The Mevalonate Pathway Is Important for Growth, Spore Production, and the Virulence of Phytophthora sojae

The mevalonate (MVA) pathway in eukaryotic organisms produces isoprenoids, sterols, ubiquinone, and dolichols. These molecules are vital for diverse cellular functions, ranging from signaling to membrane integrity, and from post-translational modification to energy homeostasis. However, information on the MVA pathway in Phytophthora species is limited. In this study, we identified the MVA pathway genes and reconstructed the complete pathway in Phytophthora sojae in silico. We characterized the function of the MVA pathway of P. sojae by treatment with enzyme inhibitor lovastatin, deletion of the geranylgeranyl diphosphate synthase gene (PsBTS1), and transcriptome profiling analysis. The MVA pathway is ubiquitously conserved in Phytophthora species. Under lovastatin treatment, mycelial growth, spore production, and virulence of P. sojae were inhibited but the zoospore encystment rate increased. Heterozygous mutants of PsBTS1 showed slow growth, abnormal colony characteristics, and mycelial morphology. Mutants showed decreased numbers of sporangia and oospores as well as reduced virulence. RNA sequencing analysis identified the essential genes in sporangia formation were influenced by the enzyme inhibitor lovastatin. Our findings elucidate the role of the MVA pathway in P. sojae and provide new insights into the molecular mechanisms underlying the development, reproduction, and virulence of P. sojae and possibly other oomycetes. Our results also provide potential chemical targets for management of plant Phytophthora diseases.

7-Ketocholesterol Induces Lipid Metabolic Reprogramming and Enhances Cholesterol Ester Accumulation in Cardiac Cells

7-Ketocholesterol (7KCh) is a major oxidized cholesterol product abundant in lipoprotein deposits and atherosclerotic plaques. Our previous study has shown that 7KCh accumulates in erythrocytes of heart failure patients, and further investigation centered on how 7KCh may affect metabolism in cardiomyocytes. We applied metabolomics to study the metabolic changes in cardiac cell line HL-1 after treatment with 7KCh. Mevalonic acid (MVA) pathway-derived metabolites, such as farnesyl-pyrophosphate and geranylgeranyl-pyrophosphate, phospholipids, and triacylglycerols levels significantly declined, while the levels of lysophospholipids, such as lysophosphatidylcholines (lysoPCs) and lysophosphatidylethanolamines (lysoPEs), considerably increased in 7KCh-treated cells. Furthermore, the cholesterol content showed no significant change, but the production of cholesteryl esters was enhanced in the treated cells. To explore the possible mechanisms, we applied mRNA-sequencing (mRNA-seq) to study genes differentially expressed in 7KCh-treated cells. The transcriptomic analysis revealed that genes involved in lipid metabolic processes, including MVA biosynthesis and cholesterol transport and esterification, were differentially expressed in treated cells. Integrated analysis of both metabolomic and transcriptomic data suggests that 7KCh induces cholesteryl ester accumulation and reprogramming of lipid metabolism through altered transcription of such genes as sterol O-acyltransferase- and phospholipase A2-encoding genes. The 7KCh-induced reprogramming of lipid metabolism in cardiac cells may be implicated in the pathogenesis of cardiovascular diseases.

Comparative Transcriptome Analysis of Genes Involved in Sesquiterpene Alkaloid Biosynthesis in Trichoderma longibrachiatum MD33 and UN32

