Glutaminyl cyclase inhibitor contributes to the regulation of HSP70, HSP90, actin, and ribosome on gene and protein levels in vitro.

Because of the crucial roles of upregulated glutaminyl cyclase (QC) in the initiation and development of Alzheimer's disease (AD), QC inhibitors are supposed as disease-modifying agents for the treatment of AD. And reported compounds encourage this hypothesis greatly based on the remarkable anti-AD effects in vivo. To illustrate the mechanism in detail, the actions of a selected QC inhibitor (23) were assessed firstly in a cell system here. It was demonstrated that QC activities and the generation of pyroglutamate-modified ß-amyloids in PC12 cells were both inhibited obviously after the treatment of 23. A total of 13 and 15 genes were up- and downregulated significantly in treated cells by RNA-sequencing analysis. Quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, WB, and immunofluorescence analysis supported the effects of 23 on the transcriptome of PC12 cells consequently. The expressions of chaperones, heat shock proteins (HSP) 70, and 90, were upreglutated, while gene expression of actin and the level of encoded protein were reduced significantly in PC12 cells with the treatment. Furthermore, the regulations of ribosome were observed after the treatment. These results indicate the potency of 23 to improve the translation, expression and folding regulation of proteins and affect the multivalent cross-linking of cytoskeletal protein and other proteins subsequently in the cell system and might contribute to the understanding of the mechanism of QC inhibitor as potential anti-AD agents.

Temporal transcriptome profiling of developing seeds reveals a concerted gene regulation in relation to oil accumulation in Pongamia (Millettia pinnata).

BACKGROUND: Pongamia (Millettia pinnata syn. Pongamia pinnata), an oilseed legume species, is emerging as potential feedstock for sustainable biodiesel production. Breeding Pongamia for favorable traits in commercial application will rely on a comprehensive understanding of molecular mechanism regulating oil accumulation during its seed development. To date, only limited genomic or transcript sequences are available for Pongamia, while a temporal transcriptome profiling of developing seeds is still lacking in this species. RESULTS: In this work, we conducted a time-series analysis of morphological and physiological characters, oil contents and compositions, as well as global gene expression profiles in developing Pongamia seeds. Firstly, three major developmental phases were characterized based on the combined evidences from embryonic shape, seed weight, seed moisture content, and seed color. Then, the gene expression levels at these three phases were quantified by RNA-Seq analyses with three biological replicates from each phase. Nearly 94% of unigenes were expressed at all three phases, whereas only less than 2% of unigenes were exclusively expressed at one of these phases. A total of 8881 differentially expressed genes (DEGs) were identified between phases. Furthermore, the qRT-PCR analyses for 10 DEGs involved in lipid metabolism demonstrated a good reliability of our RNA-Seq data in temporal gene expression profiling. We observed a dramatic increase in seed oil content from the embryogenesis phase to the early seed-filling phase, followed by a steady and moderate increase towards the maximum at the desiccation phase. We proposed that a highly active expression of most genes related to fatty acid (FA) and triacylglycerol (TAG) biosynthesis at the embryogenesis phase might trigger both the substantial oil accumulation and the membrane lipid synthesis for rapid cell proliferation at this phase, while a concerted reactivation of TAG synthesis-related genes at the desiccation phase might further promote storage lipid synthesis to achieve the maximum content of seed oils. CONCLUSIONS: This study not only built a bridge between gene expression profiles and oil accumulation in developing seeds, but also laid a foundation for future attempts on genetic engineering of Pongamia varieties to acquire higher oil yield or improved oil properties for biofuel applications.

Intrinsically Disordered Proteins as Important Players during Desiccation Stress of Soybean Radicles.

