Phytol derived from chlorophyll hydrolysis in plants is metabolized via phytenal.

Phytol is the isoprenoid alcohol bound in ester linkage to chlorophyll, the most abundant photosynthetic pigment in plants. During leaf senescence, large amounts of phytol are released by chlorophyll degradation. However, the pathway of phytol catabolism in plants is unknown. We hypothesized that phytol degradation in plants might involve its oxidation into the long-chain aldehyde phytenal. Using GC-MS for aldehyde quantification after derivatization with methylhydroxylamine, phytenal was identified in leaves, whereas other long-chain aldehydes (phytanal and pristanal) were barely detectable. We found that phytenal accumulates during chlorotic stresses, for example, salt stress, dark-induced senescence, and nitrogen deprivation. The increase in the phytenal content is mediated at least in part independently of enzyme activities, and it is independent of light. Characterization of phytenal accumulation in the pao1 mutant affected in chlorophyll degradation revealed that phytenal is an authentic phytol metabolite derived from chlorophyll breakdown. The increase in phytenal was even stronger in mutants affected in the production of other phytol metabolites including vte5-2 (tocopherol deficient) and pes1 pes2 (fatty acid phytyl ester deficient). Therefore, phytenal accumulation is controlled by competing, alternative pathways of phosphorylation (leading to tocopherol production) or esterification (fatty acid phytyl ester production). As a consequence, the content of phytenal is maintained at low levels, presumably to minimize its toxic effects caused by its highly reactive aldehyde group that can form covalent bonds with and inactivate the amino groups of proteins.

In silico structure prediction and inhibition mechanism studies of AtHDA14 as revealed by homology modeling, docking, molecular dynamics simulation.

Histone deacetylases (HDACs) play a significant role in the epigenetic mechanism by catalyzing deacetylation of lysine on histone in both animals and plants. HDACs involved in growth, development and response to stresses in plants. Arabidopsis thaliana histone deacetylase 14 (AtHDA14) is found to localize in the mitochondria and chloroplasts, and it involved in photosynthesis and melatonin biosynthesis. However, its mechanism of action was still unknowns so far. Therefore, in this study, we constructed AtHDA14 protein model using homology modeling method, validated using PROCHECK and presented using Ramachandran plots. We also performed virtual screening of AtHDA14 by docking with small molecule drugs and predicted their ADMET properties to select representative inhibitors. MD simulation for representative AtHDA14-ligand complexes was carried out to further research and reveal their stability and inhibition mechanism. Meanwhile, MM/PBSA method was utilized to obtain more valuable information about the residues energy contribution. Moreover, compared with four candidate inhibitors, we also found that compound 645533 and 6918837 might be a more potent AtHDA14 inhibitor than TSA (444732) and SAHA (5311). Therefore, compound 6445533 and 6918837 was anticipated to be a promising drug candidate for inhibition of AtHDA14.

Heat-induced unfolding of apo-CP43 studied by fluorescence spectroscopy and CD spectroscopy.

CP43 is a chlorophyll-binding protein, which acts as a conduit for the excitation energy transfer. The thermal stability of apo-CP43 was studied by intrinsic fluorescence, exogenous ANS fluorescence, and circular dichroism spectroscopy. Under heat treatment, the structure of apo-CP43 changed and existed transition state occurred between 56 and 62 °C by the intrinsic, exogenous ANS fluorescence and the analysis of hydrophobicity. Besides, the isosbestic point of the sigmoidal curve was 58.10 ± 1.02 °C by calculating α-helix transition and the Tm was 56.45 ± 0.52 and 55.59 ± 0.68 °C by calculating the unfolded fraction of tryptophan and tyrosine fluorescence, respectively. During the process of unfolding, the hydrophobic structure of C-terminal segment firstly started to expose at 40 °C, and then the hydrophobic cluster adjacent to the N-terminal segment also gradually exposed to hydrophilic environment with increasing temperature. Our results indicated that heat treatment, especially above 40 °C, has an important impact on the structural stability of apo-CP43.