Trichoderma longibrachiatum MD33, a sesquiterpene alkaloid-producing endophyte isolated from Dendrobium nobile, shows potential medical and industrial applications. To understand the molecular mechanisms of sesquiterpene alkaloids production, a comparative transcriptome analysis was performed on strain MD33 and its positive mutant UN32, which was created using Ultraviolet (UV) mutagenesis and nitrogen ion (N+) implantation. The alkaloid production of UN32 was 2.62 times more than that of MD33. One thousand twenty-four differentially expressed genes (DEGs), including 519 up-regulated and 505 down-regulated genes, were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed 139 GO terms and 87 biosynthesis pathways. Dendrobine, arguably the main sesquiterpene alkaloid the strain MD33 produced, might start synthesis through the mevalonate (MVA) pathway. Several MVA pathway enzyme-coding genes (hydroxy-methylglutaryl-CoA synthase, mevalonate kinase, and farnesyl diphosphate synthase) were found to be differentially expressed, suggesting that physical mutagenesis can disrupt genome integrity and gene expression. Some backbone post-modification enzymes and transcript factors were either discovered, suggesting the sesquiterpene alkaloid metabolism in T. longibrachiatum is a complex genetic network. Our findings help to shed light on the underlying molecular regulatory mechanism of sesquiterpene alkaloids production in T. longibrachiatum.

Towards an Improvement of Anticancer Activity of Benzyl Adenosine Analogs

N6-Isopentenyladenosine (i6A) is a naturally occurring modified nucleoside displaying in vitro and in vivo antiproliferative and pro-apoptotic properties. In our previous studies, including an in silico inverse virtual screening, NMR experiments and in vitro enzymatic assays, we demonstrated that i6A targeted farnesyl pyrophosphate synthase (FPPS), a key enzyme involved in the mevalonate (MVA) pathway and prenylation of downstream proteins, which are aberrant in several cancers. Following our interest in the anticancer effects of FPPS inhibition, we developed a panel of i6A derivatives bearing bulky aromatic moieties in the N6 position of adenosine. With the aim of clarifying molecular action of N6-benzyladenosine analogs on the FPPS enzyme inhibition and cellular toxicity and proliferation, herein we report the evaluation of the N6-benzyladenosine derivatives’ (compounds 2a–m) effects on cell viability and proliferation on HCT116, DLD-1 (human) and MC38 (murine) colorectal cancer cells (CRC). We found that compounds 2, 2a and 2c showed a persistent antiproliferative effect on human CRC lines and compound 2f exerted a significant effect in impairing the prenylation of RAS and Rap-1A proteins, confirming that the antitumor activity of 2f was related to the ability to inhibit FPPS activity.

Simvastatin induced ferroptosis for triple-negative breast cancer therapy

Triple-negative breast cancer (TNBC), a management of aggressive breast cancer, remains an unmet medical challenge. Although a wave of efforts had spurred to design novel therapeutic method of TNBC, unpredictable prognosis with lacking effective therapeutic targets along with the resistance to apoptosis seriously limited survival benefits. Ferroptosis is a non-apoptotic form of cell death that is induced by excessive lipid peroxidation, which provide an innovative way to combat cancer. Emerging evidence suggests that ferroptosis plays an important role in the treatment of TNBC cells. Herein, a novel ferroptosis nanomedicine was prepared by loading simvastatin (SIM), a ferroptosis drug, into zwitterionic polymer coated magnetic nanoparticles (Fe3O4@PCBMA) to improve the therapeutic effect of TNBC. The as-obtained Fe3O4@PCBMA-SIM nanoparticles demonstrated more cytotoxicity against MDA-MB-231 than MCF-7 due to the higher expression of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), which demonstrated that statins could effectively kill TNBC. Further experiments showed that SIM could inhibit the expression of HMGCR to downregulate the mevalonate (MVA) pathway and glutathione peroxidase 4 (GPX4), thereby inducing cancer cell ferroptosis. What’s more, PCBMA endows Fe3O4@PCBMA longer blood circulation performance to enhance their accumulation at tumor sites. Given that Fe3O4 have proven for clinical applications by the U.S. Food and Drug Administration (FDA) and SIM could induce cancer cell ferroptosis, the developed Fe3O4@PCBMA-SIM nanosystem would have great potential in clinics for overcoming the drug resistance brought about by apoptotic drugs to cancer cells.

