may therefore be lost or altered after slice preparation, as the extracellular Aβ concentration declines from the high picomolar to the low picomolar range. Hence loss of Aβ by diffusion may complicate interpretation of the effects of Aβ in experiments on acute slices from APP overexpressing mice.     

UVA irradiation induces L-isoaspartyl formation in melanoma cell proteins.

It has been reported that UVA effects are partly mediated by production of reactive oxygen species. Moreover, oxidative stress increases protein damage, involving the occurrence of isoaspartyl residues, a product of protein deamidation/isomerization reactions. This work was undertaken in order to study the effects of UVA irradiation, mediated by oxidation, on sensitive protein targets. Melanoma cells exposed to UVA rays have been chosen as a model for monitoring the occurrence of L-isoaspartyl sites. A dramatic increase of these abnormal residues, specifically recognized and methylated by the enzyme L-isoaspartate(D-aspartate) O-methyltransferase (PCMT; EC 2.1.1.77), can be detected after exposure of M14 cells to raising doses of UVA. The effect of UVA on NO and TBARS accumulation, as well as on DNA fragmentation, has also been investigated. NO formation parallels the increase in isoaspartyl formation, while lipid peroxidation occurs only at the highest UVA doses. No DNA fragmentation has been detected under the employed experimental conditions. These results, as a whole, indicate that protein damages are one of the early events on UVA-induced cell injury. The endogenous activity of PCMT remains remarkably stable under UVA treatment, suggesting that this enzyme might play a crucial role in the repair and/or disposal of damaged proteins in UVA-irradiated cells.     

Cloning and characterization of a putative TAC1 ortholog associated with leaf angle in maize (Zea mays L.)

Background

Modifying plant architecture to increase photosynthesis efficiency and reduce shade avoidance response is very important for further yield improvement when crops are grown in high density. Identification of alleles controlling leaf angle in maize is needed to provide insight into molecular mechanism of leaf development and achieving ideal plant architecture to improve grain yield.

Methodology/Principal Findings

The gene cloning was done by using comparative genomics, and then performing real-time polymerase chain reaction (RT-PCR) analysis to assay gene expression. The gene function was validated by sequence dissimilarity analysis and QTL mapping using a functional cleaved amplified polymorphism (CAP).

Conclusions

The leaf angle is controlled by a major quantitative trait locus, ZmTAC1 (Zea mays L. Leaf Angle Control 1). ZmTAC1 has 4 exons encoding a protein with 263 amino acids, and its domains are the same as those of the rice OsTAC1 protein. ZmTAC1 was found to be located in the region of qLA2 by using the CAP marker and the F_2:3 families from the cross between Yu82 and Shen137. Real-time PCR analysis revealed ZmTAC1 expression was the highest in the leaf-sheath pulvinus, less in the leaf and shoot apical meristem, and the lowest in the root. A nucleotide difference in the 5′-untranslated region (UTR) between the compact inbred line Yu82 (“CTCC”) and the expanded inbred line Shen137 (“CCCC”) influences the expression level of ZmTAC1, further controlling the size of the leaf angle. Sequence verification of the change in the 5′-UTR revealed ZmTAC1 with “CTCC” was present in 13 compact inbred lines and ZmTAC1 with “CCCC” was present in 18 expanded inbred lines, indicating ZmTAC1 had been extensively utilized in breeding with regard to the improvement of the maize plant architecture.     

Re-expression of AKAP12 inhibits progression and metastasis potential of colorectal carcinoma in vivo and in vitro

Background

AKAP12/Gravin (A kinase anchor protein 12) is one of the A-kinase scaffold proteins and a potential tumor suppressor gene in human primary cancers. Our recent study demonstrated the highly recurrent loss of AKAP12 in colorectal cancer and AKAP12 reexpression inhibited proliferation and anchorage-independent growth in colorectal cancer cells, implicating AKAP12 in colorectal cancer pathogenesis.

Methods

To evaluate the effect of this gene on the progression and metastasis of colorectal cancer, we examined the impact of overexpressing AKAP12 in the AKAP12-negative human colorectal cancer cell line LoVo, the single clone (LoVo-AKAP12) compared to mock-transfected cells (LoVo-CON).

Results

pCMV6-AKAP12-mediated AKAP12 re-expression induced apoptosis (3% to 12.7%, p<0.01), migration (89.6±7.5 cells to 31.0±4.1 cells, p<0.01) and invasion (82.7±5.2 cells to 24.7±3.3 cells, p<0.01) of LoVo cells in vitro compared to control cells. Nude mice injected with LoVo-AKAP12 cells had both significantly reduced tumor volume (p<0.01) and increased apoptosis compared to mice given AKAP12-CON. The quantitative human-specific Alu PCR analysis showed overexpression of AKAP12 suppressed the number of intravasated cells in vivo (p<0.01).

Conclusion

These results demonstrate that AKAP12 may play an important role in tumor growth suppression and the survival of human colorectal cancer.