Genomic distribution and possible functional roles of putative G-quadruplex motifs in two subspecies of Oryza sativa.

G-quadruplex is a stable, four-stranded DNA or RNA structure formed from guanine-rich regions and implicated in telomere maintenance, replication, gene regulation at transcription level or translation level, etc. Based on bioinformatics methods, we analyzed different putative G-quadruplex motifs (PGQMs) patterns in various genomic regions of two subspecies (indica and japonica) of Oryza sativa and the whole genomes of other 8 species. In total, in the 10 species we discussed, the PGQMs densities in monocots were higher than dicots. 40,483 and 31,795 PGQMs were identified with a density of 108.46 and 84.89 PGQMs/Mb, respectively, in japonica and indica genomes, 10,655 and 5420 loci were found to contain at least one PGQM in their gene bodies (with a percentage of 19% and 14%) indicating a wide distribution of G-quadruplex motifs in O. sativa genome. They preferred to locate in transcription start sites proximal regions and 5'-UTR with relative high enrichment. This phenomenon supports the hypothesis that PGQMs are involved in gene transcription and translation. In addition, we analyzed the distribution of different loop length in G-quadruplex and found the density of long loop PGQMs was less than short loop in indica's intron but it was similar in japonica. Meanwhile, we focused on the loci with PGQMs and conducted gene ontology (GO) analysis of them. As a result, many GO terms were identified and significantly correlated with the loci containing at least one PGQM. The GO analysis in the two subspecies of rice may be helpful for elucidating the functional roles of G-quadruplexes.

The structural and functional role of the three tryptophan residues in Pin1.

Pin1, the only known isomerase catalyzing phosphorylated pSer/pThr-Pro motifs in proteins, plays unique roles in human diseases notably cancers and Alzheimer's disease. Herein, site-directed mutagenesis was employed to construct the tryptophan mutants of Pin1, including W11L, W34L, and W73L. Spectral methodologies, activity measurement, and proteinase resistance analysis were used to investigate the structural and functional role of the tryptophan residues in Pin1. In general, W11 and W34 are essential to the structure and the function of Pin1, because their mutations influence the structure of WW domain of Pin1, potentially attenuate the binding affinity of Pin1 to substrates, and thus inhibit the enzymatic activity of Pin1. Particularly, W11 mutation results in significantly varied structural features of Pin1 as revealed by fluorescence and circular dichroism (CD) spectroscopies, and decreases the enzymatic activity, thermal stability, and proteinase resistance of Pin1, all of which give an explanation for the high conservation of W11 in vivo. The synchronous fluorescence spectra indicate that W11 and W34 mutations possibly block their energy transfer to Y23 or Y24, suggesting the structural rearrangement in WW domain. By contrast, W73 is of minor importance for the structure and the function of Pin1, because the parameters of W73L observed in several experiments are very similar to wild-type Pin1. These observations are very beneficial for further understanding the structural and functional characteristics of Pin1 and for unveiling the pathogenesis of Pin1-related diseases especially those caused by tryptophan mutations.

Role of pH in structural changes for Pin1 protein: an insight from molecular dynamics study.