Cloning and expression analysis of mevalonate kinase and phosphomevalonate kinase genes associated with the MVA pathway in Santalum album

Sandalwood (Santalum album L.) is highly valued for its fragrant heartwood and extracted oil. Santalols, which are the main components of that oil, are terpenoids, and these are biosynthesized via the mevalonic acid (MVA) pathway. Mevalonate kinase (MK) and phosphomevalonate kinase (PMK) are key enzymes in the MVA pathway. Little is known about the genes that encode MK and PMK in S. album or the mechanism that regulates their expression. To isolate and identify the functional genes involved in santalol biosynthesis in S. album, an MK gene designated as SaMK, and a PMK gene designated as SaPMK, were cloned from S. album. The sequences of these genes were analyzed. A bioinformatics analysis was conducted to assess the homology of SaMK and SaPMK with MK and PMK genes from other plants. The subcellular localization of SaMK and SaPMK proteins was also investigated, as was the functional complementation of SaMK and SaPMK in yeast. Our results show that the full-length cDNA sequences of SaMK and SaPMK were 1409 bp and 1679 bp long, respectively. SaMK contained a 1381 bp open reading frame (ORF) encoding a polypeptide of 460 amino acids and SaPMK contained a 1527 bp ORF encoding a polypeptide of 508 amino acids. SaMK and SaPMK showed high homology with MK and PMK genes of other plant species. Functional complementation of SaMK in a MK-deficient mutant yeast strain YMR208W and SaPMK in a PMK-deficient mutant yeast strain YMR220W confirmed that cloned SaMK and SaPMK cDNA encode a functional MK and PMK, respectively, mediating MVA biosynthesis in yeast. An analysis of tissue expression patterns revealed that SaMK and SaPMK were constitutively expressed in all the tested tissues. SaMK was highly expressed in young leaves but weakly expressed in sapwood. SaPMK was highly expressed in roots and mature leaves, but weakly expressed in young leaves. Induction experiments with several elicitors showed that SaMK and SaPMK expression was upregulated by methyl jasmonate. These results will help to further study the role of MK and PMK genes during santalol biosynthesis in S. album.

Optimization of Protein Isolation and Label-Free Quantitative Proteomic Analysis in Four Different Tissues of Korean Ginseng

Korean ginseng is one of the most valuable medicinal plants worldwide. However, our understanding of ginseng proteomics is largely limited due to difficulties in the extraction and resolution of ginseng proteins because of the presence of natural contaminants such as polysaccharides, phenols, and glycosides. Here, we compared four different protein extraction methods, namely, TCA/acetone, TCA/acetone–MeOH/chloroform, phenol–TCA/acetone, and phenol–MeOH/chloroform methods. The TCA/acetone–MeOH/chloroform method displayed the highest extraction efficiency, and thus it was used for the comparative proteome profiling of leaf, root, shoot, and fruit by a label-free quantitative proteomics approach. This approach led to the identification of 2604 significantly modulated proteins among four tissues. We could pinpoint differential pathways and proteins associated with ginsenoside biosynthesis, including the methylerythritol 4–phosphate (MEP) pathway, the mevalonate (MVA) pathway, UDP-glycosyltransferases (UGTs), and oxidoreductases (CYP450s). The current study reports an efficient and reproducible method for the isolation of proteins from a wide range of ginseng tissues and provides a detailed organ-based proteome map and a more comprehensive view of enzymatic alterations in ginsenoside biosynthesis.

Expressional diversity of grapevine 3-Hydroxy-3-methylglutaryl-CoA reductase (VvHMGR) in different grapes genotypes

