De-novo Biosynthesis Research Articles

De Novo Biosynthesis of 2-Phenylethanol by Metabolic Engineering the Oleaginous Yeast Rhodotorula toruloides.

Microbial production offers a sustainable route to plant- and chemical-based manufacturing. 2-Phenylethanol (2-PE) has been widely used in foods, flavors, and pharmaceuticals. Herein, de novo biosynthesis of 2-PE was achieved for the first time by rewiring the non-native producer Rhodotorula toruloides. Our results showed that the generation of phenylalanine (Phe) and the recycling of the amino group could be bypassed since the Ehrlich pathway outperformed the phenylacetaldehyde-dependent one in de novo biosynthesis of 2-PE when the adenosine 5'-triphosphate (ATP) citrate synthase (ACL) was inactivated. The 2-PE titer was enhanced to 151.7 mg/L in a shake flask by alleviating feedback inhibition, enhancing precursor availability and cofactor balance. Finally, the production of 2-PE was elevated to 1.06 g/L with a yield of 8.5 mg/g glucose and productivity of 8 mg/L/h in a 3 L bioreactor. Our results should shed light on the microbial production of other aromatic derivatives with R. toruloides.

De Novo Biosynthesis of a Bioactive Meroterpene Bakuchiol in Yeast.

Bakuchiol (BAK), a specialized meroterpene, is known for its valuable biological properties and has recently gained prominence in cosmetology for its retinol-like functionality. However, low abundance in natural sources leads to environmentally unfriendly and unsustainable practices associated with crop-based manufacturing and chemical synthesis. Here, we identified a prenyltransferase (PT) from Psoralea corylifolia that catalyzes the reverse geranylation of a nonaromatic carbon in para-coumaric acid (p-CA), coupled with a decarboxylation step to form BAK. Given that the biosynthesis pathway of BAK is well elucidated, we engineered Saccharomyces cerevisiae to produce BAK, starting from glucose. To enhance the titer of BAK, we employed a multifaceted approach that included increasing the supply of precursors, balancing the fluxes in the two parallel biosynthetic pathways, engineering of prenyltransferase, and fusing enzymes. Consequently, the engineered yeast strains showed a marked improvement of 117.3-fold in BAK production, reaching a titer of 9.28 mg/L from glucose. Our work provides a viable approach for the sustainable microbial production of complex natural meroterpenes.

De Novo Biosynthesis of L-Azetidine-2-Carboxylic Acid in Escherichia coli Strains for Powdery Mildew Treatment.

L-Azetidine-2-carboxylic acid (L-Aze), a natural nonproteinogenic amino acid found widely in plants, has recently been identified as an environmentally friendly agent for controlling powdery mildew with low toxicity. In this study, a biological route for L-Aze production via the methionine salvage pathway (Yang Cycle) was first in silico designed for Escherichia coli. Subsequently, systematic engineering strategies were employed to enhance the production efficiency, including the enhancement of the 5-phosphoribosyl 1-pyrophosphate (PRPP) supply, construction of the ATP-adenine cycle, and engineering of the strain's resistance to L-Aze. The final strain produced L-Aze from glucose with a titer of 568.5 mg/L. The antifungal activity of the produced L-Aze in the fermentation broth was also confirmed for treating powdery mildew in cucurbits. This approach not only provides a sustainable and green route for pesticide production to control powdery mildew but also expands our understanding of the exogenous construction of the Yang Cycle in E. coli.

Abstract Mo109: Importance of Myocyte Serine De Novo Biosynthesis and its Downregulation in Heart Failure