Pin1 protein is closely associated with the pathogenesis of cancers and Alzheimer's disease (AD). Previously, we have shown the acid-induced denaturation of Pin1 was determined by means of fluorescence emission, synchronous fluorescence etc., indicating an intermediate state around chromophores in Pin1 at about 4.0. Molecular dynamics simulations for the wild type Pin1 and its mutants were performed to explore the role of pH in the conformation changes of Pin1 protein. Our present study shows that one protein domain (PPIase domain) is more sensitive than the other one (WW domain) in Pin1 protein, and also our study shows that the integrality of the two conserve tryptophan in one domain (WW) is important in response to low pH. We arrive at the last result with the analysis of the protein root mean square distance and the analysis of the radius of gyration. The analysis of protein solvent accessible surface area values have proven our previous experiment result that there is an intermediate state around tryptophan residues at about pH 4.0. Moreover, acidic states of the protein can break the alpha-helixes in Pin1, especially the alpha-helix α3 close to active sites; as a result, Pin1 loses most of its activity at low pH. The results help us to understand the role of pH in Pin1, provide us insights into the conformation change at the atomic-level and emphasize the important role of decreased pH in the pathogenesis of some Pin1-related diseases, and support the therapeutic approach for the related Pin1 diseases by targeting acidosis and modifying the intracellular pH gradients.

Soluble expression of Spinach psbC gene in Escherichia coli and in vitro reconstitution of CP43 coupled with chlorophyll a only.

CP43 is a chlorophyll a (Chl a) and ß-carotene (ß-Car) binding protein encoded by psbC gene. In this study, psbC gene isolated from Spinach was expressed in Escherichia coli in soluble state. After lysis of the cells, the apoproteins purified by nickel affinity chromatography were examined by SDS-PAGE and Western-blot. Next, reconstitution experiment was carried out in vitro and the formation of stable pigment-protein complex was analyzed by partially denaturing electrophoresis. After purifying reconstituted CP43 (rCP43) from free pigments (FPs) by sucrose gradient ultracentrifugation and subsequently ion exchange chromatography (IEC), the eluate was analyzed by partially denaturing electrophoresis to confirm stability of the reconstructed complex. Finally, analyses of spectroscopic character of the eluate revealed that in vitro reconstitution was achieved and FPs were completely removed from the pigment-protein complex. Comparison between the absorption spectra of the rCP43 and native CP43 (nCP43) showed the lack of peaks between 450 and 500 nm, illustrating that the ß-Car was stripped off rCP43. In brief, it is feasible to obtain a reconstituted protein binding Chl a only, indicating that the occupancy of the ß-Car site has small impact on the stabilization of CP43. However, ß-Car shows strong interaction with Chl a, inducing the hyperchromic effect in blue region of spectrum and the blue shift of the 438.5 nm and 673.5 nm absorption band to 437 nm and 671 nm respectively. To some extent, our research is suggestive that ß-Car, coupled loosely with CP43, contributes to the precise orientation of Chl a in vivo.

Could Pin1 help us conquer essential hypertension at an earlier stage? A promising early-diagnostic biomarker and its therapeutic implications for the disease.

Essential hypertension is a major risk factor for cardiovascular morbidity and mortality, and the early-diagnosis is very important for the prevention of essential hypertension. Previously, we found that Pin1, the only known enzyme isomerizing pSer/pThr-Pro motifs in proteins, may gradually become inactive under conditions of stress such as intracellular acidification and fever. Interestingly, essential hypertension and the dysfunction of Pin1 often synchronously occur with the increasing age. Recent evidence indicates that Pin1 primarily increases the activity of endothelial nitric oxide synthase (eNOS) and the production of nitric oxide (NO) in multiple ways, significantly promoting the relaxation response of blood vessels and preventing the elevation of blood pressure. Further, the inhibition of Pin1 results in significantly increased blood pressure in rats. So, we hypothesized and evaluated the potential of Pin1 to be a new early-diagnostic biomarker as well as a therapeutic drug for essential hypertension. The unique activity of Pin1 and some epidemiological and experimental data evidence that the decreased activity of Pin1 may be closely associated with the development of essential hypertension. The factors that may impact the activity of Pin1 and correlate with the risk of essential hypertension were also discussed. These findings indicate that Pin1 plays a key and permanent role in efficiently preventing the development of essential hypertension, and that Pin1 may be a promising early-diagnostic biomarker as well as an effective therapeutic drug for the early-diagnosis, prevention, and treatment of essential hypertension, potentially decreasing the risk of cardiovascular morbidity and mortality.

