Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine...Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine,food,flavorings,and energy.However,traditional methods of plant extraction and chemical synthesis have limitations in industrial applications.Consequently,microbial cell factories have emerged as an important platform for terpenoid production.Terpene synthases(TPSs)are crucial in determining the structural and functional diversity of terpenoids.This review discussed the origin and classificationof TPSs,outlines commonly used TPS mining methods,and summarizes advances in TPS engineering.In addition,it also explores the influenceof machine learning on enzyme mining,the existing challenges and the future opportunities alongside cutting-edge technologies.展开更多
Background:Under hypoxia,exaggerated compensatory responses may lead to acute mountain sickness.The excessive vasodilatory effect of nitric oxide(NO)can lower the hypoxic pulmonary vasoconstriction(HPV)and peripheral ...Background:Under hypoxia,exaggerated compensatory responses may lead to acute mountain sickness.The excessive vasodilatory effect of nitric oxide(NO)can lower the hypoxic pulmonary vasoconstriction(HPV)and peripheral blood pressure.While NO is catalyzed by various nitric oxide synthase(NOS)isoforms,the regulatory roles of these types in the hemodynamics of pulmonary and systemic circulation in living hypoxic animals remain unclear.Therefore,this study aims to investigate the regu-latory effects of different NOS isoforms on pulmonary and systemic circulation in hypoxic rats by employing selective NOS inhibitors and continuously monitoring hemodynamic parameters of both pulmonary and systemic circulation.Methods:Forty healthy male Sprague–Dawley(SD)rats were randomly divided into four groups:Control group(NG-nitro-D-arginine methyl ester,D-NAME),L-NAME group(non-selective NOS inhibitor,NG-nitro-L-arginine methyl ester),AG group(in-ducible NOS inhibitor group,aminoguanidine),and 7-NI group(neurological NOS in-hibitor,7-nitroindazole).Hemodynamic parameters of rats were monitored for 10 min after inhibitor administration and 5 min after induction of hypoxia[15%O2,2200 m a.sl.,582 mmHg(76.5 kPa),Xining,China]using the real-time dynamic monitoring model for pulmonary and systemic circulation hemodynamics in vivo.Serum NO concentra-tions and blood gas analysis were measured.Results:Under normoxia,mean arterial pressure and total peripheral vascular resist-ance were increased,and ascending aortic blood flow and serum NO concentration were decreased in the L-NAME and AG groups.During hypoxia,pulmonary arterial pressure and pulmonary vascular resistance were significantly increased in the L-NAME and AG groups.Conclusions:This compensatory mechanism activated by inducible NOS and en-dothelial NOS effectively counteracts the pulmonary hemodynamic changes induced by hypoxic stress.It plays a crucial role in alleviating hypoxia-induced pulmonary arte-rial hypertension.展开更多
AIM： To determine the incidence of Epstein Barr virus associated gastric carcinoma （GC） in Brazil and compare the expressions of apoptosis related proteins and nitric oxide synthases between EBV positive and negati...AIM： To determine the incidence of Epstein Barr virus associated gastric carcinoma （GC） in Brazil and compare the expressions of apoptosis related proteins and nitric oxide synthases between EBV positive and negative gastric carcinoma. METHODS： In situ hybridization of EBV-encoded small RNA-1 （EBER-1） and PCR was performed to identify the presence of EBV in GCs. Immunohistochemistry was used to identify expressions of bcl-2, bcl-xl, bak, bax, p53, NOS-1, NOS-2, and NOS-3 proteins in 25 EBV positive GCs and in 103 EBV negative GCS. RESULTS： 12% of the cases of GC （25/208） showed EBER-1 and EBNA-1 expression. The cases were preferentially of diffuse type with intense lymphoid infiltrate in the stroma. EBV associated GCs showed higher expression of bcl-2 protein and lower expression of bak protein than in EBV negative GCs. Indeed, expressions of NOS-1 and NOS-3 were frequently observed in EBV associated GCs. CONCLUSION： Our data suggest that EBV infection may protect tumor cells from apoptosis, giving them the capacity for permanent cell cycling and proliferation. In addition, EBV positive GCs show high expression of constitutive NOS that could influence tumor progression and aggressiveness.展开更多
In the flavonoid biosynthesis pathway, Chalcone synthase (CHS) is involved in the formation of the pigment and has been shown to be a rate-limiting enzyme for the synthesis of flavonoids. In this study, a PCR approach...In the flavonoid biosynthesis pathway, Chalcone synthase (CHS) is involved in the formation of the pigment and has been shown to be a rate-limiting enzyme for the synthesis of flavonoids. In this study, a PCR approach was used to clone a Chalcone synthases cDNA from flower of sweet osmanthus “Chenghong Dangui” and it was designated as OfCHS (O. fragrans, CHS). The cDNA was 1383 bp long and a coding sequence (CDS) of 1173 bp encoding a polypeptide of 391 amino acids with an estimated molecular mass of 39.9 kDa. The theoretical isoelectric point was 6.23. Phylogenetic analysis demonstrated that OfCHS clustered with Olea europaea, Solenostemon scutellarioides, Perilla frutescens, Antirrhinum majus and Digitalis lanata. We also detected the expression of OfCHS in different tissues in “Dangui” and in two cultivars with varied coloration, “Zi Yingui” and “Chenghong Dangui” at different floral stages using quantitative real-time PCR. We observed that OfCHS transcript was higher in leaves than in petals in “Dangui”. The transcripts of OfCHS in “Zi Yingui” petals were higher than those in “Dangui” at three stages especially at xianyan stage and there was no significant difference between the two cultivars in the full flowering stage. “Chenghong Dangui” has a relatively high anthocyanin content compared to “Zi Yingui”. The relative amount of anthocyanin of “Chenghong Dangui” initially increases, and then decreases during the bloom period. However, the expression of CHS is the highest at the initial flowering stage. These data suggest that the OfCHS does not play a key role in the accumulation of total flavonoid in this cultivar. These data could contribute to explain the different accumulation of flavonoids in petals of the two cultivars.展开更多
Two chimeric sesterterpene synthases(Aa TPS1 and Aa TPS2)were functionally characterized from Alternaria alternata MB-30 isolated from the leaves of a sesterterpenoid-producing Lamiaceae plant Leucosceptrum canum.Aa T...Two chimeric sesterterpene synthases(Aa TPS1 and Aa TPS2)were functionally characterized from Alternaria alternata MB-30 isolated from the leaves of a sesterterpenoid-producing Lamiaceae plant Leucosceptrum canum.Aa TPS1 generated a 5/8/6/5 tetracyclic sesteraltererol(1)and its absolute stereochemistry was determined by X-ray crystallographic analysis of its derivative 10,11-epoxysesteraltererol(2),which enabled revision of the absolute configuration of C7 of sesterfisherol produced by Nf SS and PTTS014 characterized previously and its derivative 10,11-epoxysesterfisherol.Aa TPS2 produced a 5/15 bicyclic preterpestacin I(3).Site-directed mutagenesis suggested that F192 in Aa TPS1 was likely involved in controlling of the hydroxylation of C12,and eight amino acids were important for the enzyme activity of Aa TPS1 and Aa TPS2.The engineered Escherichia coli and Saccharomyces cerevisiae strains were constructed for the productions of compounds 1 and 3,and the highest titer of compound 1 reached 62.3 mg/L in shake-flask culture.Both compounds 1 and 2 showed anti-adipogenic activity.展开更多