Introduction: The failing heart exhibits metabolic inflexibility and dysregulated metabolism. Serine is a non-essential amino acid that contributes to purine synthesis, redox, and amino acid synthesis. It is transported into cells from the circulation or formed by de novo synthesis (SdS). We have found human failing myocardium has reduced serine (Circ 2023;147:1147) and lower expression of SdS genes (Circ 2021;143:120). Yet, little is known about the role of SdS or its depression in myocardium. Hypothesis: Reduced serine biosynthesis impairs myocyte energy and redox balance that could contribute to heart failure. Methods/Results: Immunoblots of human HF myocardium found phosphoglycerate dihydrogen-ase (PHGDH, rate limiting SdS enzyme) and other SdS enzymes were reduced in HF, and moreso in HF with preserved ejection fraction. Expression of plasmalemmal serine transporters SLC1A4, SLC1A5, SLC12A4 were consistently depressed in HFpEF. To assess the relative contribution of exogenous serine versus SdS, we inhibited PHGDH by siRNA or an inhibitor (NCT503). The former reduced serine by 71% despite exogenous sources, and both induced greater cytotoxicity than from solely depleting exogenous sources. Inhibiting PHGDH in serine/glycine-free media induced cytotoxicity after 1hr, whereas with exogenous serine, this occurred by 24 hours. 24 hr PHGDH knockdown reduced ATP and GTP levels, mitochondrial DNA. In response to endothelin-1 stimulation, basal and maximal mitochondrial O 2 consumption increased along with PHGDH gene expression. These changes in mitochondrial respiration were essentially prevented by gene silencing of PHDGH coupled to a decline in TCA metabolites. PHGDH inhibition also increased oxidative stress (increased GSSG/GSH) and reduced NAD levels. The cytotoxicity induced by 24 hr SdS inhibition by PHGDH knockdown could be offset by supplementing myocytes with ribose (purine precursor) and DTT (antioxidant). Conclusion: This study identifies SdS as a pivotal metabolic pathway in myocyte energy metabolism homeostasis, and its depression may play a role in HF, particularly HFpEF. These results support enhancing serine metabolic pathways as a therapeutic strategy for these diseases.

Liver biochemical indexes and cholesterol metabolism in cystic fibrosis patients with F508del/CFTR variant genotype after elexacaftor/tezacaftor/ivacaftor treatment

Modulators of cystic fibrosis transmembrane conductance regulator (CFTR) improved cystic fibrosis (CF) patients’ outcome. The elexacaftor/tezacaftor/ivacaftor (ETI) combination was safe and effective improving lung function in patients with different CFTR genotypes, including at least one F508del mutation. However, cases with liver damage were reported. We describe 105 CF patients heterozygous for F508del in trans with another CFTR mutation, treated for 1 year with ETI. We analyzed liver biochemical parameters and cholesterol metabolism, including lathosterol and phytosterols, surrogate markers of cholesterol de-novo synthesis and absorption, respectively. The treatment significantly improved sweat chloride, body mass index and forced expiratory volume in 1 s, whereas it caused a significant increase of total and conjugated bilirubin, ALT and GGT, even if no patients developed CF liver disease. Such alterations were less relevant than those previously observed in ETI-treated F508del homozygous patients. Furthermore, ETI treatment significantly increased serum cholesterol by enhancing its absorption (correlation between serum cholesterol and phytosterols). Whereas, we observed a normalization of de-novo biosynthesis (lathosterol reduction) that was not observed in homozygous patients. These data suggest that the second mutation in trans with the F508del contributes to reduce the liver cholesterol accumulation and thus, the triggering of liver inflammation. However, no differences in the alteration of biochemical indexes were observed between CF patients with and without liver steatosis, and between patients with different mutations in trans with the F508del. Such data suggest to further investigate the effects of ETI therapy on liver function indexes and new predictive biomarkers.

De Novo Biosynthesis of Difucosyllactose by Artificial Pathway Construction and α1,3/4-Fucosyltransferase Rational Design in Escherichia coli.

Difucosyllactose (DFL) is a significant and plentiful oligosaccharide found in human breast milk. In this study, an artificial metabolic pathway of DFL was designed, focusing on the de novo biosynthesis of GDP-fucose from only glycerol. This was achieved by engineering Escherichia coli to endogenously overexpress genes manB, manC, gmd, and wcaG and heterologously overexpress a pair of fucosyltransferases to produce DFL from lactose. The introduction of α-1,2-fucosyltransferase from Helicobacter pylori (FucT2) along with α-1,3/4-fucosyltransferase (HP3/4FT) addressed rate-limiting challenges in enzymatic catalysis and allowed for highly efficient conversion of lactose into DFL. Based on these results, molecular modification of HP3/4FT was performed based on computer-assisted screening and structure-based rational design. The best-performing mutant, MH5, containing a combination of five mutated sites (F49K/Y131D/Y197N/E338D/R369A) of HP3/4FT was obtained. The best strain BLC09-58 harboring MH5 yielded 45.81 g/L of extracellular DFL in 5-L fed-batch cultures, which was the highest titer reported to date.