Insight into the structural stability of wild type and mutants of the tobacco etch virus protease with molecular dynamics simulations.

The efficiency and high specificity of tobacco etch virus protease (TEVp) has made it widely used for cleavage of recombinant fusion proteins. However, TEVp suffers from a few intrinsic defects such as self-cleavage, poorly expressed in E. coli and less soluble. So some mutants were designed to improve it, such as S219V, T17S/N68D/I77V and L56V/S135G etc. MD simulations for the WT TEVp and its mutants were performed to explore the underlying dynamic effects of mutations on TEVp. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of TEVp, including the elongation of ß-sheet, conversion of loop to helix and the flexibility of active core. Our present study indicates that the three mutants for TEVp can change their secondary structure and tend to form more helixes and sheets to improve stability. The study also helps us to understand the effects of some mutations on TEVp, provides us insights into the change of them at the atomic level and gives a potential rational method to design an improved protein.

Aluminum(III) interferes with the structure and the activity of the peptidyl-prolyl cis-trans isomerase (Pin1): a new mechanism contributing to the pathogenesis of Alzheimer's disease and cancers?

The enzyme peptidyl-prolyl cis-trans isomerase (Pin1) may play an important role in preventing the development of Alzheimer's disease (AD). The structural and functional stability of Pin1 is extremely important. Previously, we have determined the stability of Pin1 under stressed conditions, such as thermal treatment and acidic-pH. Considering that aluminum (Al(III)) is well known for its potential neurotoxicity in the pathogenesis of AD, we examined whether Al(III) affects the structure and function of Pin1, by means of a PPIase activity assay, intrinsic fluorescence, circular dichroism (CD) spectroscopy, FTIR, and differential scanning calorimetry (DSC). The intrinsic tryptophan fluorescence measurements mainly show that Al(III) may bind to the clusters nearby W11 and W34 in the WW domain of Pin1, quenching the intrinsic fluorescence of the two tryptophan residues, which possibly results in the decreased binding affinity of Pin1 to substrates. The secondary structural analysis as revealed by FTIR and CD measurements indicate that Al(III) induces the increase in ß-sheet and the decrease in α-helix in Pin1. Furthermore, the changes of the thermodynamic parameters for Pin1 as monitored by DSC confirm that the thermal stability of Pin1 significantly increases in the presence of Al(III). The Al(III)-induced structural changes of Pin1 result in a sharp decrease of the PPIase activity of Pin1. To some extent, our research is suggestive that Al(III) may inhibit the isomerization activity of Pin1 in vivo, which may contribute to the pathogenesis of AD.

The acidic pH-induced structural changes in Pin1 as revealed by spectral methodologies.

Pin1 is closely associated with the pathogenesis of cancers and Alzheimer's disease (AD). Previously, we have shown the characteristics of the thermal denaturation of Pin1. Herein, the acid-induced denaturation of Pin1 was determined by means of fluorescence emission, synchronous fluorescence, far-UV CD, ANS fluorescence and RLS spectroscopies. The fluorescence emission spectra and the synchronous fluorescence spectra suggested the partially reversible unfolding (approximately from pH 7.0 to 4.0) and refolding (approximately from pH 4.0 to 1.0) of the structures around the chromophores in Pin1, apparently with an intermediate state at about pH 4.0-4.5. The far-UV CD spectra indicated that acidic pH (below pH 4.0) induced the structural transition from α-helix and random coils to ß-sheet in Pin1. The ANS fluorescence and the RLS spectra further suggested the exposure of the hydrophobic side-chains of Pin1 and the aggregation of it especially below pH 2.3, and the aggregation possibly resulted in the formation of extra intermolecular ß-sheet. The present work primarily shows that acidic pH can induce kinds of irreversible structural changes in Pin1, such as the exposure of the hydrophobic side-chains, the transition from α-helix to ß-sheet and the aggregation of Pin1, and also explains why Pin1 loses most of its activity below pH 5.0. The results emphasize the important role of decreased pH in the pathogenesis of some Pin1-related diseases, and support the therapeutic approach for them by targeting acidosis and modifying the intracellular pH gradients.