Intrinsically disordered proteins (IDPs) play a variety of important physiological roles in all living organisms. However, there is no comprehensive analysis of the abundance of IDPs associated with environmental stress in plants. Here, we show that a set of heat-stable proteins (i.e., proteins that do not denature after boiling at 100 °C for 10 min) was present in R0mm and R15mm radicles (i.e., before radicle emergence and 15 mm long radicles) of soybean (Glycine max) seeds. This set of 795 iTRAQ-quantified heat-stable proteins contained a high proportion of wholly or highly disordered proteins (15%), which was significantly higher than that estimated for the whole soybean proteome containing 55,787 proteins (9%). The heat-stable proteome of soybean radicles that contain many IDPs could protect lactate dehydrogenase (LDH) during freeze-thaw cycles. Comparison of the 795 heat-stable proteins in the R0mm and R15mm soybean radicles revealed that many of these proteins changed abundance during seedling growth with 170 and 89 proteins being more abundant in R0mm and R15mm, respectively. KEGG analysis identified 18 proteins from the cysteine and methionine metabolism pathways and nine proteins from the phenylpropanoid biosynthesis pathway. As an important type of IDP related to stress, 30 late embryogenesis abundant proteins were also found. Ten selected proteins with high levels of predicted intrinsic disorder were able to efficiently protect LDH from the freeze-thaw-induced inactivation, but the protective ability was not correlated with the disorder content of these proteins. These observations suggest that protection of the enzymes and other proteins in a stressed cell can be one of the biological functions of plant IDPs.

A plant cell model of polyglutamine aggregation: Identification and characterisation of macromolecular and small-molecule anti-protein aggregation activity in vivo.

In vitro studies have shown that LEA proteins from plants and invertebrates protect and stabilise other proteins under conditions of water stress, suggesting a role in stress tolerance. However, there is little information on LEA protein function in whole plants or plant cells, particularly with respect to their anti-aggregation activity. To address this, we expressed in tobacco BY-2 suspension cells an aggregation-prone protein based on that responsible for Huntington's disease (HD). In HD, abnormally long stretches of polyglutamine (polyQ) in huntingtin (Htt) protein cause aggregation of Htt fragments within cells. We constructed stably transformed BY-2 cell lines expressing enhanced green fluorescent protein (EGFP)-HttQ23 or EGFP-HttQ52 fusion proteins (encoding 23 or 52 glutamine residues, pertaining to the normal and disease states, respectively), as well as an EGFP control. EGFP-HttQ52 protein aggregated in the cytoplasm of transformed tobacco cells, which showed slow growth kinetics; in contrast, EGFP-HttQ23 or EGFP did not form aggregates and cells expressing these constructs grew normally. To test the effect of LEA proteins on protein aggregation in plant cells, we constructed cell lines expressing both EGFP-HttQ52 and LEA proteins (PM1, PM18, ZLDE-2 or AavLEA1) or a sHSP (PM31). Of these, AavLEA1 and PM31 reduced intracellular EGFP-HttQ52 aggregation and alleviated the associated growth inhibition, while PM18 and ZLDE-2 partially restored growth rates. Treatment of EGFP-HttQ52-expressing BY2 cells with the polyphenol epigallocatechin-3-gallate (EGCG) also reduced EGFP-HttQ52 aggregation and improved cell growth rate. The EGFP-HttQ52 cell line therefore has potential for characterising both macromolecular and small molecule inhibitors of protein aggregation in plant cells.

Effects of secondary metabolite extract from Phomopsis occulta on ß-amyloid aggregation.

Inhibition of ß-amyloid (Aß) aggregation is an attractive therapeutic and preventive strategy for the discovery of disease-modifying agents in Alzheimer's disease (AD). Phomopsis occulta is a new, salt-tolerant fungus isolated from mangrove Pongamia pinnata (L.) Pierre. We report here the inhibitory effects of secondary metabolites from Ph. occulta on the aggregation of Aß42. It was found that mycelia extracts (MEs) from Ph. occulta cultured with 0, 2, and 3 M NaCl exhibited inhibitory activity in an E. coli model of Aß aggregation. A water-soluble fraction, ME0-W-F1, composed of mainly small peptides, was able to reduce aggregation of an Aß42-EGFP fusion protein and an early onset familial mutation Aß42E22G-mCherry fusion protein in transfected HEK293 cells. ME0-W-F1 also antagonized the cytotoxicity of Aß42 in the neural cell line SH-SY5Y in dose-dependent manner. Moreover, SDS-PAGE and FT-IR analysis confirmed an inhibitory effect of ME0-W-F1 on the aggregation of Aß42 in vitro. ME0-W-F1 blocked the conformational transition of Aß42 from α-helix/random coil to ß-sheet, and thereby inhibited formation of Aß42 tetramers and high molecular weight oligomers. ME0-W-F1 and other water-soluble secondary metabolites from Ph. occulta therefore represent new candidate natural products against aggregation of Aß42, and illustrate the potential of salt tolerant fungi from mangrove as resources for the treatment of AD and other diseases.

Expression of a vacuole-localized BURP-domain protein from soybean (SALI3-2) enhances tolerance to cadmium and copper stresses.