De Novo Biosynthesis of Lutein in Yarrowia lipolytica.

Lutein is a high-value tetraterpenoid carotenoid that is widely used in feed, cosmetics, food, and drugs. Microbial synthesis of lutein is an important method for green and sustainable production, serving as an alternative to plant extraction methods. However, an inadequate precursor supply and low catalytic efficiency of key pathway enzymes are the main reasons for the low efficacy of microbial synthesis of lutein. In this study, some strategies, such as enhancing the MVA pathway and localizing α-carotene synthase OluLCY within the subcellular organelles in Yarrowia lipolytica, were adopted to enhance the synthesis of precursor α-carotene, which resulted in a 10.50-fold increase in α-carotene titer, reaching 38.50 mg/L. Subsequently, by improving hydroxylase activity with truncated N-terminal transport peptide and locating hydroxylases to subcellular organelles, the final strain L9 producing 75.25 mg/L lutein was obtained. Eventually, a lutein titer of 675.40 mg/L (6.13 mg/g DCW) was achieved in a 5 L bioreactor by adding the antioxidant 2,6-ditert-butyl-4-methylphenol. This study realizes de novo synthesis of lutein in Y. lipolytica for the first time and achieves the highest lutein titer reported so far.

Systematic Engineering of Saccharomyces cerevisiae for the De Novo Biosynthesis of Genistein and Glycosylation Derivatives.

Isoflavones are predominantly found in legumes and play roles in plant defense and prevention of estrogen-related diseases. Genistein is an important isoflavone backbone with various biological activities. In this paper, we describe how a cell factory that can de novo synthesize genistein was constructed in Saccharomyces cerevisiae. Different combinations of isoflavone synthase, cytochrome P450 reductase, and 2-hydroxyisoflavone dehydratase were tested, followed by pathway multicopy integration, to stably de novo synthesize genistein. The catalytic activity of isoflavone synthase was enhanced by heme supply and an increased intracellular NADPH/NADP+ ratio. Redistribution of the malonyl-CoA flow and balance of metabolic fluxes were achieved by adjusting the fatty acid synthesis pathway, yielding 23.33 mg/L genistein. Finally, isoflavone glycosyltransferases were introduced into S. cerevisiae, and the optimized strain produced 15.80 mg/L of genistin or 10.03 mg/L of genistein-8-C-glucoside. This is the first de novo synthesis of genistein-8-C-glucoside in S. cerevisiae, which is advantageous for the green industrial production of isoflavone compounds.

De Novo Biosynthesis of 4-Vinylanisole in Engineered Escherichia coli.

4-Vinylanisole is an aggregation pheromone of the locust. Both gregarious and solitary locusts exhibit a strong attraction toward 4-vinylanisole, irrespective of gender or age. Therefore, 4-vinylanisole can be used for trapping and monitoring locusts. In this study, the construction of a de novo 4-vinylanisole pathway in Escherichia coli has been demonstrated for the first time. Subsequently, by increasing the supply of precursor substrates, we further improved the biosynthesis of 4-vinylanisole. Finally, a two-phase organic overlay culture was used to increase the titer to 206 mg/L. It presents a sustainable and ecofriendly alternative for the synthesis of 4-vinylanisole.

Efficient De Novo Biosynthesis of Curcumin in Escherichia coli by Optimizing Pathway Modules and Increasing the Malonyl-CoA Supply.

