A reference-grade genome of the xerophyte Ammopiptanthus mongolicus sheds light on its evolution history in legumes and drought-tolerance mechanisms.

Plants that grow in extreme environments represent unique sources of stress-resistance genes and mechanisms. Ammopiptanthus mongolicus (Leguminosae) is a xerophytic evergreen broadleaf shrub native to semi-arid and desert regions; however, its drought-tolerance mechanisms remain poorly understood. Here, we report the assembly of a reference-grade genome for A. mongolicus, describe its evolutionary history within the legume family, and examine its drought-tolerance mechanisms. The assembled genome is 843.07 Mb in length, with 98.7% of the sequences successfully anchored to the nine chromosomes of A. mongolicus. The genome is predicted to contain 47 611 protein-coding genes, and 70.71% of the genome is composed of repetitive sequences; these are dominated by transposable elements, particularly long-terminal-repeat retrotransposons. Evolutionary analyses revealed two whole-genome duplication (WGD) events at 130 and 58 million years ago (mya) that are shared by the genus Ammopiptanthus and other legumes, but no species-specific WGDs were found within this genus. Ancestral genome reconstruction revealed that the A. mongolicus genome has undergone fewer rearrangements than other genomes in the legume family, confirming its status as a "relict plant". Transcriptomic analyses demonstrated that genes involved in cuticular wax biosynthesis and transport are highly expressed, both under normal conditions and in response to polyethylene glycol-induced dehydration. Significant induction of genes related to ethylene biosynthesis and signaling was also observed in leaves under dehydration stress, suggesting that enhanced ethylene response and formation of thick waxy cuticles are two major mechanisms of drought tolerance in A. mongolicus. Ectopic expression of AmERF2, an ethylene response factor unique to A. mongolicus, can markedly increase the drought tolerance of transgenic Arabidopsis thaliana plants, demonstrating the potential for application of A. mongolicus genes in crop improvement.

Proteomic Analysis of Circulating Extracellular Vesicles Identifies Potential Biomarkers for Lymph Node Metastasis in Oral Tongue Squamous Cell Carcinoma.

Lymph node metastasis is the most reliable indicator of a poor prognosis for patients with oral tongue cancers. Currently, there are no biomarkers to predict whether a cancer will spread in the future if it has not already spread at the time of diagnosis. The aim of this study was to quantitatively profile the proteomes of extracellular vesicles (EVs) isolated from blood samples taken from patients with oral tongue squamous cell carcinoma with and without lymph node involvement and non-cancer controls. EVs were enriched using size exclusion chromatography (SEC) from pooled plasma samples of patients with non-nodal and nodal oral tongue squamous cell carcinoma (OTSCC) and non-cancer controls. Protein cargo was quantitatively profiled using isobaric labelling (iTRAQ) and two-dimensional high-performance liquid chromatography followed by tandem mass spectrometry. We identified 208 EV associated proteins and, after filtering, generated a short list of 136 proteins. Over 85% of the EV-associated proteins were associated with the GO cellular compartment term "extracellular exosome". Comparisons between non-cancer controls and oral tongue squamous cell carcinoma with and without lymph node involvement revealed 43 unique candidate EV-associated proteins with deregulated expression patterns. The shortlisted EV associated proteins described here may be useful discriminatory biomarkers for differentiating OTSCC with and without nodal disease or non-cancer controls.

High-Throughput Mass Spectrometric Analysis of the Whole Proteome and Secretome From Sinorhizobium fredii Strains CCBAU25509 and CCBAU45436.