The deterioration in fruit quality of commercial tomatoes is a major concern of modern tomato breeding.However,the metabolism and genetics of fruit quality are poorly understood.Here,we performed transgenic and molecu...The deterioration in fruit quality of commercial tomatoes is a major concern of modern tomato breeding.However,the metabolism and genetics of fruit quality are poorly understood.Here,we performed transgenic and molecular biology experiments to reveal that tomato phytoene synthase 1(SlPSY1)is responsible for the accumulation of an important flavor chemical,6-methyl-5-hepten-2-one(MHO).To dissect the function of SlPSY1 in regulating fruit quality,we generated and analyzed a dataset encompassing over 2000 compounds detected by GC-MS and LC-MS/MS along with transcriptomic data.The combined results illustrated that SlPSY1 deficiency imparts novel flavor to yellow tomatoes with 236 volatiles significantly changed and improves fruit firmness,possibly due to accumulation of seven cutins.Further analysis indicated SlPSY1 is essential for carotenoid-derived metabolite biosynthesis by catalyzing prephytoene-PP(PPPP)to 15-cis-phytoene.Notably,we showed that SlPSY1 can influence the metabolic flux between carotenoid and flavonoid pathways,and this metabolic flux was confirmed by silencing SlCHS1.Our study provided insights into the multiple effects of SlPSY1 on tomato fruit metabolome and highlights the potential to produce high-quality fruit by rational design of SlPSY1 expression.展开更多
Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary ce...Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.展开更多
To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthasc isoforms （nNOS and iNOS）, rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia...To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthasc isoforms （nNOS and iNOS）, rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen glucose deprivation （OGD） for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD （P〈0.05）. The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase （LDH） efflux into the incubation solution. The resuits suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.展开更多
Seedlings of the gymnosperm, Pinus edulis Engelm., have a distinctive pattern of starch accumulation following germination; however, the enzymes involved in starch synthesis have not been studied in gymnosperm sp...Seedlings of the gymnosperm, Pinus edulis Engelm., have a distinctive pattern of starch accumulation following germination; however, the enzymes involved in starch synthesis have not been studied in gymnosperm species. In this study, enzymes and starch were extracted from P. edulis seedlings germinated in the dark at room temperature. Granule_bound proteins of 58 kD and 91 kD were recognized by a pea SS Ⅱ antiserum. The 58 kD granule_bound protein was purified and identified as granule_bound starch synthase Ⅰ by alignment of the N_terminal sequence with that of granule_bound starch synthase Ⅰ from several angiosperms. Elution of soluble starch synthase activity from a DEAE_Sepharose column showed two starch synthase activity peaks, indicating at least two isoforms of soluble starch synthases. Primer affinities of soluble starch synthases were investigated. Glycogen from rabbit was the best primer for soluble starch synthase. The enzymological properties of Pinus starch synthases appear to be similar to those reported for angiosperms.展开更多
Sesquiterpenoids play an import role in the direct or indirect defense of plants.Farnesyl pyrophosphate synthases(FPSs)catalyze the biosynthesis of farnesyl pyrophosphate,which is a key precursor of farnesol and(E)-β...Sesquiterpenoids play an import role in the direct or indirect defense of plants.Farnesyl pyrophosphate synthases(FPSs)catalyze the biosynthesis of farnesyl pyrophosphate,which is a key precursor of farnesol and(E)-β-farnesene.In the current study,two FPS genes in Gossypium hirsutum,GhFPS1 and GhFPS2,were heterologously cloned and functionally characterized in a greenhouse setting.The open reading frames for full-length GhFPS1 and GhFPS2 were each 1029 nucleotides,and encoded two proteins of 342 amino acids with molecular weights of 39.4 kDa.The deduced amino acid sequences of GhFPS1–2 showed high identity to FPSs of other plants.Quantitative real-time PCR analysis revealed that GhFPS1 and GhFPS2 were highly expressed in G.hirsutum leaves,and were upregulated in methyl jasmonate(MeJA)-,methyl salicylate(MeSA)-and aphid infestation-treated cotton plants.The recombinant proteins of either GhFPS1 or GhFPS2 plus calf intestinal alkaline phosphatase could convert geranyl diphosphate(GPP)or isopentenyl diphosphate(IPP)to one major product,farnesol.Moreover,in electrophysiological response and Y-tube olfactometer assays,farnesol showed obvious attractiveness to female Aphidius gifuensis,which is an important parasitic wasp of aphids.Our findings suggest that two GhFPSs are involved in farnesol biosynthesis and they play a crucial role in indirect defense of cotton against aphid infestation.展开更多
Polysaccharides(carbohydrate polymers)have longbeen recognised as both structural and energy storagemolecules,and are critical components of plants.Plantcell walls are rich in polysaccharides,and play importantroles i...Polysaccharides(carbohydrate polymers)have longbeen recognised as both structural and energy storagemolecules,and are critical components of plants.Plantcell walls are rich in polysaccharides,and play importantroles in determining the size and shape of cells throughcontrolling the rate and direction of cell expansion·Wallsare also involved in ahsorption of nutrients,transductionof sigmals and initial resistance to pathogen attack oncells.Wall polysaccharides from different plants are alsofood sources,used as building and paper-making materi-als and have other industrial applications.展开更多
Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the'cineole cassette'comprising 1,8-cineole,limonene,myrcene,α-pinene,β-pinene,sabinene,andα-terpineol.We succes...Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the'cineole cassette'comprising 1,8-cineole,limonene,myrcene,α-pinene,β-pinene,sabinene,andα-terpineol.We successfully isolated genes of Nicotiana alata and Nicotiana langsdorfii that encoded enzymes,which produced the characteristic monoter-penes of this'cineole cassette'withα-terpineol being most abundant in the volatile spectra.The amino acid sequences of both terpineol synthases were 99%identical.The enzymes cluster in a monophyletic branch together with the closely related cineole synthase of Nicotiana suaveolens and monoterpene synthase 1 of Solanum lycopersicum.The cyclization reactions(α-terpineol to 1,8-cineole)of the terpineol synthases of N.alata and N.langsdorfii were less efficient compared to the'cineole cassette'monoterpene synthases of Arabidopsis thaliana,N.suaveolens,Salvia fruticosa,Salvia officinalis,and Citrus unshiu.The terpineol synthases of IV.alata and N.langsdorfii were localized in pistils and in the adaxial and abaxial epidermis of the petals.The enzyme activities reached their maxima at the second day after anthesis when flowers were fully opened and the enzyme activity in N.alata was highest at the transition from day to night(diurnal rhythm).展开更多
Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,w...Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,were only recently reported.These aromatic abietanes are presumably derived from ent-miltiradiene,but such biosynthetic capacity has not yet been reported in O.sativa.While wild rice has been reported to contain such an enzyme,specifically ent-kaurene synthase-like 10(KSL10),the only characterized ortholog from O.sativa(OsKSL10),specifically from the well-studied cultivar(cv.)Nipponbare,instead has been shown to make ent-sandaracopimaradiene,precursor to the oryzalexins.Notably,in many other cultivars,OsKSL10 is accompanied by a tandem duplicate,termed here OsKSL14.Biochemical characterization of OsKLS14 from cv.Kitaake demonstrates that this produces the expected abietoryzin precursor ent-miltiradiene.Strikingly,phylogenetic analysis of OsKSL10 across the rice pan-genome reveals that from cv.Nipponbare is an outlier,whereas the alleles from most other cultivars group with those from wild rice,suggesting that these also might produce ent-miltiradiene.Indeed,OsKSL10 from cv.Kitaake exhibits such activity as well,consistent with its production of abietoryzins but not oryzalexins.Similarly consistent with these results is the lack of abietoryzin production by cv.Nipponbare.Although their equivalent product outcome might suggest redundancy,OsKSL10 and OsKSL14 were observed to exhibit distinct expression patterns,indicating such differences may underlie retention of these duplicated genes.Regardless,the results reported here clarify abietoryzin biosynthesis and provide insight into the evolution of rice diterpenoid phytoalexins.展开更多
Monoterpenoids are typically present in the secretory tissues of higher plants,and their biosynthesis is catalyzed by the action of monoterpene synthases(MTSs).However,the knowledge about these enzymes is restricted i...Monoterpenoids are typically present in the secretory tissues of higher plants,and their biosynthesis is catalyzed by the action of monoterpene synthases(MTSs).However,the knowledge about these enzymes is restricted in a few plant species.MTSs are responsible for the complex cyclization of monoterpene precursors,resulting in the production of diverse monoterpene products.These enzymatic reactions are considered exceptionally complex in nature.Therefore,it is crucial to understand the catalytic mechanism of MTSs to elucidate their ability to produce diverse or specific monoterpenoid products.In our study,we analyzed thirteen genomes of Dipterocarpaceae and identified 38 MTSs that generate a variety of monoterpene products.By focusing on four MTSs with different product spectra and analyzing the formation mechanism of acyclic,monocyclic and bicyclic products in MTSs,we observed that even a single amino acid mutation can change the specificity and diversity of MTS products,which is due to the synergistic effect between the shape of the active cavity and the stabilization of carbon-positive intermediates that the mutation changing.Notably,residues N340,I448,and phosphoric acid groups were found to be significant contributors to the stabilization of intermediate terpinyl and pinene cations.Alterations in these residues,either directly or indirectly,can impact the synthesis of single monoterpenes or their mixtures.By revealing the role of key residues in the catalytic process and establishing the interaction model between specific residues and complex monoterpenes in MTSs,it will be possible to reasonably design and engineer different catalytic activities into existing MTSs,laying a foundation for the artificial design and industrial application of MTSs.展开更多
The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions.Therefore,discovering tools that aid crystallization is crucial.In this review,the versatility of fragment-antig...The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions.Therefore,discovering tools that aid crystallization is crucial.In this review,the versatility of fragment-antigen binding domains(F_(ab)s)as protein crystallization chaperones is discussed.F_(ab)s have aided the crystallization of membrane-bound and soluble proteins as well as RNA.The ability to bind three F_(ab)s onto a single protein target has demonstrated their potential for crystallization of challenging proteins.We describe a high-throughput workflow for identifying F_(ab)s to aid the crystallization of a protein of interest(POI)by leveraging phage display technologies and differential scanning fluorimetry(DSF).This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases(PKSs),which harbor flexible domains and assume transient conformations.PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds.Despite years of research studying these megasynthases,their overall topology has remained elusive.One F ab in particular,1B2,has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic(cryoEM)analyses of multiple modules from distinct assembly-line PKSs.Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM,thereby enabling the visualization of intact PKS modules at near-atomic(3–5Å)resolution.The identification of PKS-binding F_(ab)s can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.展开更多
Long noncoding RNA and microRNA are regulatory noncoding RNAs that are implicated in Alzheimer's disease, but the role of long noncoding RNA-associated competitive endogenous RNA has not been fully elucidated. The...Long noncoding RNA and microRNA are regulatory noncoding RNAs that are implicated in Alzheimer's disease, but the role of long noncoding RNA-associated competitive endogenous RNA has not been fully elucidated. The long noncoding RNA growth arrest-specific 5(GAS5) is a member of the 5′-terminal oligopyrimidine gene family that may be involved in neurological disorders, but its role in Alzheimer's disease remains unclear. This study aimed to investigate the function of GAS5 and construct a GAS5-associated competitive endogenous RNA network comprising potential targets. RNA sequencing results showed that GAS5 was upregulated in five familial Alzheimer's disease(5×FAD) mice, APPswe/PSEN1dE9(APP/PS1) mice, Alzheimer's disease-related APPswe cells, and serum from patients with Alzheimer's disease. Functional experiments with targeted overexpression and silencing demonstrated that GAS5 played a role in cognitive dysfunction and multiple Alzheimer's disease-associated pathologies, including tau hyperphosphorylation, amyloid-beta accumulation, and neuronal apoptosis. Mechanistic studies indicated that GAS5 acted as an endogenous sponge by competing for microRNA-23b-3p(miR-23b-3p) binding to regulate its targets glycogen synthase kinase 3beta(GSK-3β) and phosphatase and tensin homologue deleted on chromosome 10(PTEN) expression in an Argonaute 2-induced RNA silencing complex(RISC)-dependent manner. GAS5 inhibited miR-23b-3p-mediated GSK-3β and PTEN cascades with a feedforward PTEN/protein kinase B(Akt)/GSK-3β linkage. Furthermore, recovery of GAS5/miR-23b-3p/GSK-3β/PTEN pathways relieved Alzheimer's disease-like symptoms in vivo, indicated by the amelioration of spatial cognition, neuronal degeneration, amyloid-beta load, and tau phosphorylation. Together, these findings suggest that GAS5 promotes Alzheimer's disease pathogenesis. This study establishes the functional convergence of the GAS5/miR-23b-3p/GSK-3β/PTEN pathway on multiple pathologies, suggesting a candidate therapeutic target in Alzheimer's disease.展开更多
Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid （MIA） anti- cancer agents： vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid an...Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid （MIA） anti- cancer agents： vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate （GPP）, the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate （IPP） and dimethylallyl diphosphate （DMAPP） by GPP synthase （GPPS）. Here, we report three genes encoding proteins with sequence similarity to large subunit （CrGPPS.LSU） and small subunit （CrGPPS.SSU） of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate （GGPP）, CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coil resulted in heteromeric enzyme with enhanced activity producing only GPR While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate （MelA） treatment of leaves sig- nificantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.展开更多
Plant isoprenoids(also known as terpenes or terpenoids)are a wide family of primary and secondary metabolites with multiple functions.In particular,most photosynthesis-related isoprenoids(including carotenoids and chl...Plant isoprenoids(also known as terpenes or terpenoids)are a wide family of primary and secondary metabolites with multiple functions.In particular,most photosynthesis-related isoprenoids(including carotenoids and chlorophylls)as well as diterpenes and polyterpenes derive from geranylgeranyl diphosphate(GGPP)produced by GGPP synthase(GGPPS)enzymes in several cell compartments.Plant genomes typically harbor multiple copies of differentially expressed genes encoding GGPPS-like pro-teins.While sequence comparisons allow to identify potential GGPPS candidates,experimental evidence is required to ascertain their enzymatic activity and biologi cal function.Actually,functional analyses of the full set of potential GGPPS paralogs are only available for a handful of plant species.Here we review our current knowledge on the GGPPS families of the model plant Arabidopsis thaliana and the crop species rice(0ryza sativa),pepper(Capsicum annuum)and tomato(Solanum lycopersicum).The results indicate that a major determinant of the biological role of particular GGPPS paralogs is the expression profile of the corresponding genes even though specific interactions with other proteins(including GGPP-consuming enzymes)might also contribute to subfunctionalization.In some species,however,a single GGPPS isoforms appears to be responsible for the production of most if not all GGPP required for cell functions.Deciphering the mechanisms regulating GGPPS activity in particular cell compartments,tissues,organs and plant species will be very useful for future metabolic engineering approaches aimed to manipulate the accumulation of particular GGPP-derived products of interest without negatively impacting the levels of other isoprenoids required to sustain essential cell functions.展开更多
Background Exposure of adult mice to more than 95% O_2 produces a lethal injury by 72 hours. Nitric oxide synthase （NOS） is thought to contribute to the pathophysiology of murine hyperoxia-induced acute lung injury ...Background Exposure of adult mice to more than 95% O_2 produces a lethal injury by 72 hours. Nitric oxide synthase （NOS） is thought to contribute to the pathophysiology of murine hyperoxia-induced acute lung injury （ALI）. Osteopontin （OPN） is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase （iNOS）, which generates large amounts of nitric oxide production. However, the relationship between nitric oxide and endogenous OPN in lung tissue during hyperoxia-induced ALI has not yet been elucidated, thus we examined the role that OPN plays in the hyperoxia-induced lung injury and its relationships with NOS.Methods One hundred and forty-four osteopontin knock-out （KO） mice and their matched wild type background control （WT） were exposed in sealed cages 〉95% oxygen or room air for 24-72 hours, and the severity of lung injury was assessed; expression of OPN, endothelial nitric oxide synthase （eNOS） and iNOS mRNA in lung tissues at 24,48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction （RT-PCR）; immunohistochemistry （IHC） was performed for the detection of iNOS, eNOS, and OPN protein in lung tissues.Results OPN KO mice developed more severe acute lung injury at 72 hours of hyperoxia. The wet/dry weight ratio increased to 6.85±0.66 in the KO mice at 72 hours of hyperoxia as compared to 5.31±0.92 in the WT group （P〈0.05）. iNOS mRNA （48 hours： 1.04±0.08 vs. 0.63±0.09, P〈0.01; 72 hours： 0.89±0.08 vs. 0.72±0.09, P〈0.05） and eNOS mRNA （48 hours： 0.62±0.08 vs. 0.43±0.09, P〈0.05; 72 hours： 0.67±0.08 vs. 0.45±0.09, P〈0.05） expression was more significantly increased in OPN KO mice than their matched WT mice when exposed to hyperoxia. IHC study showed higher expression of iNOS （20.54±3.18 vs. 12.52±2.46, P 〈0.05） and eNOS （19.83±5.64 vs. 9.45±3.82, P 〈0.05） in lung tissues of OPN KO mice at 72 hours of hyperoxia. Conclusion OPN can protect against hyperoxia-induced lung injury by inhibiting NOS.展开更多
Class I terpene synthase(TPS)generates bioactive terpenoids with diverse backbones.Sesterterpene synthase(sester-TPS,C25),a branch of class I TPSs,was recently identified in Brassicaceae.However,the catalytic mechanis...Class I terpene synthase(TPS)generates bioactive terpenoids with diverse backbones.Sesterterpene synthase(sester-TPS,C25),a branch of class I TPSs,was recently identified in Brassicaceae.However,the catalytic mechanisms of sester-TPSs are not fully understood.Here,we first identified three nonclustered functional sester-TPSs(AtTPS06,AtTPS22,and AtTPS29)in Arabidopsis thaliana.AtTPS06 utilizes a type-B cyclization mechanism,whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism.We then determined the crystal structure of the AtTPS18–FSPP complex to explore the cyclization mechanism of plant sester-TPSs.We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism:(1)mainly due to the outward shift of helix G,plant sester-TPSs have a larger catalytic pocket than do mono-,sesqui-,and di-TPSs to accommodate GFPP;(2)type-A sester-TPSs have more aromatic residues(five or six)in their catalytic pocket than classic TPSs(two or three),which also determines whether the type-A or type-B cyclization mechanism is active;and(3)the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs.Altogether,this study improves our understanding of the catalytic mechanism of plant sester-TPS,which ultimately enables the rational engineering of sesterterpenoids for future applications.展开更多
基金supported by the National Key Research and Development Program of China(2020YFA0908300)the Natural Science Foundation of China(22138006,22278240).