Curcumin is a natural phenylpropanoid compound with various biological activities and is widely used in food and pharmaceuticals. A de novo curcumin biosynthetic pathway was constructed in Escherichia coli BL21(DE3). Optimization of the curcumin biosynthesis module achieved a curcumin titer of 26.8 ± 0.6 mg/L. Regulating the metabolic fluxes of the β-oxidation pathway and fatty acid elongation cycle and blocking the endogenous malonyl-CoA consumption pathway increased the titer to 113.6 ± 7.1 mg/L. Knockout of endogenous curcumin reductase (curA) and intermediate product detoxification by heterologous expression of the solvent-resistant pump (srpB) increased the titer to 137.5 ± 3.0 mg/L. A 5 L pilot-scale fermentation, using a three-stage pH alternation strategy, increased the titer to 696.2 ± 20.9 mg/L, 178.5-fold higher than the highest curcumin titer from de novo biosynthesis previously reported, thereby laying the foundation for efficient biosynthesis of curcumin and its derivatives.

Metabolome and transcriptome analysis provides insights into the mechanism of postharvest defence in fresh-cut broccoli

Fresh-cut produce yields mechanical damage and limits quality preservation. This research identifies the defence responses of fresh-cut broccoli (W (whole flower), F (floret), QF (quarterly-cut floret), FS (shredded floret)) during storage (0, 1, 2, 3 days) at room temperature. Consequently, changes during storage mainly occurred earlier (day 1), while fresh-cut changes primarily appeared at intense damage (FS). Uniquely accumulated quinic acid, 4-methoxyglucobrassicin, etc. were found in FS, while citric acid and glucose were exceptionally formed in W, F and QF. Importantly, a different glucosinolate breakdown system was identified in broccoli storage, encoded by myrosinases instead of TGGs in the pre-harvest system. Significant changes were also observed in the glucosinolates breakdown and de-novo biosynthesis as regulated by genes like PEN2/3, NIT, MYB122 and CYP81F1. Conclusively, candidate metabolites and genes identified in this study provide a reference for the precise preservation of cruciferous vegetables.

Abstract 3680: Multiomics analysis of triple negative breast cancer identifies potential metabolic vulnerability for overcoming the resistance of neoadjuvant therapy

Abstract Triple-negative breast cancer (TNBC) is a heterogeneous disease that remains currently medically incurable. We comprehensively analyzed clinical, proteomic, and metabolomic data of a cohort of 12 highly selected primary TNBCs. Among them, 7 patients after neoadjuvant chemotherapy had residual disease and micro- or macro-metastases that were non-pathologically complete response (non-pCR); 5 patients had pathologically complete response (pCR). Interestingly, both proteomic and metabolic profiling results revealed dramatically different molecular signatures in non-pCR relative to pCR. Non-pCR were enriched in metabolites involving aspartic acid, fumarate, nicotinamide, and adenosine that are critical intermediates for nitrogen recycling and tricarboxylic acid (TCA) cycle, which are essential fuels for tumor biomass. Moreover, non-pCR were enriched in urea cycle, oxidative phosphorylation, glycolysis, sirtuin signaling, nucleotide De Novo biosynthesis, which are resistance-associated. Putative therapeutic targets were identified in non-pCR patients. Collectively, our multiomics analysis demonstrated the heterogeneity of TNBCs at multi-levels and enabled the development of personalized therapies targeting unique tumor metabolic profiles. Citation Format: Zuen Ren, Kiran Kurmi, Robert Morris, Shakchhi Joshi, Eric Zaniewski, Johannes Kreuzer, Gabrielle Elena Gioia, Wilhelm Haas, Marcia C. Haigis, Leif W. Ellisen. Multiomics analysis of triple negative breast cancer identifies potential metabolic vulnerability for overcoming the resistance of neoadjuvant therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3680.

De Novo Biosynthesis of Anisyl Alcohol and Anisyl Acetate in Engineered Escherichia coli.

Anisyl alcohol and its ester anisyl acetate are both important fragrance compounds and have a wide range of applications in the cosmetics, perfumery, and food industries. The currently commercially available anisyl alcohol and anisyl acetate are based on chemical synthesis. However, consumers increasingly prefer natural fragrance compounds. Therefore, it is of great significance to construct microbial cell factories to produce anisyl alcohol and anisyl acetate. In this study, we first established a biosynthetic pathway in engineered Escherichia coli MG1655 for the production of anisyl alcohol from simple carbon sources. We further increased the anisyl alcohol production to 355 mg/L by the increasing availability of erythrose-4-phosphate and phosphoenolpyruvate. Finally, we further demonstrated the production of anisyl acetate by overexpressing alcohol acetyltransferase ATF1 for the subsequent acetylation of anisyl alcohol to produce anisyl acetate. To our knowledge, this is the first report on the biosynthesis of anisyl alcohol and anisyl acetate directly from a renewable carbon source.