Sinorhizobium fredii is a dominant rhizobium on alkaline-saline land that can induce nitrogen-fixing symbiotic root nodules in soybean. Two S. fredii strains, CCBAU25509 and CCBAU45436, were used in this study to facilitate in-depth analyses of this species and its interactions with soybean. We have previously completed the full assembly of the genomes and detailed transcriptomic analyses for these two S. fredii strains, CCBAU25509 and CCBAU45436, that exhibit differential compatibility toward some soybean hosts. In this work, we performed high-throughput Orbitrap analyses of the whole proteomes and secretomes of CCBAU25509 and CCBAU45436 at different growth stages. Our proteomic data cover coding sequences in the chromosome, chromid, symbiotic plasmid, and other accessory plasmids. In general, we found higher levels of protein expression by genes in the chromosomal genome, whereas proteins encoded by the symbiotic plasmid were differentially accumulated in bacteroids. We identified secreted proteins from the extracellular medium, including seven and eight Nodulation Outer Proteins (Nops) encoded by the symbiotic plasmid of CCBAU25509 and CCBAU45436, respectively. Differential host restriction of CCBAU25509 and CCBAU45436 is regulated by the allelic type of the soybean Rj2(Rfg1) protein. Using sequencing data from this work and available in public databases, our analysis confirmed that the soybean Rj2(Rfg1) protein has three major allelic types (Rj2/rfg1, rj2/Rfg1, rj2/rfg1) that determine the host restriction of some Bradyrhizobium diazoefficiens and S. fredii strains. A mutant defective in the type 3 protein secretion system (T3SS) in CCBAU25509 allowed this strain to nodulate otherwise-incompatible soybeans carrying the rj2/Rfg1 allelic type, probably by disrupting Nops secretion. The allelic forms of NopP and NopI in S. fredii might be associated with the restriction imposed by Rfg1. By swapping the NopP between CCBAU25509 and CCBAU45436, we found that only the strains carrying NopP from CCBAU45436 could nodulate soybeans carrying the rj2/Rfg1 allelic type. However, no direct interaction between either forms of NopP and Rfg1 could be observed.

Mechanisms of Soybean Roots' Tolerances to Salinity Revealed by Proteomic and Phosphoproteomic Comparisons Between Two Cultivars.

Understanding molecular mechanisms underlying plant salinity tolerance provides valuable knowledgebase for effective crop improvement through genetic engineering. Current proteomic technologies, which support reliable and high-throughput analyses, have been broadly used for exploring sophisticated molecular networks in plants. In the current study, we compared phosphoproteomic and proteomic changes in roots of different soybean seedlings of a salt-tolerant cultivar (Wenfeng07) and a salt-sensitive cultivar (Union85140) induced by salt stress. The root samples of Wenfeng07 and Union85140 at three-trifoliate stage were collected at 0 h, 0.5 h, 1 h, 4 h, 12 h, 24 h, and 48 h after been treated with 150 mm NaCl. LC-MS/MS based phosphoproteomic analysis of these samples identified a total of 2692 phosphoproteins and 5509 phosphorylation sites. Of these, 2344 phosphoproteins containing 3744 phosphorylation sites were quantitatively analyzed. Our results showed that 1163 phosphorylation sites were differentially phosphorylated in the two compared cultivars. Among them, 10 MYB/MYB transcription factor like proteins were identified with fluctuating phosphorylation modifications at different time points, indicating that their crucial roles in regulating flavonol accumulation might be mediated by phosphorylated modifications. In addition, the protein expression profiles of these two cultivars were compared using LC MS/MS based shotgun proteomic analysis, and expression pattern of all the 89 differentially expressed proteins were independently confirmed by qRT-PCR. Interestingly, the enzymes involved in chalcone metabolic pathway exhibited positive correlations with salt tolerance. We confirmed the functional relevance of chalcone synthase, chalcone isomerase, and cytochrome P450 monooxygenase genes using soybean composites and Arabidopsis thaliana mutants, and found that their salt tolerance were positively regulated by chalcone synthase, but was negatively regulated by chalcone isomerase and cytochrome P450 monooxygenase. A novel salt tolerance pathway involving chalcone metabolism, mostly mediated by phosphorylated MYB transcription factors, was proposed based on our findings. (The mass spectrometry raw data are available via ProteomeXchange with identifier PXD002856).

GmSAL1 hydrolyzes inositol-1,4,5-trisphosphate and regulates stomatal closure in detached leaves and ion compartmentalization in plant cells.