文摘Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine,food,flavorings,and energy.However,traditional methods of plant extraction and chemical synthesis have limitations in industrial applications.Consequently,microbial cell factories have emerged as an important platform for terpenoid production.Terpene synthases(TPSs)are crucial in determining the structural and functional diversity of terpenoids.This review discussed the origin and classificationof TPSs,outlines commonly used TPS mining methods,and summarizes advances in TPS engineering.In addition,it also explores the influenceof machine learning on enzyme mining,the existing challenges and the future opportunities alongside cutting-edge technologies.
基金This work was supported by the National Natural Science Foundation of China(grant numbers 81560301 and 81160012)the Natural Science Foundation of Qinghai Province(grant number 2022-ZJ-905)‘2022 Qinghai Province Kunlun Talents High-end Innovation and Entrepreneurship Talents’Outstanding Talent Project.
文摘Background:Under hypoxia,exaggerated compensatory responses may lead to acute mountain sickness.The excessive vasodilatory effect of nitric oxide(NO)can lower the hypoxic pulmonary vasoconstriction(HPV)and peripheral blood pressure.While NO is catalyzed by various nitric oxide synthase(NOS)isoforms,the regulatory roles of these types in the hemodynamics of pulmonary and systemic circulation in living hypoxic animals remain unclear.Therefore,this study aims to investigate the regu-latory effects of different NOS isoforms on pulmonary and systemic circulation in hypoxic rats by employing selective NOS inhibitors and continuously monitoring hemodynamic parameters of both pulmonary and systemic circulation.Methods:Forty healthy male Sprague–Dawley(SD)rats were randomly divided into four groups:Control group(NG-nitro-D-arginine methyl ester,D-NAME),L-NAME group(non-selective NOS inhibitor,NG-nitro-L-arginine methyl ester),AG group(in-ducible NOS inhibitor group,aminoguanidine),and 7-NI group(neurological NOS in-hibitor,7-nitroindazole).Hemodynamic parameters of rats were monitored for 10 min after inhibitor administration and 5 min after induction of hypoxia[15%O2,2200 m a.sl.,582 mmHg(76.5 kPa),Xining,China]using the real-time dynamic monitoring model for pulmonary and systemic circulation hemodynamics in vivo.Serum NO concentra-tions and blood gas analysis were measured.Results:Under normoxia,mean arterial pressure and total peripheral vascular resist-ance were increased,and ascending aortic blood flow and serum NO concentration were decreased in the L-NAME and AG groups.During hypoxia,pulmonary arterial pressure and pulmonary vascular resistance were significantly increased in the L-NAME and AG groups.Conclusions:This compensatory mechanism activated by inducible NOS and en-dothelial NOS effectively counteracts the pulmonary hemodynamic changes induced by hypoxic stress.It plays a crucial role in alleviating hypoxia-induced pulmonary arte-rial hypertension.
基金Supported by the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo grant 04/15650-1
文摘AIM： To determine the incidence of Epstein Barr virus associated gastric carcinoma （GC） in Brazil and compare the expressions of apoptosis related proteins and nitric oxide synthases between EBV positive and negative gastric carcinoma. METHODS： In situ hybridization of EBV-encoded small RNA-1 （EBER-1） and PCR was performed to identify the presence of EBV in GCs. Immunohistochemistry was used to identify expressions of bcl-2, bcl-xl, bak, bax, p53, NOS-1, NOS-2, and NOS-3 proteins in 25 EBV positive GCs and in 103 EBV negative GCS. RESULTS： 12% of the cases of GC （25/208） showed EBER-1 and EBNA-1 expression. The cases were preferentially of diffuse type with intense lymphoid infiltrate in the stroma. EBV associated GCs showed higher expression of bcl-2 protein and lower expression of bak protein than in EBV negative GCs. Indeed, expressions of NOS-1 and NOS-3 were frequently observed in EBV associated GCs. CONCLUSION： Our data suggest that EBV infection may protect tumor cells from apoptosis, giving them the capacity for permanent cell cycling and proliferation. In addition, EBV positive GCs show high expression of constitutive NOS that could influence tumor progression and aggressiveness.
文摘In the flavonoid biosynthesis pathway, Chalcone synthase (CHS) is involved in the formation of the pigment and has been shown to be a rate-limiting enzyme for the synthesis of flavonoids. In this study, a PCR approach was used to clone a Chalcone synthases cDNA from flower of sweet osmanthus “Chenghong Dangui” and it was designated as OfCHS (O. fragrans, CHS). The cDNA was 1383 bp long and a coding sequence (CDS) of 1173 bp encoding a polypeptide of 391 amino acids with an estimated molecular mass of 39.9 kDa. The theoretical isoelectric point was 6.23. Phylogenetic analysis demonstrated that OfCHS clustered with Olea europaea, Solenostemon scutellarioides, Perilla frutescens, Antirrhinum majus and Digitalis lanata. We also detected the expression of OfCHS in different tissues in “Dangui” and in two cultivars with varied coloration, “Zi Yingui” and “Chenghong Dangui” at different floral stages using quantitative real-time PCR. We observed that OfCHS transcript was higher in leaves than in petals in “Dangui”. The transcripts of OfCHS in “Zi Yingui” petals were higher than those in “Dangui” at three stages especially at xianyan stage and there was no significant difference between the two cultivars in the full flowering stage. “Chenghong Dangui” has a relatively high anthocyanin content compared to “Zi Yingui”. The relative amount of anthocyanin of “Chenghong Dangui” initially increases, and then decreases during the bloom period. However, the expression of CHS is the highest at the initial flowering stage. These data suggest that the OfCHS does not play a key role in the accumulation of total flavonoid in this cultivar. These data could contribute to explain the different accumulation of flavonoids in petals of the two cultivars.