Apoptosis induced by genipin in human leukemia K562 cells: involvement of c-Jun N-terminal kinase in G2/M arrest.

AIM: To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms. METHODS: The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using DNA ladder, propidium iodide (PI)-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of genipin on cell cycle distribution was determined using PI staining. Caspase 3 activity was analyzed to detect apoptosis at different time points. Protein levels of phospho-c-Jun, phosphor-c-Jun N-terminal kinase (p-JNK), phosphor-p38, Fas-L, p63, and Bax and the release of cytochrome c were detected using Western blot analysis. RESULTS: Genipin reduced the viability of K562 cells with an IC(50) value of approximately 250 µmol/L. Genipin 200-400 µmol/L induced formation of typical apoptotic bodies and DNA fragmentation. Additionally, genipin 400 µmol/L significantly increased the caspase 3 activity from 8-24 h and arrested the cells in the G2/M phase. After stimulation with genipin 500 µmol/L, the levels of p-JNK, p-c-Jun, Fas-L, Bax, and cytochrome c were remarkably upregulated, but there were no obvious changes of p-p38. Genipin 200-500 µmol/L significantly upregulated the Fas-L expression and downregulated p63 expression. Dicoumarol 100 µmol/L, a JNK1/2 inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 µmol/L. CONCLUSION: These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand.

Spectroscopic studies on the irreversible heat-induced structural transition of Pin1.

Previously, the mechanism of the thermal unfolding of Pin1 (on-line measurements) was studied, revealing that Pin1 has a relatively high thermal stability. However, it is still questionable whether the unfolding of Pin1 is reversible. In the present work, intrinsic tryptophan fluorescence, ANS fluorescence, RLS, FTIR and CD spectroscopies are used to evaluate the reversibility of the thermal unfolding of Pin1. Intrinsic tryptophan fluorescence studies indicate that structural changes around tryptophan motifs in Pin1 are possibly reversible after heat treatment (even above 98°C), for no significant change in the intensity or λ(max) of the spectra was observed. ANS fluorescence measurements indicate the irreversible exposure of the hydrophobic clusters in Pin1 after heat treatment at 98°C, with increase in the fluorescence intensity and blue shift in λmax. Also, RLS signals of the Pin1-ANS system increased after heat treatment, possibly implying both the unfolding and the aggregation of Pin1. In addition, FTIR and CD results confirmed the irreversible unfolding of the secondary structure in Pin1 after heat treatment above 90°C, showing decreases in both α-helix and ß-sheet. In summary, the present work mainly suggests that heat treatment, especially above 90°C, has an important impact on the structural stability of Pin1, and the structural unfolding induced by heat was proved to be irreversible.

Kunitz-type trypsin inhibitor with high stability from Spinacia oleracea L. seeds.

The trypsin inhibitor SOTI was isolated from Spinacia oleracea L. seeds through ammonium sulfate precipitation, Sepharose 4B-trypsin affinity chromatography, and Sephadex G-75 chromatography. This typical Kunitz inhibitor showed remarkable stability to heat, pH, and denaturant. It retained 80% of its activity against trypsin after boiling for 20 min, and more than 90% activity when treated with 6 M guanidine hydrochloride. The formation of stable SOTI-trypsin complex (K(i) = 2.3x10(-6) M) is consistent with significant inhibitory activity of SOTI against trypsin-like proteinases present in the larval midgut of Pieris rapae. Sequences of SOTI fragments showed homology with other inhibitors.

A trypsin inhibitor from Cassia obtusifolia seeds: isolation, characterization and activity against Pieris rapae.