Inositol polyphosphatases are important regulators since they control the catabolism of phosphoinositol derivatives, which are often signaling molecules for cellular processes. Here we report on the characterization of one of their members in soybean, GmSAL1. In contrast to the substrate specificity of its Arabidopsis homologues (AtSAL1 and AtSAL2), GmSAL1 only hydrolyzes inositol-1,4,5-trisphosphate (IP3) but not inositol-1,3,4-trisphosphate or inositol-1,4-bisphosphate.The ectopic expression of GmSAL1 in transgenic Arabidopsis thaliana led to a reduction in IP3 signals, which was inferred from the reduction in the cytoplasmic signals of the in vivo biomarker pleckstrin homology domain-green florescent protein fusion protein and the suppression of abscisic acid-induced stomatal closure. At the cellular level, the ectopic expression of GmSAL1 in transgenic BY-2 cells enhanced vacuolar Na(+) compartmentalization and therefore could partially alleviate salinity stress.

GmPHD5 acts as an important regulator for crosstalk between histone H3K4 di-methylation and H3K14 acetylation in response to salinity stress in soybean.

BACKGROUND: Accumulated evidence suggest that specific patterns of histone posttranslational modifications (PTMs) and their crosstalks may determine transcriptional outcomes. However, the regulatory mechanisms of these "histone codes" in plants remain largely unknown. RESULTS: In this study, we demonstrate for the first time that a salinity stress inducible PHD (plant homeodomain) finger domain containing protein GmPHD5 can read the "histone code" underlying the methylated H3K4. GmPHD5 interacts with other DNA binding proteins, including GmGNAT1 (an acetyl transferase), GmElongin A (a transcription elongation factor) and GmISWI (a chromatin remodeling protein). Our results suggest that GmPHD5 can recognize specific histone methylated H3K4, with preference to di-methylated H3K4. Here, we illustrate that the interaction between GmPHD5 and GmGNAT1 is regulated by the self-acetylation of GmGNAT1, which can also acetylate histone H3. GmGNAT1 exhibits a preference toward acetylated histone H3K14. These results suggest a histone crosstalk between methylated H3K4 and acetylated H3K14. Consistent to its putative roles in gene regulation under salinity stress, we showed that GmPHD5 can bind to the promoters of some confirmed salinity inducible genes in soybean. CONCLUSION: Here, we propose a model suggesting that the nuclear protein GmPHD5 is capable of regulating the crosstalk between histone methylation and histone acetylation of different lysine residues. Nevertheless, GmPHD5 could also recruit chromatin remodeling factors and transcription factors of salt stress inducible genes to regulate their expression in response to salinity stress.

Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord and placenta: implication in the migration.

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC- and P-MSC possess immunophenotypic and functional characteristics similar to BM-MSC. However, under defined conditions, the migration capacity of BM- and P-MSC was found to be 5.9- and 3.2-folds higher than that of UC-MSC, respectively. By the use of 2-DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Interestingly, the expression levels of those proteins reflect perfectly the migration capacity of corresponding MSC, which is also proved by in vitro overexpression and silencing techniques. Our study indicates that a bunch of migration-related proteins are pivotal in governing the migration capacity of MSC.

Identification of histone methylation multiplicities patterns in the brain of senescence-accelerated prone mouse 8.

