Enzyme In Biosynthesis Pathway Research Articles

N-Methyl Protoporphyrin IX: An Understudied Porphyrin.

N-Methyl protoporphyrin IX (NmePPIX) is a derivative of protoporphyrin IX (PPIX) and the lattice of heme. Certain xenobiotics strongly induce NmePPIX production in the liver. The existence of endogenous NmePPIX in untreated animal liver has also been reported. The detailed mechanisms of NmePPIX biosynthesis remain unclear, but cytochrome P450 enzymes are thought to be critical in xenobiotic-induced NmePPIX production. High levels of NmePPIX cause PPIX accumulation because NmePPIX is a potent inhibitor (Ki = 7 nM) of ferrochelatase, the last enzyme in the heme biosynthesis pathway that converts PPIX to heme. NmePPIX is also involved in several other physiological processes, including inhibition of nitric oxide production and promotion of lamin aggregation. Compared to the two well-characterized porphyrins, PPIX and heme, NmePPIX is understudied regarding the mechanism of formation, fate, and physiological functions. This Review summarizes the current understanding of NmePPIX and provides perspectives on areas of future research on NmePPIX.

Transcriptome reveals BCAAs biosynthesis pathway is influenced by lovastatin and can act as a potential control target in Phytophthora sojae.

Lovastatin has been indicated to impair growth and development of Phytophthora sojae. Therefore, this study was performed to understand the inhibitory mechanism of lovastatin and investigate the metabolic pathway potentially served as a new control target for this plant pathogen. Whole transcriptome analysis of lovastatin-treated P. sojae was performed by RNA-sequencing. The results revealed that 84 genes were upregulated and 58 were downregulated with more than fourfold changes under treatment. Kyoto Encyclopaedia of Genes and Genomes analysis indicated that the branched-chain amino acids (BCAAs) biosynthesis pathway was abundantly enriched. All enzymes in the BCAAs biosynthesis pathway were identified in the P. sojae genome. Moreover, the study found that the herbicide flumetsulam targeting acetohydroxyacid synthase (AHAS) of the BCAAs biosynthesis pathway could effectively inhibit mycelial growth of P. sojae. Lovastatin treatment significantly influences the BCAAs biosynthesis pathway in P. sojae. Moreover, the herbicide flumetsulam targets AHAS and inhibits growth of P. sojae. The present study revealed that BCAAs biosynthesis pathway was influenced by lovastatin treatment and its key enzyme AHAS was identified as a potential new control target, which provides clues for exploring more oomycetes to control plant diseases caused by P. sojae.

Analysis of the Antioxidant Mechanism of Tamarix ramosissima Roots under NaCl Stress Based on Physiology, Transcriptomic and Metabolomic.

There is a serious problem with soil salinization that affects the growth and development of plants. Tamarix ramosissima Ledeb (T. ramosissima), as a halophyte, is widely used for afforestation in salinized soils. At present, there are few reports on the antioxidant mechanism of T. ramosissima under NaCl stress. In this study, we learned about the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and hydrogen peroxide (H2O2) and malondialdehyde (MDA) content changes in T. ramosissima. We also mined the relevant metabolic pathways in the antioxidant mechanism, candidate key genes, and their related differential metabolites and verified them using quantitative real-time PCR (qRT-PCR). The results show that the SOD, POD, and CAT activities, and the H2O2 and MDA content reached the highest values in the roots of T. ramosissima. Simultaneously, 92 differentially expressed genes (DEGs) related to antioxidant enzyme activities changed during 48 and 168 h of NaCl stress, and these DEGs were mainly upregulated in 168 h. Based on the association analysis of transcriptomic and metabolomic data, we found Unigene0089358 and Unigene0007782 as genes related to key enzymes in the flavonoid biosynthesis pathway. They were located in the upstream positive regulation at 48 and 168 h under NaCl stress, and their respective related metabolites (phloretin and pinocembrin) were involved in resistance to NaCl stress, and they were significantly correlated with their respective metabolites. In conclusion, at 48 and 168 h under NaCl stress, the roots of T. ramosissima resist NaCl stress by enhancing enzymatic and nonenzymatic antioxidant mechanisms, scavenging ROS generated by high-salt stress, alleviating NaCl toxicity, and maintaining the growth of T. ramosissima. This study provides genetic resources and a scientific theoretical basis for further breeding of salt-tolerant Tamarix plants and the molecular mechanism of antioxidants to alleviate NaCl toxicity.