A trypsin inhibitor was isolated from Cassia obtusifolia by ammonium sulfate precipitation, Sepharose 4B-trypsin affinity and Sephadex G-75 chromatography. The inhibitor consisted of a single polypeptide chain with a molecular mass of 19, 812.55 Da. It was stable from pH 2 to 12 for 24 h, whereas it was unstable either above 70 degrees C for 10 min or under reduced conditions. The inhibitor, which inhibited trypsin activity with an apparent Ki of 0.3 microM, had one reactive site involving a lysine residue. The native inhibitor was resistant to pepsin digestion, whereas the heated inhibitor produced 40% degree of susceptibility. The disulfide linkage and lysine residue were important in maintaining its conformation. Partial amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz inhibitor family. Moreover, the inhibitor showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Pieris rapae and could suppress the growth of larvae.

Purification and Characterization of the Antenna Protein CP29 from Spinach Photosystem II.

A Chla/b-binding protein, CP29, was purified from PS II core complex of spinach by DEAE-Toyopearl-650S anion-exchange chromatography, after treatment with the mild nonionic detergent beta-dodecyl maltoside and high concentration of LiClO(4). At room temperature, purified CP29 had a maximum absorption at 677 nm and a fluorescence maximum at 681 nm and doublet CD signals which indicated the presence of excitonic interactions between chlorophylls. The pigment content of the CP29 was 5 D7 Chla molecules and 2 D3 Chlb molecules per CP29 polypeptide, which was determined by spectroscopic method. These results suggested that the purified CP29 was in a native state. The conformational contents of CP29 were analyzed with the help of the room temperature CD spectra of CP29. The secondary structure of CP29 was predicted by using Chou-Fasman method.

Studies on Light Harvesting Complexes of Photosystem II in a Chlorophyll-deficient Barley Mutant NYB.

As compared with wild type barley, the LHCII content of NYB barley mutant reduced markedly and its polypeptide components also changed: the contents of 24 kD, 27 kD and 30 kD polypeptides reduced and 26 kD polypeptide was missing. Northern blot analysis with specific cab gene probe showed that there was no difference in the LHCII mRNA level between the wild and mutant barley, indicating the decreased LHCII level in mutant barley was not due to a reduced transcription or accumulation of its mRNA. The rudimentary development state of thylakoids in NYB barley may be correlated with the loss of 26 kD polypeptide.

Purification and Characterization of the Core Antenna CP43 of Photosystem II.

The core antenna CP43 of photosystem II was purified from the PSII core complex of spinach by DEAE-Toyopearl-650S anion-exchange chromatography, using the mild nonionic detergent beta-dodecyl maltoside and high concentration of LiClO(4). At room temperature, the purified CP43 has a maximum absorption at 671 nm, a fluorescence maximum at 683 nm and doublet CD signals which indicated the presence of excitonic interactions between some chlorophylls. The pigment content of the CP43 was 20 - 21 Chl a per CP43 polypeptide as determined by a spectroscopic method. The results suggested that the purified CP43 was still in a native state. Moreover, the specific antiserum was obtained and identified.

Purification and Characterization of a DTT-sensitive Protease Associated with PS II Particles.

A DTT-sensitive protease was purified by hydrophobic chromatography on butyl-Toyopearl 650 M and anion-exchange chromatography on DEAE-Sephadex A-50 from the 1 M NaCl extract of PS II particles. The results of SDS-PAGE and gel-filtration chromatography on Superose 12 Have showed that it is a monomeric protease with a MW of 37 000. The protease generated polypeptides of 13.2 kD, 12 kD and 10.5 kD fragments from 18 kD protein, and those of 23 kD, 22 kD and 20 kD fragments from 24 kD protein, respectively. The protease had maximum activity at pH 8.0, and showed to be sensitive to DTT and beta-Me but insensitive to the ionic strength of NaCl. Studies with protease inhibitors suggested that this enzyme is not a serine-protease.