Histone post-translational modifications (PTMs) are involved in diverse biological processes and methylation was regarded as a long-term epigenetic mark. Though aging represented one of the major risk factors for neurodegenerative diseases, no systematic investigations had correlated the patterns of histone PTMs in the brain with aging and the roles of such concerted histone PTMs in brain aging are still unknown. In this study, enzyme digestion, nano-LC, MALDI-TOF/TOF MS analysis and Western blotting were combined to investigate the defined methylation of core histones (H2A, H2B, H3 and H4) in the brain of 12-month-old senescence accelerated mouse prone 8 (SAMP8). The expression of several modified histones in the brain of 3-, and 12-month-old SAMP8 mice as well as that of the age-matched control senescence accelerated-resistant mouse (SAMR1) was compared. In the brain of 12-month-old SAMP8 mice, seven methylation sites (H3K24, H3K27, H3K36, H3K79, H3R128, H4K20 and H2A R89) were detected and most PTMs sites were located on histone H3. Mono-methylated H4K20 decreased significantly in the brain of 12-month-old SAMP8 mice. Methylated H3K27 and H3K36 coexisted in the aged brain with different methylation multiplicities. Di-methylated H3K79 expressed in the neurons of cerebral cortex and hippocampus. This study showed histone methylation patterns in the aged SAMP8 mice brain and provided the experimental evidences for further research on histone PTMs in the aged brain. We hope these results could initiate a platform for the exchange of comprehensive information concerning aging or neurodegenerative disease and help us interpret the change of gene expression and DNA repair ability at epigenetic level.

Proteomic characterization of Phragmites communis in ecotypes of swamp and desert dune.

Phragmites communis Trin. (common reed) is a recognized model plant for studying its adaptation to contrasting and harsh environments. To understand the inherent molecular basis for its remarkable resistance to combined stresses, we performed a comprehensive proteomic analysis of the leaf proteins from two ecotypes, i.e. swamp and desert dune, naturally growing in the desert region of northwestern China. First, a proteome reference map of Phragmites was established based on the swamp ecotype. Proteins were resolved by 2-D/SDS-PAGE and identified by MALDI-TOF/TOF MS. In total, 177 spots were identified corresponding to 51 proteins. The major proteins identified are proteins involved in photosynthesis, glutathione and ascorbic acid metabolism as well as protein synthesis and quality control. Second, the 2-DE profiles of the two ecotypes were compared quantitatively via DIGE analysis. Compared with swamp ecotype, 51 proteins spots are higher-expressed and 58 protein spots are lower-expressed by twofold or more in desert dune ecotype. Major differences were found for the proteins involved in light reaction of photosynthesis, protein biosynthesis and quality control and antioxidative reactions. The physiological significance of such differences is discussed in the context of a flow of complex events in relation to plant adaptation to combined environmental stresses.

Mass spectrometry analysis of the variants of histone H3 and H4 of soybean and their post-translational modifications.

BACKGROUND: Histone modifications and histone variants are of importance in many biological processes. To understand the biological functions of the global dynamics of histone modifications and histone variants in higher plants, we elucidated the variants and post-translational modifications of histones in soybean, a legume plant with a much bigger genome than that of Arabidopsis thaliana. RESULTS: In soybean leaves, mono-, di- and tri-methylation at Lysine 4, Lysine 27 and Lysine 36, and acetylation at Lysine 14, 18 and 23 were detected in HISTONE H3. Lysine 27 was prone to being mono-methylated, while tri-methylation was predominant at Lysine 36. We also observed that Lysine 27 methylation and Lysine 36 methylation usually excluded each other in HISTONE H3. Although methylation at HISTONE H3 Lysine 79 was not reported in A. thaliana, mono- and di-methylated HISTONE H3 Lysine 79 were detected in soybean. Besides, acetylation at Lysine 8 and 12 of HISTONE H4 in soybean were identified. Using a combination of mass spectrometry and nano-liquid chromatography, two variants of HISTONE H3 were detected and their modifications were determined. They were different at positions of A31F41S87S90 (HISTONE variant H3.1) and T31Y41H87L90 (HISTONE variant H3.2), respectively. The methylation patterns in these two HISTONE H3 variants also exhibited differences. Lysine 4 and Lysine 36 methylation were only detected in HISTONE H3.2, suggesting that HISTONE variant H3.2 might be associated with actively transcribing genes. In addition, two variants of histone H4 (H4.1 and H4.2) were also detected, which were missing in other organisms. In the histone variant H4.1 and H4.2, the amino acid 60 was isoleucine and valine, respectively. CONCLUSION: This work revealed several distinct variants of soybean histone and their modifications that were different from A. thaliana, thus providing important biological information toward further understanding of the histone modifications and their functional significance in higher plants.