基金supported financially by the National Natural Science Foundation of China(Nos.21937006 and 22107103)the Yunnan Key Research and Development Program(No.2019ZF011–2)the“Western Light”Program of the CAS(to Y.Liu)。
文摘Two chimeric sesterterpene synthases(Aa TPS1 and Aa TPS2)were functionally characterized from Alternaria alternata MB-30 isolated from the leaves of a sesterterpenoid-producing Lamiaceae plant Leucosceptrum canum.Aa TPS1 generated a 5/8/6/5 tetracyclic sesteraltererol(1)and its absolute stereochemistry was determined by X-ray crystallographic analysis of its derivative 10,11-epoxysesteraltererol(2),which enabled revision of the absolute configuration of C7 of sesterfisherol produced by Nf SS and PTTS014 characterized previously and its derivative 10,11-epoxysesterfisherol.Aa TPS2 produced a 5/15 bicyclic preterpestacin I(3).Site-directed mutagenesis suggested that F192 in Aa TPS1 was likely involved in controlling of the hydroxylation of C12,and eight amino acids were important for the enzyme activity of Aa TPS1 and Aa TPS2.The engineered Escherichia coli and Saccharomyces cerevisiae strains were constructed for the productions of compounds 1 and 3,and the highest titer of compound 1 reached 62.3 mg/L in shake-flask culture.Both compounds 1 and 2 showed anti-adipogenic activity.
基金supported by the National Natural Science Foundation of China(Grant Nos.31991185,31902019,32102384)National Key Research and Development Program of China(Grant No.2021YFF1000103)+2 种基金Key Research and Development Program of Guangdong Province(Grant No.2021B0707010005)Taishan Scholars Program of Shandong Province,China(2016-2020)supported by the Youth innovation Program of Chinese Academy of Agricultural Sciences(Grant No.Y2023QC05)。
文摘The deterioration in fruit quality of commercial tomatoes is a major concern of modern tomato breeding.However,the metabolism and genetics of fruit quality are poorly understood.Here,we performed transgenic and molecular biology experiments to reveal that tomato phytoene synthase 1(SlPSY1)is responsible for the accumulation of an important flavor chemical,6-methyl-5-hepten-2-one(MHO).To dissect the function of SlPSY1 in regulating fruit quality,we generated and analyzed a dataset encompassing over 2000 compounds detected by GC-MS and LC-MS/MS along with transcriptomic data.The combined results illustrated that SlPSY1 deficiency imparts novel flavor to yellow tomatoes with 236 volatiles significantly changed and improves fruit firmness,possibly due to accumulation of seven cutins.Further analysis indicated SlPSY1 is essential for carotenoid-derived metabolite biosynthesis by catalyzing prephytoene-PP(PPPP)to 15-cis-phytoene.Notably,we showed that SlPSY1 can influence the metabolic flux between carotenoid and flavonoid pathways,and this metabolic flux was confirmed by silencing SlCHS1.Our study provided insights into the multiple effects of SlPSY1 on tomato fruit metabolome and highlights the potential to produce high-quality fruit by rational design of SlPSY1 expression.
文摘Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.
文摘To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthasc isoforms （nNOS and iNOS）, rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen glucose deprivation （OGD） for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD （P〈0.05）. The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase （LDH） efflux into the incubation solution. The resuits suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.
文摘Seedlings of the gymnosperm, Pinus edulis Engelm., have a distinctive pattern of starch accumulation following germination; however, the enzymes involved in starch synthesis have not been studied in gymnosperm species. In this study, enzymes and starch were extracted from P. edulis seedlings germinated in the dark at room temperature. Granule_bound proteins of 58 kD and 91 kD were recognized by a pea SS Ⅱ antiserum. The 58 kD granule_bound protein was purified and identified as granule_bound starch synthase Ⅰ by alignment of the N_terminal sequence with that of granule_bound starch synthase Ⅰ from several angiosperms. Elution of soluble starch synthase activity from a DEAE_Sepharose column showed two starch synthase activity peaks, indicating at least two isoforms of soluble starch synthases. Primer affinities of soluble starch synthases were investigated. Glycogen from rabbit was the best primer for soluble starch synthase. The enzymological properties of Pinus starch synthases appear to be similar to those reported for angiosperms.
基金This work was supported by the National Natural Science Foundation of China(31772176,31672038 and 31621064)and the National Key Research and Development Program of China(2017YFDO201900 and 2017YFD0200400).
文摘Sesquiterpenoids play an import role in the direct or indirect defense of plants.Farnesyl pyrophosphate synthases(FPSs)catalyze the biosynthesis of farnesyl pyrophosphate,which is a key precursor of farnesol and(E)-β-farnesene.In the current study,two FPS genes in Gossypium hirsutum,GhFPS1 and GhFPS2,were heterologously cloned and functionally characterized in a greenhouse setting.The open reading frames for full-length GhFPS1 and GhFPS2 were each 1029 nucleotides,and encoded two proteins of 342 amino acids with molecular weights of 39.4 kDa.The deduced amino acid sequences of GhFPS1–2 showed high identity to FPSs of other plants.Quantitative real-time PCR analysis revealed that GhFPS1 and GhFPS2 were highly expressed in G.hirsutum leaves,and were upregulated in methyl jasmonate(MeJA)-,methyl salicylate(MeSA)-and aphid infestation-treated cotton plants.The recombinant proteins of either GhFPS1 or GhFPS2 plus calf intestinal alkaline phosphatase could convert geranyl diphosphate(GPP)or isopentenyl diphosphate(IPP)to one major product,farnesol.Moreover,in electrophysiological response and Y-tube olfactometer assays,farnesol showed obvious attractiveness to female Aphidius gifuensis,which is an important parasitic wasp of aphids.Our findings suggest that two GhFPSs are involved in farnesol biosynthesis and they play a crucial role in indirect defense of cotton against aphid infestation.
文摘Polysaccharides(carbohydrate polymers)have longbeen recognised as both structural and energy storagemolecules,and are critical components of plants.Plantcell walls are rich in polysaccharides,and play importantroles in determining the size and shape of cells throughcontrolling the rate and direction of cell expansion·Wallsare also involved in ahsorption of nutrients,transductionof sigmals and initial resistance to pathogen attack oncells.Wall polysaccharides from different plants are alsofood sources,used as building and paper-making materi-als and have other industrial applications.