The plant-specific BURP family proteins play diverse roles in plant development and stress responses, but the function mechanism of these proteins is still poorly understood. Proteins in this family are characterized by a highly conserved BURP domain with four conserved Cys-His repeats and two other Cys, indicating that these proteins potentially interacts with metal ions. In this paper, an immobilized metal affinity chromatography (IMAC) assay showed that the soybean BURP protein SALI3-2 could bind soft transition metal ions (Cd(2+), Co(2+), Ni(2+), Zn(2+) and Cu(2+)) but not hard metal ions (Ca(2+) and Mg(2+)) in vitro. A subcellular localization analysis by confocal laser scanning microscopy revealed that the SALI3-2-GFP fusion protein was localized to the vacuoles. Physiological indexes assay showed that Sali3-2-transgenic Arabidopsis thaliana seedlings were more tolerant to Cu(2+) or Cd(2+) stresses than the wild type. An inductively coupled plasma optical emission spectrometry (ICP-OES) analysis illustrated that, compared to the wild type seedlings the Sali3-2-transgenic seedlings accumulated more cadmium or copper in the roots but less in the upper ground tissues when the seedlings were exposed to excessive CuCl2 or CdCl2 stress. Therefore, our findings suggest that the SALI3-2 protein may confer cadmium (Cd(2+)) and copper (Cu(2+)) tolerance to plants by helping plants to sequester Cd(2+) or Cu(2+) in the root and reduce the amount of heavy metals transported to the shoots.

Chaetoglobosin Y, a new cytochalasan from Chaetomium globosum.

Chaetoglobosin Y (1), was isolated from the endolichenic fungal strain Chaetomium globosum (No. 64-5-8-2), along with related six known cytochalasans, chaetoglobosin Fex (2), chaetoglobosin E (3), isochaetoglobosin D (4), chaetoglobosin G (5), cytoglobosin B (6), and cytoglobosin C (7). Their structures were determined by detailed spectroscopic analyses and comparison with those of the closely related compounds previously reported. The cytotoxicity to HCT-116 cell line of 2-7 was evaluated in vitro with doxorubicin as positive control.

Critique of the use of fluorescence-based reporters in Escherichia coli as a screening tool for the identification of peptide inhibitors of Aß42 aggregation.

High-throughput screens that dispense with the need for expensive synthetic Aß peptide would be invaluable for identifying novel anti-aggregants as potential treatments for Alzheimer's disease. A biosynthetic in vivo approach, using a recombinant fluorescent green fluorescent protein (GFP) reporter for the aggregation state of Aß in Escherichia coli, has been reported by other workers. Here, inducible Aß-GFP expression in E. coli was coupled to the concurrent constitutive production of a quasi-random peptide library to screen for anti-aggregant activity. To attempt to introduce greater robustness, mCherry was also co-expressed as an internal fluorescence standard to allow ratiometric comparison between samples. However, fluctuations in mCherry expression levels, as well as a low dynamic range of GFP output between positive and negative anti-aggregant peptides, highlighted limitations with the approach. Despite this, two novel peptides were identified that showed an equivalent in vitro anti-aggregant activity to that of epigallocatechin-3-gallate. Thus, although biosynthetic in vivo strategies show promise as screens for novel activities, unforeseen problems can arise because of the variability inherent in any biological system.

[Application of fluorescence spectroscopy in studying the protective function of 11-amino acid motif of group3 late embryogenesis abundant protein].

Late embryogenesis abundant protein (LEA) can enhance the tolerance of organisms under drought, low-temperature and other stress conditions. However, their protection mechanisms are still unclear. In the present paper, the peptides consisting of multi-copies of 11-amino acid motif, such as PM2D and PM2E are proved to protect the activity of the lactate dehydrogenase (LDH) from freeze-thaw by using ultroviolet spectrometry. Furthermore, the fluorescence spectroscopic results show that the peptide consisting of multi-copies of 11-amino acid can stabilize the structure of LDH through synergy and multi-sites binding. However, the peptides consisting of less copies of 11-amino acid, such as PM2F and PM2G bind to LDH through one site, and the binding is weak. They thus can not protect the activity of LDH. In addition, the peptides consisted of multi-copies of 11-amino acid protect LDH by acting with trehalose synergically, and the protection mechanisms of LEA and trehalose on LDH are different.

Polyproline II structure is critical for the enzyme protective function of soybean Em (LEA1) conserved domains.