Glucose-regulated protein 78 is an intracellular antiviral factor against hepatitis B virus.

Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to analyze the host response to HBV using an inducible HBV-producing cell line, HepAD38. Twenty-three proteins were identified as differentially expressed with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real time RT-PCR and Western blotting as well as in HBV-infected human liver biopsies by immunohistochemistry. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium. Conversely overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-beta1 (IFN-beta1). In this connection, the IFN-beta1-mediated 2',5'-oligoadenylate synthetase and RNase L signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p = 0.019) as compared with their counterpart pretreatment liver biopsies. In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via the IFN-beta1-2',5'-oligoadenylate synthetase-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection.

Computational identification of protein methylation sites through bi-profile Bayes feature extraction.

Protein methylation is one type of reversible post-translational modifications (PTMs), which plays vital roles in many cellular processes such as transcription activity, DNA repair. Experimental identification of methylation sites on proteins without prior knowledge is costly and time-consuming. In silico prediction of methylation sites might not only provide researches with information on the candidate sites for further determination, but also facilitate to perform downstream characterizations and site-specific investigations. In the present study, a novel approach based on Bi-profile Bayes feature extraction combined with support vector machines (SVMs) was employed to develop the model for Prediction of Protein Methylation Sites (BPB-PPMS) from primary sequence. Methylation can occur at many residues including arginine, lysine, histidine, glutamine, and proline. For the present, BPB-PPMS is only designed to predict the methylation status for lysine and arginine residues on polypeptides due to the absence of enough experimentally verified data to build and train prediction models for other residues. The performance of BPB-PPMS is measured with a sensitivity of 74.71%, a specificity of 94.32% and an accuracy of 87.98% for arginine as well as a sensitivity of 70.05%, a specificity of 77.08% and an accuracy of 75.51% for lysine in 5-fold cross validation experiments. Results obtained from cross-validation experiments and test on independent data sets suggest that BPB-PPMS presented here might facilitate the identification and annotation of protein methylation. Besides, BPB-PPMS can be extended to build predictors for other types of PTM sites with ease. For public access, BPB-PPMS is available at http://www.bioinfo.bio.cuhk.edu.hk/bpbppms.

Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord, and placenta: implication in the migration.

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC- and P-MSC possess immunophenotypic and functional characteristics similar to BM-MSC. However, their migration capacity, which is indispensable during tissue regeneration process, is unclear. Under defined conditions, the migration capacity of BM- and P-MSC was found 5.9- and 3.2-folds higher than that of UC-MSC, respectively. By the use of 2-DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Consistent with their migration capacity, the levels of migration enhancing proteins including cathepsin B, cathepsin D and prohibitin,were significantly lower in UC-MSC when compared with those in BM- and P-MSC. For the migration inhibiting proteins such as plasminogen activator inhibitor-1 (PAI-1) and manganese superoxide dismutase, higher expression was found in the UC-MSC. We also showed that the overexpression of the PAI-1 impaired the migration capacity of BM- and P-MSC while silencing of PAI-1 enhanced the migration capacity of UC-MSC. Our study indicates that PAI-1 and other migration-related proteins are pivotal in governing the migration capacity of MSC.

Fusion core structure of the severe acute respiratory syndrome coronavirus (SARS-CoV): in search of potent SARS-CoV entry inhibitors.