文摘Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the'cineole cassette'comprising 1,8-cineole,limonene,myrcene,α-pinene,β-pinene,sabinene,andα-terpineol.We successfully isolated genes of Nicotiana alata and Nicotiana langsdorfii that encoded enzymes,which produced the characteristic monoter-penes of this'cineole cassette'withα-terpineol being most abundant in the volatile spectra.The amino acid sequences of both terpineol synthases were 99%identical.The enzymes cluster in a monophyletic branch together with the closely related cineole synthase of Nicotiana suaveolens and monoterpene synthase 1 of Solanum lycopersicum.The cyclization reactions(α-terpineol to 1,8-cineole)of the terpineol synthases of N.alata and N.langsdorfii were less efficient compared to the'cineole cassette'monoterpene synthases of Arabidopsis thaliana,N.suaveolens,Salvia fruticosa,Salvia officinalis,and Citrus unshiu.The terpineol synthases of IV.alata and N.langsdorfii were localized in pistils and in the adaxial and abaxial epidermis of the petals.The enzyme activities reached their maxima at the second day after anthesis when flowers were fully opened and the enzyme activity in N.alata was highest at the transition from day to night(diurnal rhythm).
基金supported by grants from the NIH(GM131885)and USDA(2020-67013-32557)to R.J.P.
文摘Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,were only recently reported.These aromatic abietanes are presumably derived from ent-miltiradiene,but such biosynthetic capacity has not yet been reported in O.sativa.While wild rice has been reported to contain such an enzyme,specifically ent-kaurene synthase-like 10(KSL10),the only characterized ortholog from O.sativa(OsKSL10),specifically from the well-studied cultivar(cv.)Nipponbare,instead has been shown to make ent-sandaracopimaradiene,precursor to the oryzalexins.Notably,in many other cultivars,OsKSL10 is accompanied by a tandem duplicate,termed here OsKSL14.Biochemical characterization of OsKLS14 from cv.Kitaake demonstrates that this produces the expected abietoryzin precursor ent-miltiradiene.Strikingly,phylogenetic analysis of OsKSL10 across the rice pan-genome reveals that from cv.Nipponbare is an outlier,whereas the alleles from most other cultivars group with those from wild rice,suggesting that these also might produce ent-miltiradiene.Indeed,OsKSL10 from cv.Kitaake exhibits such activity as well,consistent with its production of abietoryzins but not oryzalexins.Similarly consistent with these results is the lack of abietoryzin production by cv.Nipponbare.Although their equivalent product outcome might suggest redundancy,OsKSL10 and OsKSL14 were observed to exhibit distinct expression patterns,indicating such differences may underlie retention of these duplicated genes.Regardless,the results reported here clarify abietoryzin biosynthesis and provide insight into the evolution of rice diterpenoid phytoalexins.
基金supported by the National Key R&D Program of China(2020YFA0908000)the National Natural Science Foundation of China(31901015)Science and Technology Partnership Program,Ministry of Science and Technology of China(KY202001017).
文摘Monoterpenoids are typically present in the secretory tissues of higher plants,and their biosynthesis is catalyzed by the action of monoterpene synthases(MTSs).However,the knowledge about these enzymes is restricted in a few plant species.MTSs are responsible for the complex cyclization of monoterpene precursors,resulting in the production of diverse monoterpene products.These enzymatic reactions are considered exceptionally complex in nature.Therefore,it is crucial to understand the catalytic mechanism of MTSs to elucidate their ability to produce diverse or specific monoterpenoid products.In our study,we analyzed thirteen genomes of Dipterocarpaceae and identified 38 MTSs that generate a variety of monoterpene products.By focusing on four MTSs with different product spectra and analyzing the formation mechanism of acyclic,monocyclic and bicyclic products in MTSs,we observed that even a single amino acid mutation can change the specificity and diversity of MTS products,which is due to the synergistic effect between the shape of the active cavity and the stabilization of carbon-positive intermediates that the mutation changing.Notably,residues N340,I448,and phosphoric acid groups were found to be significant contributors to the stabilization of intermediate terpinyl and pinene cations.Alterations in these residues,either directly or indirectly,can impact the synthesis of single monoterpenes or their mixtures.By revealing the role of key residues in the catalytic process and establishing the interaction model between specific residues and complex monoterpenes in MTSs,it will be possible to reasonably design and engineer different catalytic activities into existing MTSs,laying a foundation for the artificial design and industrial application of MTSs.
文摘The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions.Therefore,discovering tools that aid crystallization is crucial.In this review,the versatility of fragment-antigen binding domains(F_(ab)s)as protein crystallization chaperones is discussed.F_(ab)s have aided the crystallization of membrane-bound and soluble proteins as well as RNA.The ability to bind three F_(ab)s onto a single protein target has demonstrated their potential for crystallization of challenging proteins.We describe a high-throughput workflow for identifying F_(ab)s to aid the crystallization of a protein of interest(POI)by leveraging phage display technologies and differential scanning fluorimetry(DSF).This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases(PKSs),which harbor flexible domains and assume transient conformations.PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds.Despite years of research studying these megasynthases,their overall topology has remained elusive.One F ab in particular,1B2,has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic(cryoEM)analyses of multiple modules from distinct assembly-line PKSs.Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM,thereby enabling the visualization of intact PKS modules at near-atomic(3–5Å)resolution.The identification of PKS-binding F_(ab)s can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.
基金supported by the National Natural Science Foundation of China,Nos. 82173806 and U1803281Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science,Nos. 2021-I2M-1-030 and 2022-I2M-2-002Non-Profit Central Research Institute Fund of Chinese Academy of Medical Sciences,No. 2022-JKCS-08 (all to RL)。
文摘Long noncoding RNA and microRNA are regulatory noncoding RNAs that are implicated in Alzheimer's disease, but the role of long noncoding RNA-associated competitive endogenous RNA has not been fully elucidated. The long noncoding RNA growth arrest-specific 5(GAS5) is a member of the 5′-terminal oligopyrimidine gene family that may be involved in neurological disorders, but its role in Alzheimer's disease remains unclear. This study aimed to investigate the function of GAS5 and construct a GAS5-associated competitive endogenous RNA network comprising potential targets. RNA sequencing results showed that GAS5 was upregulated in five familial Alzheimer's disease(5×FAD) mice, APPswe/PSEN1dE9(APP/PS1) mice, Alzheimer's disease-related APPswe cells, and serum from patients with Alzheimer's disease. Functional experiments with targeted overexpression and silencing demonstrated that GAS5 played a role in cognitive dysfunction and multiple Alzheimer's disease-associated pathologies, including tau hyperphosphorylation, amyloid-beta accumulation, and neuronal apoptosis. Mechanistic studies indicated that GAS5 acted as an endogenous sponge by competing for microRNA-23b-3p(miR-23b-3p) binding to regulate its targets glycogen synthase kinase 3beta(GSK-3β) and phosphatase and tensin homologue deleted on chromosome 10(PTEN) expression in an Argonaute 2-induced RNA silencing complex(RISC)-dependent manner. GAS5 inhibited miR-23b-3p-mediated GSK-3β and PTEN cascades with a feedforward PTEN/protein kinase B(Akt)/GSK-3β linkage. Furthermore, recovery of GAS5/miR-23b-3p/GSK-3β/PTEN pathways relieved Alzheimer's disease-like symptoms in vivo, indicated by the amelioration of spatial cognition, neuronal degeneration, amyloid-beta load, and tau phosphorylation. Together, these findings suggest that GAS5 promotes Alzheimer's disease pathogenesis. This study establishes the functional convergence of the GAS5/miR-23b-3p/GSK-3β/PTEN pathway on multiple pathologies, suggesting a candidate therapeutic target in Alzheimer's disease.