Effects of Inhibiting the Expression of Chitin Synthase Gene SfCHSB on the Metabolism of Trehalose and Chitin in Spodoptera frugiperda Larvae

Spodoptera frugiperda is one of the world’s major agricultural pests and it occurs in many countries around the world. In Lepidoptera, the peritrophic membrane in the midgut protects the midgut epithelial cells and facilitates the digestion and absorption of food. Its main components are chitin and protein. Chitin synthase (CHS), as the last enzyme in the chitin biosynthesis pathway, is very important. Here, the expression of chitin synthase B (CHSB) gene identified from midgut was inhibited by RNA interference to explore its function in the metabolism of trehalose and chitin of S. frugiperda larvae. The results were as follows: SfCHSB was highly expressed in actively feeding larvae. Second, knockdown of SfCHSB resulted in decreased expression of most genes involved in chitin metabolism, including chitinase, and abnormal phenotypes of S. frugiperda were observed. In addition, membrane-bound trehalase activity and glucose content increased, and trehalose content decreased at 24 h after dsSfCHSB injection. Trehalase activities increased significantly and the glycogen content decreased at 48 h after dsSfCHSB injection. Finally, the activity of chitinase decreased and the content of chitin increased significantly. Our results indicate that SfCHSB expression is specific, SfCHSB has a large role in regulating chitin metabolism, and it has broad application prospects in the biocontrol of S. frugiperda.

Phenylalanine Ammonia Lyase GmPAL1.1 Promotes Seed Vigor under High-Temperature and -Humidity Stress and Enhances Seed Germination under Salt and Drought Stress in Transgenic Arabidopsis

Seed vigor is an important agronomic attribute, essentially associated with crop yield. High-temperature and humidity (HTH) stress directly affects seed development of plants, resulting in the decrease of seed vigor. Therefore, it is particularly important to discover HTH-tolerant genes related to seed vigor. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24) is the first rate-limiting enzyme in the phenylpropanoid biosynthesis pathway and a key enzyme involved in plant growth and development and environmental adaptation. However, the biological function of PAL in seed vigor remains unknown. Here, GmPAL1.1 was cloned from soybean, and its protein was located in the cytoplasm and cell membrane. GmPAL1.1 was significantly induced by HTH stress in developing seeds. The overexpression of GmPAL1.1 in Arabidopsis (OE) accumulated lower level of ROS in the developing seeds and in the leaves than the WT at the physiological maturity stage under HTH stress, and the activities of SOD, POD, and CAT and flavonoid contents were significantly increased, while MDA production was markedly reduced in the leaves of the OE lines than in those of the WT. The germination rate and viability of mature seeds of the OE lines harvested after HTH stress were higher than those of the WT. Compared to the control, the overexpression of GmPAL1.1 in Arabidopsis enhanced the tolerance to salt and drought stresses during germination. Our results suggested the overexpression of GmPAL1.1 in Arabidopsis promoted seed vigor at the physiological maturation period under HTH stress and increased the seeds' tolerance to salt and drought during germination.

Functional verification and characterization of a type-III geranylgeranyl diphosphate synthase gene from Sporobolomyces pararoseus NGR.

Carotenoids, a group of natural pigments, have strong antioxidant properties and act as precursors to vitamin A, which have garnered attention from industry and researchers. Sporobolomyces pararoseus represents a hyper-producer of carotenoids, mainly including β-carotene, torulene, and torularhodin. Geranylgeranyl diphosphate synthase (GGPPS) is regarded as a key enzyme in the carotenoid biosynthesis pathway. However, the precise nature of the gene encoding GGPPS in S. pararoseus has not been reported yet. Here, we cloned a cDNA copy of the GGPPS protein-encoding gene crtE from S. pararoseus NGR. The crtE full-length genomic DNA and cDNA are 1,722 and 1,134 bp, respectively, which consist of 9 exons and 8 introns. This gene encodes 377 amino acids protein with a predicted molecular mass of 42.59 kDa and a PI of 5.66. Identification of the crtE gene encoding a functional GGPPS was performed using heterologous complementation detection in Escherichia coli. In vitro enzymatic activity experiments showed that CrtE utilized farnesyl diphosphate (FPP) as an allylic substrate for the condensation reaction with isopentenyl diphosphate (IPP), generating more of the unique product GGPP compared to other allylic substrates. The predicted CrtE 3D-model was analyzed in comparison with yeast GGPPS. The condensation reaction occurs in the cavity of the subunit, and three bulky amino acids (Tyr110, Phe111, and His141) below the cavity prevent further extension of the product. Our findings provide a new source of genes for carotenoid genetic engineering.