Severe acute respiratory coronavirus (SARS-CoV) spike (S) glycoprotein fusion core consists of a six-helix bundle with the three C-terminal heptad repeat (HR2) helices packed against a central coiled-coil of the other three N-terminal heptad repeat (HR1) helices. Each of the three peripheral HR2 helices shows prominent contacts with the hydrophobic surface of the central HR1 coiled-coil. The concerted protein-protein interactions among the HR helices are responsible for the fusion event that leads to the release of the SARS-CoV nucleocapsid into the target host-cell. In this investigation, we applied recombinant protein and synthetic peptide-based biophysical assays to characterize the biological activities of the HR helices. In a parallel experiment, we employed a HIV-luc/SARS pseudotyped virus entry inhibition assay to screen for potent inhibitory activities on HR peptides derived from the SARS-CoV S protein HR regions and a series of other small-molecule drugs. Three HR peptides and five small-molecule drugs were identified as potential inhibitors. ADS-J1, which has been used to interfere with the fusogenesis of HIV-1 onto CD4+ cells, demonstrated the highest HIV-luc/SARS pseudotyped virus-entry inhibition activity among the other small-molecule drugs. Molecular modeling analysis suggested that ADS-J1 may bind to the deep pocket of the hydrophobic groove on the surface of the central coiled-coil of SARS-CoV S HR protein and prevent the entrance of the SARS-CoV into the host cells.

Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells.

Genes encoding ion transporters that regulate ion homeostasis in soybean have not been carefully investigated. Using degenerate primers, we cloned a putative chloride channel gene (GmCLC1) and a putative Na+/H+ antiporter gene (GmNHX1) from soybean. Confocal microscopic studies using yellow fluorescent fusion proteins revealed that GmCLC1 and GmNHX1 were both localized on tonoplast. The expressions of GmCLC1 and GmNHX1 were both induced by NaCl or dehydration stress imposed by polyethylene glycol (PEG). Using mitochondrial integrity and cell death as the damage indicators, a clear alleviation under NaCl stress (but not PEG stress) was observed in both GmCLC1 and GmNHX1 transgenic cells. Using fluorescent dye staining and quenching, respectively, a higher concentration of chloride ion (Cl-) or sodium ion (Na+) was observed in isolated vacuoles in the cells of GmCLC1 and of GmNHX1 transgenic lines. Our result suggested that these vacuolar-located ion transporters function to sequester ions from cytoplasm into vacuole to reduce its toxic effects.

Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CL(pro)) mediates extensive proteolytic processing of replicase polyproteins, and is considered a promising target for anti-SARS drug development. Here we present a rapid and high-throughput screening method to study the substrate specificity of SARS-CoV 3CL(pro). Six target amino acid positions flanking the SARS-CoV 3CL(pro) cleavage site were investigated. Each batch of mixed peptide substrates with defined amino acid substitutions at the target amino acid position was synthesized via the "cartridge replacement" approach and was subjected to enzymatic cleavage by recombinant SARS-CoV 3CL(pro). Susceptibility of each peptide substrate to SARS-CoV 3CL(pro) cleavage was monitored simultaneously by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The hydrophobic pocket in the P2 position at the protease cleavage site is crucial to SARS-CoV 3CL(pro)-specific binding, which is limited to substitution by hydrophobic residue. The binding interface of SARS-CoV 3CL(pro) that is facing the P1' position is suggested to be occupied by acidic amino acids, thus the P1' position is intolerant to acidic residue substitution, owing to electrostatic repulsion. Steric hindrance caused by some bulky or beta-branching amino acids in P3 and P2' positions may also hinder the binding of SARS-CoV 3CL(pro). This study generates a comprehensive overview of SARS-CoV 3CL(pro) substrate specificity, which serves as the design basis of synthetic peptide-based SARS-CoV 3CL(pro) inhibitors. Our experimental approach is believed to be widely applicable for investigating the substrate specificity of other proteases in a rapid and high-throughput manner that is compatible for future automated analysis.

Involvement of protein kinase C and E2F-5 in euxanthone-induced neurite differentiation of neuroblastoma.