文摘Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid （MIA） anti- cancer agents： vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate （GPP）, the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate （IPP） and dimethylallyl diphosphate （DMAPP） by GPP synthase （GPPS）. Here, we report three genes encoding proteins with sequence similarity to large subunit （CrGPPS.LSU） and small subunit （CrGPPS.SSU） of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate （GGPP）, CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coil resulted in heteromeric enzyme with enhanced activity producing only GPR While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate （MelA） treatment of leaves sig- nificantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.
文摘Plant isoprenoids(also known as terpenes or terpenoids)are a wide family of primary and secondary metabolites with multiple functions.In particular,most photosynthesis-related isoprenoids(including carotenoids and chlorophylls)as well as diterpenes and polyterpenes derive from geranylgeranyl diphosphate(GGPP)produced by GGPP synthase(GGPPS)enzymes in several cell compartments.Plant genomes typically harbor multiple copies of differentially expressed genes encoding GGPPS-like pro-teins.While sequence comparisons allow to identify potential GGPPS candidates,experimental evidence is required to ascertain their enzymatic activity and biologi cal function.Actually,functional analyses of the full set of potential GGPPS paralogs are only available for a handful of plant species.Here we review our current knowledge on the GGPPS families of the model plant Arabidopsis thaliana and the crop species rice(0ryza sativa),pepper(Capsicum annuum)and tomato(Solanum lycopersicum).The results indicate that a major determinant of the biological role of particular GGPPS paralogs is the expression profile of the corresponding genes even though specific interactions with other proteins(including GGPP-consuming enzymes)might also contribute to subfunctionalization.In some species,however,a single GGPPS isoforms appears to be responsible for the production of most if not all GGPP required for cell functions.Deciphering the mechanisms regulating GGPPS activity in particular cell compartments,tissues,organs and plant species will be very useful for future metabolic engineering approaches aimed to manipulate the accumulation of particular GGPP-derived products of interest without negatively impacting the levels of other isoprenoids required to sustain essential cell functions.
文摘Background Exposure of adult mice to more than 95% O_2 produces a lethal injury by 72 hours. Nitric oxide synthase （NOS） is thought to contribute to the pathophysiology of murine hyperoxia-induced acute lung injury （ALI）. Osteopontin （OPN） is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase （iNOS）, which generates large amounts of nitric oxide production. However, the relationship between nitric oxide and endogenous OPN in lung tissue during hyperoxia-induced ALI has not yet been elucidated, thus we examined the role that OPN plays in the hyperoxia-induced lung injury and its relationships with NOS.Methods One hundred and forty-four osteopontin knock-out （KO） mice and their matched wild type background control （WT） were exposed in sealed cages 〉95% oxygen or room air for 24-72 hours, and the severity of lung injury was assessed; expression of OPN, endothelial nitric oxide synthase （eNOS） and iNOS mRNA in lung tissues at 24,48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction （RT-PCR）; immunohistochemistry （IHC） was performed for the detection of iNOS, eNOS, and OPN protein in lung tissues.Results OPN KO mice developed more severe acute lung injury at 72 hours of hyperoxia. The wet/dry weight ratio increased to 6.85±0.66 in the KO mice at 72 hours of hyperoxia as compared to 5.31±0.92 in the WT group （P〈0.05）. iNOS mRNA （48 hours： 1.04±0.08 vs. 0.63±0.09, P〈0.01; 72 hours： 0.89±0.08 vs. 0.72±0.09, P〈0.05） and eNOS mRNA （48 hours： 0.62±0.08 vs. 0.43±0.09, P〈0.05; 72 hours： 0.67±0.08 vs. 0.45±0.09, P〈0.05） expression was more significantly increased in OPN KO mice than their matched WT mice when exposed to hyperoxia. IHC study showed higher expression of iNOS （20.54±3.18 vs. 12.52±2.46, P 〈0.05） and eNOS （19.83±5.64 vs. 9.45±3.82, P 〈0.05） in lung tissues of OPN KO mice at 72 hours of hyperoxia. Conclusion OPN can protect against hyperoxia-induced lung injury by inhibiting NOS.
基金supported by the National Key R&D Program of China(grant no.2018YFA0900600)the National Natural Science Foundation of China(grant nos.31970315 and 31700263)+3 种基金the“Priority Research Program”of the Chinese Academy of Sciences(grant nos.ZDRW-ZS-2019-2 and XDB27020103)the Grant-in-Aid Program for Scientific Research from the MEXT,Japan(JSPS KAKENHI grant no.JP16H06443)the State Key Laboratory of Plant Genomics of China(grant no.SKLPG2016A-13)supported by the Foundation of Youth Innovation Promotion Association of the Chinese Academy of Sciences.
文摘Class I terpene synthase(TPS)generates bioactive terpenoids with diverse backbones.Sesterterpene synthase(sester-TPS,C25),a branch of class I TPSs,was recently identified in Brassicaceae.However,the catalytic mechanisms of sester-TPSs are not fully understood.Here,we first identified three nonclustered functional sester-TPSs(AtTPS06,AtTPS22,and AtTPS29)in Arabidopsis thaliana.AtTPS06 utilizes a type-B cyclization mechanism,whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism.We then determined the crystal structure of the AtTPS18–FSPP complex to explore the cyclization mechanism of plant sester-TPSs.We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism:(1)mainly due to the outward shift of helix G,plant sester-TPSs have a larger catalytic pocket than do mono-,sesqui-,and di-TPSs to accommodate GFPP;(2)type-A sester-TPSs have more aromatic residues(five or six)in their catalytic pocket than classic TPSs(two or three),which also determines whether the type-A or type-B cyclization mechanism is active;and(3)the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs.Altogether,this study improves our understanding of the catalytic mechanism of plant sester-TPS,which ultimately enables the rational engineering of sesterterpenoids for future applications.
