Arabidopsis Squalene Epoxidase 3 （SQE3） Complements SQE1 and Is Important for Embryo Development and Bulk Squalene Epoxidase Activity

The existence of multigenic families in the mevalonate pathway suggests divergent functional roles for pathway components involved in the biosynthesis of plant sterols. Squalene epoxidases （SQEs） are key components of this pathway, and Squalene Epoxidase 1 （SQE1） has been identified as a fundamental enzyme in this biosynthetic step. In the present work, we extended the characterization of the remaining SQE family members, phylogenetically resolving between true SQEs and a subfamily of SQE-like proteins that is exclusive to Brassicaceae. Functional characterization of true SQE family members, Squalene Epox- idase 2 （SQE2） and Squalene Epoxidase 3 （SQE3）, indicates that SQE3, but not SQE2, contributes to the bulk SQE activity in Arabidopsis, with sqe3-1 mutants accumulating squalene and displaying sensitivity to ter- binafine. We genetically demonstrated that SQE3 seems to play a particularly significant role in embryo development. Also, SQE1 and SQE3 both localize in the endoplasmic reticulum, and SQE3 can functionally complement SQEI. Thus, SQE1 and SQE3 seem to be two functionally unequal redundant genes in the pro- motion of plant SQE activity in Arabidopsis.

Molecular Cloning and Expression of Squalene Epoxidase from a Medicinal Plant, Bupleurum chinense

Objective In plant, squalene epoxidase （SE） catalyzes the first oxygenation step in the biosynthetic pathway of triterpenoid and phytosterol, representing one of the rate-limiting enzymes in this pathway. Bupleurum chinense is an important medicinal herb with its major active constituents such as triterpenoid saponins and saikosaponins. In order to obtain the series of enzymatic genes involved in saikosaponin biosynthesis, a cDNA of SE, designated BcSEI, was cloned from B. chinense. Methods The BcSEI gene was cloned by homology-based PCR and 5＇/3＇ RACE methods from the adventitious roots of B. chinense. The physical and chemical parameters of BcSE1 protein were predicted by protparam. In order to discover hints in amino acid sequences on the dominant functions in the biosynthesis of saponin or phytosterol, sequences of SE from other plants were downloaded from NCBI for sequences alignment and phylogenetic analysis. BcSEI was cloned into a yeast mutant KLNI （MATa, ergl.＇：URA3, leu2, ura3, and trpl） to verify the enzyme activity of BcSE1. Additionally, the tissue-specific expression and methyl jasmonate （MeJA） inducibility of BcSEI were investigated using quantitative real-time PCR. Results The predicted protein of BcSE1 is highly similar to SEs from other plants sharing amino acid sequence identities of up to 88%. The BcSEI can functionally complement with yeast SE gene （ERGI） when expressed in the KLNI mutant （MATa, ergl：：URA3, leu2, ura3, and trpl）. Using as controls with ^-amyrin synthase （G-AS） which is presumed to catalyze the first committed step in saikosaponin biosynthesis and a cycloartenol synthase （CAS） relating to the phytosterol biosynthesis, the transcript of BcSE1 was significantly elevated by MeJA in adventitious roots of B. chinenseand the transcript of BcSElwas most abundant in the fruits and flowers of plants, followed by that in the leaves and roots, and least in stems. Conclusion It is the first time to illustrate the molecular information of SE in B. chinense and to clone the full-length SEgene in plants of genus Bupleurum L.

Squalene epoxidase promotes colorectal cancer cell proliferation through accumulating calcitriol and activating CYP24A1-mediatedMAPK signaling

Background:Colorectal cancer(CRC)is one of the most malignant tumorswith high incidence,yet its molecular mechanism is not fully understood,hindering the development of targeted therapy.Metabolic abnormalities are a hallmark of cancer.Targeting dysregulated metabolic features has become an important direction for modern anticancer therapy.In this study,we aimed to identify a new metabolic enzyme that promotes proliferation of CRC and to examine the related molecular mechanisms.Methods:We performed RNA sequencing and tissue microarray analyses of human CRC samples to identify new genes involved in CRC.Squalene epoxidase(SQLE)was identified to be highly upregulated in CRC patients.The regulatory function of SQLE in CRC progression and the therapeutic effect of SQLE inhibitors were determined by measuring CRC cell viability,colony and organoid formation,intracellular cholesterol concentration and xenograft tumor growth.Themolecularmechanism of SQLE functionwas explored by combining transcriptome and untargeted metabolomics analysis.Western blotting and realtime PCR were used to assess MAPK signaling activation by SQLE.Results:SQLE-related control of cholesterol biosynthesis was highly upregulated in CRC patients and associated with poor prognosis.SQLE promoted CRC growth in vitro and in vivo.Inhibition of SQLE reduced the levels of calcitriol(active form of vitamin D3)and CYP24A1,followed by an increase in intracellular Ca2+concentration.Subsequently,MAPK signaling was suppressed,resulting in the inhibition of CRC cell growth.Consistently,terbinafine,an SQLE inhibitor,suppressed CRC cell proliferation and organoid and xenograft tumor growth.Conclusions:Our findings demonstrate that SQLE promotes CRC through the accumulation of calcitriol and stimulation of CYP24A1-mediated MAPK signaling,highlighting SQLE as a potential therapeutic target for CRC treatment.