Euxanthone, a neuritogenic agent isolated from the medicinal herb Polygala caudata, has been shown to induce morphological differentiation and neurite outgrowth in murine neuroblastoma Neuro 2a cells (BU-1 subclone). In order to elucidate the underlying mechanisms of euxanthone-induced neurite outgrowth, a proteomic approach was employed. In the present study, two dimensional (2-D) gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry were performed to investigate the alterations in protein expression profile of euxanthone-treated BU-1 cells. Fourteen identified proteins were changed in expression levels after induction of neurite growth. These proteins included participants in transcription and cell cycle regulation, calcium influx and calcium signaling, fatty acid metabolism, cytoskeleton reorganization, casein kinase signal transduction, putative transbilayer amphipath transport and protein biosynthesis. Among the 14 identified proteins, E2F transcription factor 5 (E2F-5) was significantly up-regulated after euxanthone treatment. Go6976, a protein kinase C (PKC) alpha/betaI inhibitor, was found to inhibit neuritogenesis and expression of E2F-5 in the euxanthone-treated BU-1 cells, while SH-6, the Akt/PKB inhibitor, had no inhibitory effect. The gene silencing of E2F-5 by small interfering RNA (siRNA) was found to abolish the euxanthone-induced neurite outgrowth. In conclusion, these results indicated that the transcription factor E2F-5 was actively involved in the regulation of euxanthone-induced neurite outgrowth via PKC pathway.

SET8 recognizes the sequence RHRK20VLRDN within the N terminus of histone H4 and mono-methylates lysine 20.

Methylation of lysine 20 in histone H4 has been proven to play important roles in chromatin structure and gene regulation. SET8 is one of the methyltransferases identified to be specific for this modification. In this study, the minimal active SET domain of SET8 has been mapped to the region of amino acids 195-352. This region completely retains the same methylation activity and substrate specificity as the full-length SET8. The SET domain recognizes a stretch of specific amino acid sequence around lysine 20 of H4 for its methylation activity. Methylation assays with N terminus mutants of H4 that contain deletions and single alanine or glutamine substitutions of charged residues revealed that SET8 requires the sequence RHRK20VLRDN for methylation at lysine 20. The individual mutation of any charged residue in this sequence to alanine or glutamine abolished or greatly decreased levels of methylation of lysine 20 of H4 by SET8. Interestingly, mutation of lysine 16 to alanine, arginine, glutamine, or methionine did not affect methylation of lysine 20 by the SET domain. Mass spectrometric analysis of synthesized H4 N-terminal peptides modified by SET8 showed that SET8 selectively mono-methylates lysine 20 of H4. Taken together, our results suggested that the coordination between the amino acid sequence RHRK20VLRDN and the SET domain of SET8 determines the substrate specificity and multiplicity of methylation of lysine 20 of H4.

Synthetic peptide studies on the severe acute respiratory syndrome (SARS) coronavirus spike glycoprotein: perspective for SARS vaccine development.

BACKGROUND: The S (spike) protein of the etiologic coronavirus (CoV) agent of severe acute respiratory syndrome (SARS) plays a central role in mediating viral infection via receptor binding and membrane fusion between the virion and the host cell. We focused on using synthetic peptides for developing antibodies against SARS-CoV, which aimed to block viral invasion by eliciting an immune response specific to the native SARS-CoV S protein. METHODS: Six peptide sequences corresponding to the surface regions of SARS-CoV S protein were designed and investigated by use of combined bioinformatics and structural analysis. These synthetic peptides were used to immunize both rabbits and monkeys. Antisera collected 1 week after the second immunization were analyzed by ELISA and tested for antibody specificity against SARS-CoV by immunofluorescent confocal microscopy. RESULTS: Four of our six synthetic peptides (S2, S3, S5, and S6) elicited SARS-CoV-specific antibodies, of which S5 (residues 788-820) and S6 (residues 1002-1030) exhibited immunogenic responses similar to those found in a parallel investigation using truncated recombinant protein analogs of the SARS-CoV S protein. This suggested that our S5 and S6 peptides may represent two minimum biologically active sequences of the immunogenic regions of the SARS-CoV S protein. CONCLUSIONS: Synthetic peptides can elicit specific antibodies to SARS-CoV. The study provides insights for the future development of SARS vaccine via the synthetic-peptide-based approach.