Background 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a key enzyme in the mevalonate (MVA) pathway, which regulates the metabolism of terpenoids in the cytoplasm and determines the type and content of downstream terpenoid metabolites. Results Results showed that grapevine HMGR family has three members, such as VvHMGR1, VvHMGR2, and VvHMGR3. The expression of VvHMGRs in 'Kyoho' has tissue specificity, for example, VvHMGR1 keeps a higher expression, VvHMGR2 is the lowest, and VvHMGR3 gradually decreases as the fruit development. VvHMGR3 is closely related to CsHMGR1 and GmHMGR9 and has collinearity with CsHMGR2 and GmHMGR4. By the prediction of interaction protein, it can interact with HMG-CoA synthase, MVA kinase, FPP/GGPP synthase, diphosphate mevalonate decarboxylase, and participates in the synthesis and metabolism of terpenoids. VvHMGR3 have similar trends in expression with some of the genes of carotenoid biosynthesis and MEP pathways. VvHMGR3 responds to various environmental and phytohormone stimuli, especially salt stress and ultraviolet (UV) treatment. The expression level of VvHMGRs is diverse in grapes of different colors and aroma. VvHMGRs are significantly higher in yellow varieties than that in red varieties, whereas rose-scented varieties showed significantly higher expression than that of strawberry aroma. The expression level is highest in yellow rose-scented varieties, and the lowest in red strawberry scent varieties, especially ‘Summer Black’ and ‘Fujiminori’. Conclusion This study confirms the important role of VvHMGR3 in the process of grape fruit coloring and aroma formation, and provided a new idea to explain the loss of grape aroma and poor coloring during production. There may be an additive effect between color and aroma in the HMGR expression aspect.

Mechanism of Mutant p53 Using Three-Dimensional Culture on Breast Cancer Malignant Phenotype via SREBP-Dependent Cholesterol Synthesis Pathway

In many cancers, including hormone sensitive tumors such as breast cancer, the “gain of function” caused by mutations in the tumor suppressor gene p53 plays an important role in the acquisition of malignant phenotypes and the regulation of cancer stem cell characteristics. However, its action of molecular mechanisms, particularly in vivo conditions, has not been fully clarified. Therefore, we focused on the “gain of function” of mutant p53 and the cholesterol biosynthesis pathway, especially the mevalonate(MVA) pathway, using breast cancer cells, and clarified the interaction between them and the relationship with cancer malignancy using 3D-culture. Here, we generated knock out and knock in breast cancer cell lines for p53 using CRISPR-Cas9 system, and then confirmed malignant morphological changes by 3D-culture model. We found that the introduction of mutant p53 was solely able to mediate the malignant transformation of cancer. Next, focusing on the relationship between cancer malignant transformation and lipid metabolism pathway, we investigated the role of the MVA pathway in malignant transformation by mutation p53. When investigating the effects of the addition of HMG-CoA inhibitors and isoprenoids, intermediate metabolites were important for malignant transformation during 3D culture. Furthermore, knockdown of SREBP2, which controls the MVA pathway, suppressed malignant phenotypes, so we proceeded with analysis of the interaction between mutant p53 and SREBP2. As the result, we found that mutant p53 and SREBP2 co-localize in the nucleus and consistently mutant p53 was associated with mevalonate pathway genes in parallel with binding pattern of SREBP2. Thus, our results provide the novel insight into the potential therapeutic targets for breast cancer with poor prognosis.

Farnesyl pyrophosphate is a new danger signal inducing acute cell death

Cell death is a vital event in life. Infections and injuries cause lytic cell death, which gives rise to danger signals that can further induce cell death, inflammation, and tissue damage. The mevalonate (MVA) pathway is an essential, highly conserved and dynamic metabolic pathway. Here, we discover that farnesyl pyrophosphate (FPP), a metabolic intermediate of the MVA pathway, functions as a newly identified danger signal to trigger acute cell death leading to neuron loss in stroke. Harboring both a hydrophobic 15-carbon isoprenyl chain and a heavily charged pyrophosphate head, FPP leads to acute cell death independent of its downstream metabolic pathways. Mechanistically, extracellular calcium influx and the cation channel transient receptor potential melastatin 2 (TRPM2) exhibit essential roles in FPP-induced cell death. FPP activates TRPM2 opening for ion influx. Furthermore, in terms of a mouse model constructing by middle cerebral artery occlusion (MCAO), FPP accumulates in the brain, which indicates the function of the FPP and TRPM2 danger signal axis in ischemic injury. Overall, our data have revealed a novel function of the MVA pathway intermediate metabolite FPP as a danger signal via transient receptor potential cation channels.