Transcriptome-Wide Identification and Functional Analysis of PgSQE08-01 Gene in Ginsenoside Biosynthesis in Panax ginseng C.A.Mey.

Panax ginseng C.A.Mey.is an important plant species used in traditional Chinese medicine,whose primary active ingredient is a ginsenoside.Ginsenoside biosynthesis is not only regulated by transcription factors but also controlled by a variety of structural genes.Nonetheless,the molecular mechanism underlying ginsenoside biosynthesis has always been a topic in the discussion of ginseng secondary metabolites.Squalene epoxidase(SQE)is a key enzyme in the mevalonic acid pathway,which affects the biosynthesis of secondary metabolites such as terpenoid.Using ginseng transcriptome,expression,and ginsenoside content databases,this study employed bioinformatic methods to systematically analyze the genes encoding SQE in ginseng.We first selected six PgSQE candidates that were closely involved in ginsenoside biosynthesis and then identified PgSQE08-01 to be highly associated with ginsenoside biosynthesis.Next,we constructed the overexpression vector pCAMBIA3301-PgSQE08-01 and the RNAi vector pART27-PgSQE08-01 and transformed ginseng adventitious roots using Agrobacterium rhizogenes,to obtain positive hairy-root clones.Thereafter,quantitative reverse transcriptionpolymerase chain reaction and high-performance liquid chromatography were used to determine the expression of relevant genes and ginsenoside content,respectively.Then,we focused on the function of PgSQE08-01 gene,which was noted to be involved in ginsenoside biosynthesis.Thus,these findings not only provided a molecular basis for the identification of important functional genes in ginseng but also enriched genetic resources for the biosynthesis of ginsenosides using synthetic biology.

Mycorrhizas Affect Polyphyllin Accumulation of Paris polyphylla var.yunnanensis through Promoting PpSE Expression

Paris polyphylla var.yunnanensis is a traditional Chinese medicinal plant,in which polyphyllin as the main medicinal component is an important secondary metabolite with bioactivity.Arbuscular mycorrhizal fungi(AMF)have multiple positive effects on plants,while it is not clear whether AMF increase the content of medicinal components in medicinal plants.In this study,a total of nine AMF treatments were laid to analyze the mycorrhizal effect on polyphyllin accumulation and PpHMGR and PpSE expression of P.polyphylla var.yunnanensis.AMF increased the content of polyphyllin in the cultivated variety with low relation to the increase of inoculation intensity.Polyphyllin I,II,and VII were identified and partly improved by AMF inoculation,dependent on AMF treatments and culture environments.Similarly,the PpHMGR and PpSE expression was induced by mycorrhization,dependent on AMF species,whilst the induction was more obvious in PpSE than in PpHMGR after mycorrhization.It concluded that the symbiotic relationship between P.polyphylla var.yunnanensis and AMF increased polyphyllin content level in the plant,which was associated with the up-regulation of PpSE transcripts.

Antifungal Activity of Aspidin BB from Dryopteris fragrans against Trichophyton rubrum Involved Inhibition of Ergosterol Biosynthesis

Objectives Aspidin BB, a typical phloroglucinol derivative from Dryopteris fragrans, possesses significant antifungal property. This study aimed to investigate potential mechanism of antifungal activity of Aspidin BB against Trichophyton rubrum which is the most common pathogens responsible for chronic dermatophytosis. Methods The minimum inhibitory concentration （MIC） ofAspidin BB against strains was determined by broth microdilution. The effects of Aspidin BB on ergosterol biosynthesis were investigated by content determination based on UPLC method. Besides, the effects of drugs on squalene epoxidase （SE） in T. rubrum cell membrane were analyzed. Results MIC value of Aspidin BB against T. rubrum was 25.0 IJg/mL. Aspidin BB reduced ergosterol content significantly, but no notable effect on squalene epoxidase activity. Conclusion The results suggested that Aspidin BB inhibited ergosterol biosynthesis. However, it was not squalene epoxidase but other components may sever as possible targets in ergosterol biosynthesis pathway.

Effect of light quality on total gypenosides accumulation and related key enzyme gene expression in Gynostemma pentaphyllum

Objective： Light quality has effect on the accumulation of gypenosides in the medicinal plant Gynosternma pentaphyllum in the family Cucurbitaceae, while the squalene synthase （SS） and squalene epoxidase （SE） are the key enzymes for gypenoside biosynthesis, The objective of this study was to elucidate the rela- tionship between light quality and biosynthesis key enzyme involving the regulation of gypenoside accu- mulation. Methods： The content of total gypenosides was measured by colorimetric method and the expression of SS and SE gene was determined by quantitative Real-time PCR in the seedlings of G. pentaphyllum which were grown with different light quality. Results： Light quality showed remarkable impacts on the accumulation of total gypenosides. The highest content of total gypenosides in the plant under red light condition was determined, followed by blue light and white light, while the lowest content was recorded under dark condition, qRT-PCR analysis proved that the expression levels of SS and SE genes were also affected by light quality. The high-level gene expressions of SS and SE were found in the plant under red light condition, followed by blue light, with the least content in darkness. The statistical analysis revealed that the total gypenosides were significantly different in different light treatment and the content of total gypenosides was positively related to the expression of SS and SE genes. Conclusions： Light quality regulates gypenoside accumulation via altering the expression of SS and SE in G. pentaphyllum.