Late-season biosynthesis of leaf fatty acids and n-alkanes of a mature beech (Fagus sylvatica) tree traced via 13CO2 pulse-chase labelling and compound-specific isotope analysis.

Leaf cuticular waxes play an important role in reducing evapotranspiration via diffusion. However, the ability of mature trees to regulate the biosynthesis of waxes to changing conditions (e.g., drought, light exposition) remain an open question, especially during the late growing season. This holds also true for one of the most widely distributed trees in Central Europe, the European beech tree (Fagus sylvatica L.). In order to investigate the ongoing formation of wax constituents like alkanes and fatty acids, we conducted a 13CO2 pulse-chase labelling experiment on sun-exposed and shaded branches of a mature beech tree during the late summer 2018. The 13C-label was traced via compound-specific δ13C isotope analysis of n-alkanes and fatty acids to determine the de-novo biosynthesis within these compound classes. We did not observe a significant change in lipid concentrations during the late growing season, but we found higher n-alkane concentrations in sun-exposed compared to shaded leaves in August and September. The n-alkane and fatty acid composition showed ongoing modifications during the late growing season. Together with the uptake and following subsequent decrease of the 13C-label, this suggests ongoing de-novo biosynthesis, especially of fatty acids in European beech leaves. Moreover, there is a high variability in the 13C-label among individual branches and between sun-exposed and shaded leaves. At the same time, sun-exposed leaves invest more of the assimilated C into secondary metabolites such as lipids than shaded leaves. This indicates that the investigated mature beech tree could adjust its lipid production and composition in order to acclimate to changes in microclimates within the tree crown and during the investigated period.

Efficient De Novo Biosynthesis of Heme by Membrane Engineering in Escherichia coli

Heme is of great significance in food nutrition and food coloring, and the successful launch of artificial meat has greatly improved the application of heme in meat products. The precursor of heme, 5-aminolevulinic acid (ALA), has a wide range of applications in the agricultural and medical fields, including in the treatment of corona virus disease 2019 (COVID-19). In this study, E. coli recombinants capable of heme production were developed by metabolic engineering and membrane engineering. Firstly, by optimizing the key genes of the heme synthesis pathway and the screening of hosts and plasmids, the recombinant strain EJM-pCD-AL produced 4.34 ± 0.02 mg/L heme. Then, the transport genes of heme precursors CysG, hemX and CyoE were knocked out, and the extracellular transport pathways of heme Dpp and Ccm were strengthened, obtaining the strain EJM-ΔCyoE-pCD-AL that produced 9.43 ± 0.03 mg/L heme. Finally, fed-batch fermentation was performed in a 3-L fermenter and reached 28.20 ± 0.77 mg/L heme and 303 ± 1.21 mg/L ALA. This study indicates that E. coli recombinant strains show a promising future in the field of heme and ALA production.

Reprogramming alternative macrophage polarization by GATM-mediated endogenous creatine synthesis: A potential target for HDM-induced asthma treatment.