DhMYB2 and DhbHLH1 regulates anthocyanin accumulation via activation of late biosynthesis genes in Phalaenopsis-type Dendrobium.

Phalaenopsis-type Dendrobium is a popular orchid with good ornamental and market value. Despite their popularity, molecular regulation of anthocyanin biosynthesis during flower development remains poorly understood. In this study, we systematically investigated the regulatory roles of the transcription factors DhMYB2 and DhbHLH1 in anthocyanins biosynthesis. Gene expression analyses indicated that both DhMYB2 and DhbHLH1 are specifically expressed in flowers and have similar expression patterns, showing high expression in purple floral tissues with anthocyanin accumulation. Transcriptomic analyses showed 29 differentially expressed genes corresponding to eight enzymes in anthocyanin biosynthesis pathway have similar expression patterns to DhMYB2 and DhbHLH1, with higher expression in the purple lips than the yellow petals and sepals of Dendrobium 'Suriya Gold'. Further gene expression analyses and Pearson correlation matrix analyses of Dendrobium hybrid progenies revealed expression profiles of DhMYB2 and DhbHLH1 were positively correlated with the structural genes DhF3'H1, DhF3'5'H2, DhDFR, DhANS, and DhGT4. Yeast one-hybrid and dual-luciferase reporter assays revealed DhMYB2 and DhbHLH1 can bind to promoter regions of DhF3'H1, DhF3'5'H2, DhDFR, DhANS and DhGT4, suggesting a role as transcriptional activators. These results provide new evidence of the molecular mechanisms of DhMYB2 and DhbHLH1 in anthocyanin biosynthesis in Phalaenopsis-type Dendrobium.

STEM-21. REPURPOSING CLEMASTINE TO SUPPRESS GLIOBLASTOMA STEM CELLS

Abstract Glioblastoma stem cells (GSCs), also known as brain tumor initiating cells (BTICs), drive tumor progression, heterogeneity, and resistance to treatments, posing formidable challenges to advancing effective treatments against glioblastoma (GBM). We postulated that inducing BTIC differentiation can serve as a solution to diminishing their stem-like features. Here, we report that clemastine, an over-the-counter oral medication for allergy relief, attenuates stemness and proliferation of BTICs. These effects of clemastine were accompanied by altered transcriptional programs suggestive of a shift from maintaining stem cell identity to differentiation, resonating with the ability of clemastine to promote oligodendrocyte precursor cell (OPC) differentiation to mature oligodendrocytes. Genetic perturbation and small-molecule inhibition of putative pharmacological targets of clemastine revealed that Emopamil-binding protein (EBP), an enzyme in the sterol biosynthesis pathway, played a pivotal role in mediating the differentiating and anti-tumor effects of clemastine. Notably, loss-of-function assays showed that EBP expression was indispensable for BTIC propagation. In contrast, overexpression of EBP stimulated BTIC proliferation, thus uncovering a previously unknown role of sterol metabolism in BTIC maintenance. Finally, we demonstrated that a mouse neural stem cell-derived glioma model was similarly susceptible to clemastine treatment. Taken together, our study identifies pathways essential for the perpetuation of stemness in GBM, and implicates a non-oncology drug with a well-established safety profile that can be repurposed to mitigate the stem-like properties of GBM.

Identification of natural small molecule modulators of MurB from Salmonella enterica serovar Typhi Ty2 strain using computational and biophysical approaches.