Group 1 late embryogenesis-abundant (LEA1) proteins protect enzyme activity from dehydration and are structurally conserved with three different 20 amino acid motifs in the N-terminal, middle and C-terminal domains. Three soybean Em (LEA1) domain peptides (Em-N, Em-2M and Em-C) covering these respective motifs were constructed and had differential protective ability on lactate dehydrogenase against freeze-thaw: Em-C > Em-2M > Em-N. CD spectroscopy revealed that Em-2M and Em-C contained both polyproline II (PII) helical structure and α-helix, while Em-N had a high potential to form α-helix but did not contain PII structure. The PII helical structure between the third and fifth glycine in the middle motif was shown, through site mutation, to be critical for the enzyme protective function of soybean Em (LEA1) conserved domain under freezing stress.

Both plant and animal LEA proteins act as kinetic stabilisers of polyglutamine-dependent protein aggregation.

LEA (late embryogenesis abundant) proteins are intrinsically disordered proteins that contribute to stress tolerance in plants and invertebrates. Here we show that, when both plant and animal LEA proteins are co-expressed in mammalian cells with self-aggregating polyglutamine (polyQ) proteins, they reduce aggregation in a time-dependent fashion, showing more protection at early time points. A similar effect was also observed in vitro, where recombinant LEA proteins were able to slow the rate of polyQ aggregation, but not abolish it altogether. Thus, LEA proteins act as kinetic stabilisers of aggregating proteins, a novel function in protein homeostasis consistent with a proposed role as molecular shields.

Effect of treatment of rosacea in females by Chibixiao Recipe in combination with minocycline and spironolactone.

OBJECTIVE: To observe the clinical efficacy of Chibixiao Recipe (CBX) in combination with minocycline and spironolactone in treating rosacea in females. METHODS: Sixty-eight women with rosacea were randomly assigned to the treated group (48 cases) and the control group (20 cases), both of which were treated with minocycline and spironolactone taken orally, but to the treated group, the Chinese herbal recipe, CBX was given additionally. Besides, cryotherapy with liquid nitrogen was applied to those with apparent capillary dilatation. The therapeutic course for both groups was 8 weeks. The levels of serum testosterone before and after treatment were determined by radioimmunoassay (RIA), and a 4-month follow-up was conducted. RESULTS: In the treated group the cure-markedly effective rate was 87.5% and the recurrent rate was 6.5%, while in the control group, they were 45.0% and 41.2% respectively. Comparisons in the indexes between the two groups all showed significant difference (both P<0.01), with the cure-markedly effective rate higher, and the recurrent rate lower in the treated group. The serum level of testosterone got lowered in both groups ( P<0.05 and P<0.01), but the lowering in the treated group was more significant, showing significance when compared with that in the control group ( P<0.01). CONCLUSION: CBX in combination with Western medicine has effect in treating rosacea superior to that of Western medicine alone, and could effectively reduce recurrent rate and the serum level of testosterone. female rosacea, testosterone, Chibixiao Recipe, minocycline, spironolactone

PM2, a group 3 LEA protein from soybean, and its 22-mer repeating region confer salt tolerance in Escherichia coli.

To have knowledge of the effect of soybean PM2 protein in protecting dehydrated cells and its functional region, PM2 cDNA was isolated from soybean immature seeds. The recombinants expressing full-length PM2, truncated polypeptides of PM2A (aa 1-262) or PM2B (aa 129-262, 22-mer repeating region), or artificial polypeptide PM2C (duplication of 22-mer repeating region) were constructed. By using SDS-PAGE and mass spectrometry approaches, these fusion polypeptides were identified and proved to be hydrophilic and heat-stable. Spot assays of BL/PM2 and BL/pET28 (as control) showed that protein PM2 increased salt tolerance (500 mM NaCl or 500 mM KCl) of Escherichia coli, rather than osmotic tolerance (1100 mM sorbitol). In addition, comparing the survival ratios of the transformants under 500 mM NaCl or 500 mM KCl stresses, the results showed that: (1) the survival ratios of BL/PM2 and BL/PM2B were quite similar, both showing much higher values than those of BL/pET28. (2) The survival ratios of BL/PM2C were much higher than those of BL/PM2, BL/PM2A, and BL/PM2B. This provides the first experimental evidence that PM2 polypeptide enhances salt tolerance of E. coli cells, and the 22-mer repeat region is an important functional region.