Cellular energy metabolism plays a crucial role in the regulation of macrophage polarization and in the execution of immune functions. A recent study showed that Slc6a8-mediated creatine uptake from exogenous supplementation modulates macrophage polarization, yet little is known about the role of the de novo creatine de novobiosynthesis pathway in macrophage polarization. Here, we observed that glycine amidinotransferase (GATM), the rate-limiting enzyme for creatine synthesis, was upregulated in alternative (M2) polarized macrophages, and was dependent on the transcriptional factor STAT6, whereas GATM expression was suppressed in the classical polarized (M1) macrophage. Next, we revealed that exogenous creatine supplementation enhanced IL-4-induced M2 polarization, confirming recent work. Furthermore, we revealed that genetic ablation of GATM did not affect expression of M1 marker genes (Nos2, IL1b, IL12b) or the production of nitric oxide in both peritoneal macrophages (PMs) and bone marrow-derived macrophages (BMDMs). By contrast, expression levels of M2 markers (Arg1, Mrc1, Ccl17 and Retnla) were lower following GATM deletion. Moreover, we found that deletion of GATM in resident alveolar macrophages (AMs) significantly blocked M2 polarization but with no obvious effect on the number of cells in knockout mice. Lastly, an upregulation of GATM was found in lung tissue and bronchoalveolar lavage fluid macrophages from HDM-induced asthmatic mice. Our study uncovers a previously uncharacterized role for the de novo creatine biosynthesis enzyme GATM in M2 macrophage polarization, which may be involved in the pathogenesis of related inflammatory diseases such as an T helper 2 (Th2)-associated allergic asthma.

Facile Synthesis of Sugar Nucleotides from Common Sugars by the Cascade Conversion Strategy.

Sugar nucleotides are essential glycosylation donors in the carbohydrate metabolism. Naturally, most sugar nucleotides are derived from a limited number of common sugar nucleotides by de novo biosynthetic pathways, undergoing single or multiple reactions such as dehydration, epimerization, isomerization, oxidation, reduction, amination, and acetylation reactions. However, it is widely believed that such complex bioconversions are not practical for synthetic use due to the high preparation cost and great difficulties in product isolation. Therefore, most of the discovered sugar nucleotides are not readily available. Here, based on de novo biosynthesis mainly, 13 difficult-to-access sugar nucleotides were successfully prepared from two common sugars D-Man and sucrose in high yields, at a multigram scale, and without the need for tedious purification manipulations. This work demonstrated that de novo biosynthesis, although undergoing complex reactions, is also practical and cost-effective for synthetic use by employing a cascade conversion strategy.

De Novo Biosynthesis of D-p-Hydroxyphenylglycine by a Designed Cofactor Self-Sufficient Route and Co-culture Strategy.

d-p-Hydroxyphenylglycine (D-HPG) is an important intermediate for the synthesis of β-lactam antibiotics with an annual market demand of thousands of tons. Currently, the main production processes are via chemical approaches. Although enzymatic conversion has been investigated for D-HPG production, synthesis of the substrate DL-hydroxyphenylhydantoin is still chemically based, which suffers from high pollution and harsh reaction conditions. In this study, one cofactor self-sufficient route for D-HPG production from l-phenylalanine was newly designed and the artificial pathway was functionalized by selecting suitable enzymes and adjusting their expressions in strain Pseudomonas putida KT2440. Notably, a new R-mandelate dehydrogenase from Lactococcus lactis with relatively high activity under pH neutral conditions was successfully mined to demonstrate the biosynthetic pathway in vivo. The performance of the engineered P. putida strain was further increased by enhancing cellular NAD availability and blocking l-phenylalanine consumption. Coupled with the l-phenylalanine producer, Escherichia coli strain ATCC 31884, a stable and interactive co-culture process was also developed by engineering a "cross-link auxotrophic" system to produce D-HPG directly from glucose. Thus, this study is the first approach for the de novo biosynthesis of D-HPG by engineering a non-natural pathway and lays the foundation for further improving the efficiency of D-HPG production via a green and sustainable route.

Novel Insights Into Sterol Uptake and Intracellular Cholesterol Trafficking During Eimeria bovis Macromeront Formation.