The increase of antibiotic-resistant bacterial pathogens has created challenges in treatment and warranted the design of antibiotics against comparatively less exploited targets. The peptidoglycan (PG) biosynthesis delineates unique pathways for the design and development of a novel class of drugs. Mur ligases are an essential component of bacterial cell wall synthesis that play a pivotal role in PG biosynthesis to maintain internal osmotic pressure and cell shape. Inhibition of these enzymes can interrupt bacterial replication and hence, form attractive targets for drug discovery. In the present work, we focused on the PG biosynthesis pathway enzyme, UDP-N-acetylpyruvylglucosamine reductase, from Salmonella enterica serovar Typhi (stMurB). Biophysical characterization of purified StMurB was performed to gauge the molecular interactions and estimate thermodynamic stability for determination of attributes for possible therapeutic intervention. The thermal melting profile of MurB was monitored by circular dichroism and validated through differential scanning calorimetry experiment. Frequently used chemical denaturants, GdmCl and urea, were employed to study the chemical-induced denaturation of stMurB. In the search for natural compound-based inhibitors, against this important drug target, an in silico virtual screening based investigation was conducted with modeled stMurB structure. The three top hits (quercetin, berberine, and scopoletin) returned were validated for complex stability through molecular dynamics simulation. Further, fluorescence binding studies were undertaken for the selected natural compounds with stMurB alone and with NADPH bound form. The compounds scopoletin and berberine, displayed lesser binding to stMurB whereas quercetin exhibited stronger binding affinity than NADPH. This study suggests that quercetin can be evolved as an inhibitor of stMurB enzyme.

AML-144 Uroporphyrinogen Decarboxylase (UROD) Is a Therapeutically Targetable Dependency in Acute Erythroid Leukemia

Acute erythroid leukemia (AEL) is a disease of erythroid lineage that commonly exhibits TP53 mutations, a complex karyotype, and poor prognosis irrespective of currently available therapies. To identify AEL dependencies and novel therapeutic targets in AEL using genome-wide CRISPR/Cas9 knockout screens in mouse models of AEL. Analysis of the screens performed using the Brie library identified the Urod gene encoding uroporphyrinogen decarboxylase as a critical dependency in two Trp53/Bcor mutated mouse AEL cell lines. UROD is a cytosolic enzyme in the heme biosynthesis pathway involved in converting cytosolic uroporphyrinogen III into the key heme biosynthetic intermediate, coproporphyrinogen III. In an analysis of RNA-seq data from over 2,000 acute leukemia samples, UROD was significantly overexpressed and active by a data-driven network inference algorithm in AEL compared to other leukemia subtypes. We validated UROD as a dependency by electroporation of Cas9 and synthetic sgRNA ribonucleoprotein complex in the two screened cell lines, additional mouse leukemia cell lines, and two commercially available human AEL cell lines. Targeted knockout of UROD resulted in decreased tumor cell growth and viability compared to the non-targeting guide control cells. Sequencing analysis showed high editing efficiency (>85%) followed by recovery of the wildtype cells, indicating that UROD is a dependency in these cells. These results correlated with a significant increase of necrotic cells preceding loss of editing in the two tested human cell lines. We also observed an increase in ALAS1 expression, which is negatively regulated by cellular heme, indicating that UROD knockout leukemic cells cannot generate heme, leading to necrotic cell death. Moreover, mitochondrial function assays showed significant decreases in basal respiration, ATP production, maximal respiration, and spare capacity in a time-dependent manner, followed by the rescue of mitochondrial function after loss of editing efficiency and re-expression of UROD. We have recently developed homozygous UROD-dTAG engineered human AEL cell lines to perform in-vivo survival studies. Through whole-genome CRISPR knockout screening and functional studies, we show that UROD is a dependency in AEL and provides a basis for exploring its therapeutic inhibition.

Analysis of Cyp51 protein sequences shows 4 major Cyp51 gene family groups across fungi.

Azole drugs target fungal sterol biosynthesis and are used to treat millions of human fungal infections each year. Resistance to azole drugs has emerged in multiple fungal pathogens including Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, and Aspergillus fumigatus. The most well-studied resistance mechanism in A. fumigatus arises from missense mutations in the coding sequence combined with a tandem repeat in the promoter of cyp51A, which encodes a cytochrome P450 enzyme in the fungal sterol biosynthesis pathway. Filamentous members of Ascomycota such as A. fumigatus have either 1 or 2 of 3 Cyp51 paralogs (Cyp51A, Cyp51B, and Cyp51C). Most previous research in A. fumigatus has focused on Cyp51A due to its role in azole resistance. We used the A. fumigatus Cyp51A protein sequence as the query in database searches to identify Cyp51 proteins across fungi. We found 435 Cyp51 proteins in 295 species spanning from early-diverging fungi (Blastocladiomycota, Chytridiomycota, Zoopagomycota, and Mucormycota) to late-diverging fungi (Ascomycota and Basidiomycota). We found these sequences formed 4 major Cyp51 groups: Cyp51, Cyp51A, Cyp51B, and Cyp51C. Surprisingly, we found all filamentous Ascomycota had a Cyp51B paralog, while only 50% had a Cyp51A paralog. We created maximum likelihood trees to investigate the evolution of Cyp51 in fungi. Our results suggest Cyp51 is present in all fungi with 3 paralogs emerging in Pezizomycotina, including Cyp51C which appears to have diverged from the progenitor of the Cyp51A and Cyp51B groups.

Identification of Novel Natural Inhibitors to Human 3-Phosphoglycerate Dehydrogenase (PHGDH) for Cancer Treatment.

Targeting the serine biosynthesis pathway enzymes has turned up as a novel strategy for anti-cancer therapeutics. 3- Phosphoglycerate dehydrogenase (PHGDH) is the rate-limiting enzyme that catalyzes the conversion of 3-Phosphoglyceric acid (3-PG) into 3-Phosphohydroxy pyruvate (3-PPyr) in the first step of serine synthesis pathway and perform a critical role in cancer progression. PHGDH has been reported to be overexpressed in different types of cancers and emerged as a novel target for cancer therapeutics. During this study, virtual screening tools were used for the identification of inhibitors of PHGDH. A library of phenolic compounds was docked against two binding sites of PHGDH using Molegro Virtual Docker (MVD) software. Out of 169 virtually tested compounds, Salvianolic acid C and Schizotenuin F possess good binding potential to co-factor binding site of PHGDH while Salvianolic acid I and Chicoric acid were identified as the best binding compounds toward the substrate binding site of PHGDH. The top selected compounds were evaluated for different physiochemical and ADMET properties, the obtained results showed that none of these hit compounds violated the Pfizer Rule and they possess acceptable ADMET profiles. Further, a commercially available hit compound, Chicoric acid, was evaluated for its anti-cancer potential against PHGDH-expressing gastric cancer cell lines (MGC-803 and SGC-7901) as well as cell lines with low expression of PHGDH (MCF-7 and MDA-MB2-31), which demonstrated that Chicoric acid possesses selective cytotoxicity toward PHGDH expressing cancer cell lines. Thus, this study has unveiled the potential of phenolic compounds, which could serve as novel candidates for the development of PHGDH inhibitors as anti-cancer agents.

A novel formamidase is required for riboflavin biosynthesis in invasive bacteria

Biosynthesis of riboflavin (RF), the precursor of the redox cofactors FMN and FAD, was thought to be well understood in bacteria, with all the pathway enzymes presumed to be known and essential. Our previous research has challenged this view by showing that, in the bacterium Sinorhizobium meliloti, deletion of the ribBA gene encoding the enzyme that catalyzes the initial steps on the RF biosynthesis pathway only causes a reduction in flavin secretion rather than RF auxotrophy. This finding led us to hypothesize that RibBA participates in the biosynthesis of flavins destined for secretion, whereas S. meliloti has another enzyme that performs this function for internal cellular metabolism. Here, we identify and biochemically characterize a novel formamidase (SMc02977) involved in the production of RF for intracellular functions in S. meliloti. This catalyst, which we named Sm-BrbF, releases formate from the early RF precursor 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate to yield 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate. We show that homologs of this enzyme are present in many bacteria, are highly abundant in the Rhizobiales order, and that sequence homologs from Brucella abortus and Liberobacter solanacearum complement the RF auxotrophy of the Sm1021ΔSMc02977 mutant. Furthermore, we show that the B. abortus enzyme (Bab2_0247, Ba-BrbF) is also an 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate formamidase, and that the bab2_0247 mutant is a RF auxotroph exhibiting a lower level of intracellular infection than the wildtype strain. Finally, we show that Sm-BrbF and Ba-BrbF directly interact with other RF biosynthesis pathway enzymes. Together, our results provide novel insight into the intricacies of RF biosynthesis in bacteria.

Identification of the 12-oxophytoeienoic acid reductase (OPR) gene family in pepper (Capsicum annuum L.) and functional characterization of CaOPR6 in pepper fruit development and stress response.