Apicomplexan parasites are considered as defective in cholesterol synthesis. Consequently, they need to scavenge cholesterol from the host cell by either enhancing the uptake of extracellular cholesterol sources or by upregulating host cellular de-novo biosynthesis. Given that Eimeria bovis macromeront formation in bovine lymphatic endothelial host cells in vivo is a highly cholesterol-demanding process, we here examined host parasite interactions based on host cellular uptake of different low-density lipoprotein (LDL) types, i.e., of non-modified (LDL), oxidized (oxLDL), and acetylated LDL (acLDL). Furthermore, the expression of lipoprotein-oxidized receptor 1 (LOX-1), which mediates acLDL and oxLDL internalization, was monitored throughout first merogony, in vitro and ex vivo. Moreover, the effects of inhibitors blocking exogenous sterol uptake or intracellular transport were studied during E. bovis macromeront formation in vitro. Hence, E. bovis-infected primary bovine umbilical vein endothelial cells (BUVEC) were treated with inhibitors of sterol uptake (ezetimibe, poly-C, poly-I, sucrose) and of intracellular sterol transport and release from endosomes (progesterone, U18666A). As a read-out system, the size and number of macromeronts as well as merozoite I production were estimated. Overall, the internalization of all LDL modifications (LDL, oxLDL, acLDL) was observed in E. bovis-infected BUVEC but to different extents. Supplementation with oxLDL and acLDL at lower concentrations (5 and 10 µg/ml, respectively) resulted in a slight increase of both macromeront numbers and size; however, at higher concentrations (25–50 µg/ml), merozoite I production was diminished. LOX-1 expression was enhanced in E. bovis-infected BUVEC, especially toward the end of merogony. As an interesting finding, ezetimibe treatments led to a highly significant blockage of macromeront development and merozoite I production confirming the relevance of sterol uptake for intracellular parasite development. Less prominent effects were induced by non-specific inhibition of LDL internalization via sucrose, poly-I, and poly-C. In addition, blockage of cholesterol transport via progesterone and U18666A treatments resulted in significant inhibition of parasite development. Overall, current data underline the relevance of exogenous sterol uptake and intracellular cholesterol transport for adequate E. bovis macromeront development, unfolding new perspectives for novel drug targets against E. bovis.

Proteomic Analysis of KCNK3 Loss of Expression Identified Dysregulated Pathways in Pulmonary Vascular Cells.

The physiopathology of pulmonary arterial hypertension (PAH) is characterized by pulmonary artery smooth muscle cell (PASMC) and endothelial cell (PAEC) dysfunction, contributing to pulmonary arterial obstruction and PAH progression. KCNK3 loss of function mutations are responsible for the first channelopathy identified in PAH. Loss of KCNK3 function/expression is a hallmark of PAH. However, the molecular mechanisms involved in KCNK3 dysfunction are mostly unknown. To identify the pathological molecular mechanisms downstream of KCNK3 in human PASMCs (hPASMCs) and human PAECs (hPAECs), we used a Liquid Chromatography-Tandem Mass Spectrometry-based proteomic approach to identify the molecular pathways regulated by KCNK3. KCNK3 loss of expression was induced in control hPASMCs or hPAECs by specific siRNA targeting KCNK3. We found that the loss of KCNK3 expression in hPAECs and hPASMCs leads to 326 and 222 proteins differentially expressed, respectively. Among them, 53 proteins were common to hPAECs and hPASMCs. The specific proteome remodeling in hPAECs in absence of KCNK3 was mostly related to the activation of glycolysis, the superpathway of methionine degradation, and the mTOR signaling pathways, and to a reduction in EIF2 signaling pathways. In hPASMCs, we found an activation of the PI3K/AKT signaling pathways and a reduction in EIF2 signaling and the Purine Nucleotides De Novo Biosynthesis II and IL-8 signaling pathways. Common to hPAECs and hPASMCs, we found that the loss of KCNK3 expression leads to the activation of the NRF2-mediated oxidative stress response and a reduction in the interferon pathway. In the hPAECs and hPASMCs, we found an increased expression of HO-1 (heme oxygenase-1) and a decreased IFIT3 (interferon-induced proteins with tetratricopeptide repeats 3) (confirmed by Western blotting), allowing us to identify these axes to understand the consequences of KCNK3 dysfunction. Our experiments, based on the loss of KCNK3 expression by a specific siRNA strategy in control hPAECs and hPASMCs, allow us to identify differences in the activation of several signaling pathways, indicating the key role played by KCNK3 dysfunction in the development of PAH. Altogether, these results allow us to better understand the consequences of KCNK3 dysfunction and suggest that KCNK3 loss of expression acts in favor of the proliferation and migration of hPASMCs and promotes the metabolic shift and apoptosis resistance of hPAECs.