The 12-oxophytoeienoic acid reductase (OPR) is a kind of enzyme in the octadecanoid biosynthesis pathway that determines the biosynthesis of jasmonic acid. Although the roles of OPRs have been extensively studied in several crop plants, little is known about the biological functions of OPR-encoding genes in Capsicum annuum plants. In this study, seven OPR family genes (CaOPR1-7) were identified from the C. annuum genome. The physical and chemical properties of CaOPR1-7 were further analyzed, including gene expression patterns, promoter elements, and chromosomal locations. The results showed that the seven CaOPR homologues could be divided into two subgroups, and CaOPR6 was highly similar to AtOPR3 in Arabidopsis. The expression of CaOPR6 was significantly induced by various stresses such as cold, salt, and pathogen infection, indicating that CaOPR6 plays important roles in response to abiotic and biotic stresses. Overall, these findings improve the understanding of the biological functions of CaOPR6 in the development of pepper fruit and stress response of pepper plants, and facilitate further studies on the molecular biology of OPR proteins in Solanaceae vegetables.

Metabolic engineering of the diosgenin biosynthesis pathway in Trigonella foenum-graceum hairy root cultures

Diosgenin as a triterpene with numbers of pharmaceutical applications has been identified in Trigonella foenum-graceum. In this survey, in order to scale up the amount of diosgenin in Fenugreek as a promising alternative of yam, ∆24-reductase as a rate limiting enzyme in diosgenin biosynthesis pathway has been overexpressed by utilizing pBI121 expression plasmid in hairy roots culture platform. The recombinant binary vector pBI121-∆24-reductase was transformed into R. rhizogenes strain ATCC 15834 to induce transgenic hairy roots in “Hamedan” as a low-diosgenin production genotype. In the transgenic hairy roots, the ∆24-reductase expression level was significantly 8.15 times overexpressed comparing to the non-transgenic hairy roots, Nonetheless the Sterol-methyltransferase, as a competitive enzyme, was 6 times downregulated. Furthermore, the expression rate of Squalene synthase, Cycloartenol synthase, C26-Hydroxylase were also increased 1.5, 1.7, 2.9 times higher than those of the non-transgenic hairy roots, respectively. The diosgenin content in the transgenic hairy root was raised 3 times up comparing to the non-transgenic hairy roots, besides it was scaled up 25-fold comparing to the diosgenin amount in “Hamedan” Leaf. As a result, the first metabolic engineering on this pathway was clearly revealed the impact of ∆24 -reductase gene in diosgenin content enhancement.

Abstract P3051: Yap Regulates Serine Biosynthesis During Compensatory Cardiac Hypertrophy

Introduction: In response to increased workload, cardiomyocytes increase in size, and the heart undergoes hypertrophy. The initial hypertrophy is a compensatory process that adds contractile units but reduces wall stress to help maintain heart function. Yes-associated protein (YAP), a major effector of the Hippo pathway, is transiently activated in response to acute pressure overload (PO). Cardiac-specific heterozygous downregulation of YAP inhibits hypertrophy but promotes heart failure, suggesting that endogenous YAP is salutary and mediates compensatory hypertrophy during the acute phase of PO. However, the mechanism by which YAP mediates compensatory hypertrophy is not well understood. We have shown previously that 3-phosphoglyceric acid and serine accumulate in the heart during acute PO in a YAP-dependent manner. The serine biosynthetic pathway provides precursors for the TCA cycle and antioxidant and purine biosynthesis. Hypothesis: YAP induces serine synthesis, thereby promoting compensatory cardiac hypertrophy in the presence of acute PO. Methods and Results: We evaluated the levels of intermediate enzymes in the serine synthesis pathway in YAP-overexpressing neonatal rat ventricular myocytes (NRVMs). Furthermore, we investigated whether YAP-induced cardiomyocyte hypertrophy is inhibited by siRNA targeting serine biosynthesis enzymes. Immunofluorescent staining of NRVMs with cardiac troponin T was conducted to evaluate cardiomyocyte size, which was assessed using ImageJ software. YAP upregulated protein levels of phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), phosphoserine phosphatase (PSPH), and serine hydroxymethyltransferase 1 (SHMT1) in NRVMs. siRNA-mediated downregulation of PHGDH, PSAT1, PSPH, or SHMT1 significantly inhibited YAP-induced cardiomyocytes hypertrophy. Conclusions: YAP upregulates the expression of enzymes in the serine biosynthesis pathway, thereby inducing hypertrophy in NRVMs. Metabolites of the serine biosynthesis pathway may play an important role in mediating YAP-induced compensatory cardiac hypertrophy.

MetA is a "thermal fuse" that inhibits growth and protects Escherichia coli at elevated temperatures.

Adaptive stress resistance in microbes is mostly attributed to the expression of stress response genes, including heat-shock proteins. Here, we report a response of E.coli to heat stress caused by degradation of an enzyme in the methionine biosynthesis pathway (MetA). While MetA degradation can inhibit growth, which by itself is detrimental for fitness, we show that it directly benefits survival at temperatures exceeding 50°C, increasing survival chances by more than 1,000-fold. Using both experiments and mathematical modeling, we show quantitatively how protein expression, degradation rates, and environmental stressors cause long-term growth inhibition in otherwise habitable conditions. Because growth inhibition can be abolished with simple mutations, namely point mutations of MetA and protease knockouts, we interpret the breakdown of methionine synthesis as a system that has evolved to halt growth at high temperatures, analogous to "thermal fuses" in engineering that shut off electricity to prevent overheating.

In Silico Design Gene Encoding CYP71AV1 for Expression in Escherichia coli

Cytochrome P450 monooxygenase (CYP71AV1) is a crucial enzyme in the artemisinin biosynthesis pathway. This enzyme oxidized Amorpha 4,11 diene to produce artemisinic acid. This study aimed to in silico design high-level expression of CYP71AV1 in the E.coli system. In silico techniques are highly suitable for designing protein recombinant production before entering the laboratory. The amino acid sequence of CYP71AV1 was back-translated to the DNA sequence and adapt to E.coli codon usage by using Gene Designer. The DNA sequence of optimized CYP71AV1 was analyzed using Rare Codon Analysis to assess the expression efficiency in E.coli. The protein solubility prediction was determined using the SoDoPe tool. The optimized CYP71AV1 was determined to have a CAI 0.81, a GC content of 53.08 %, CFD with low frequency, and no negative cis or repeat elements. The result of the probability of solubility of CYP71AV1 was 0.6207 when expressed in E.coli. The MBP fusion partner can be used to increase the solubility of CYP71AV1. The in silico results showed the possibility of high-level protein expression of optimized CYP71AV1 in the E.coli system.

Molecular Characterization of UDP-N-Acetylglucosamine Pyrophosphorylase and Its Role in the Growth and Development of the White-Backed Planthopper Sogatella furcifera (Hemiptera: Delphacidae).

UDP-N-acetylglucosamine pyrophosphorylase (UAP) is a key enzyme in the chitin biosynthesis pathway of insects. Here, we described the gene SfUAP in the white-backed planthopper Sogatella furcifera (Horváth) with an open reading frame of 1470 bp. Quantitative real-time polymerase chain reaction (qPCR) suggested that SfUAP exhibits a different developmental expression pattern and a higher expression after molting. The highest expression of SfUAP was observed in the integument tissues of adults, whereas head tissues showed negligible expression. RNAi-based gene silencing decreased the mRNA transcript levels in S. furcifera nymphs injected with double-stranded RNA of SfUAP. Finally, SfUAP silencing led to 84% mortality and malformed phenotypes in nymphs. Thus, our results can help better understand the role of SfUAP in S. furcifera.

Inferred ancestry of scytonemin biosynthesis proteins in cyanobacteria indicates a response to Paleoproterozoic oxygenation.

Protection from radiation damage is an important adaptation for phototrophic microbes. Living in surface, shallow water, and peritidal environments, cyanobacteria are especially exposed to long-wavelength ultraviolet (UVA) radiation. Several groups of cyanobacteria within these environments are protected from UVA damage by the production of the pigment scytonemin. Paleontological evidence of cyanobacteria in UVA-exposed environments from the Proterozoic, and possibly as early as the Archaean, suggests a long evolutionary history of radiation protection within this group. We show that phylogenetic analyses of enzymes in the scytonemin biosynthesis pathway support this hypothesis and reveal a deep history of vertical inheritance of this pathway within extant cyanobacterial diversity. Referencing this phylogeny to cyanobacterial molecular clocks suggests that scytonemin production likely appeared during the early Proterozoic, soon after the Great Oxygenation Event. This timing is consistent with an adaptive scenario for the evolution of scytonemin production, wherein the threat of UVA-generated reactive oxygen species becomes significantly greater once molecular oxygen is more pervasive across photosynthetic environments.