Enzymes In Metabolic Pathways Research Articles

The biochemical mechanism of enhancing the conversion of chicken manure to biogenic methane using coal slime as additive

To improve the efficiency of methane production from chicken manure (CM) anaerobic digestion, the mechanism of coal slime (CS) as an additive on methane production characteristics were investigated. The results showed that adding an appropriate amount of CS quickened the start of the fermentation and effectively increased the methane yield. In addition, the pH changed in a stable manner in the liquid phase, and the concentrations of total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) were reduced. Moreover, organic matter was decomposed and volatile fatty acids (VFAs) were consumed effectively. The abundance of Bacteroides in the bacterial community and Methanosarcina in the archaea was increased. In addition, the reduction of CO2 was the main methanogenic pathway, and adding CS raised the abundance of genes for key enzymes in metabolic pathways during methane metabolism. The results provide a novel method for the efficient methane production from CM.

Metagenomic analysis provides functional insights into seasonal change of a non-cyanobacterial prokaryotic community in temperate coastal waters.

The taxonomic compositions of marine prokaryotic communities are known to follow seasonal cycles, but functional metagenomic insights into this seasonality is still limited. We analyzed a total of 22 metagenomes collected at 11 time points over a 14-month period from two sites in Sendai Bay, Japan to obtain seasonal snapshots of predicted functional profiles of the non-cyanobacterial prokaryotic community. Along with taxonomic composition, functional gene composition varied seasonally and was related to chlorophyll a concentration, water temperature, and salinity. Spring phytoplankton bloom stimulated increased abundances of putative genes that encode enzymes in amino acid metabolism pathways. Several groups of functional genes, including those related to signal transduction and cellular communication, increased in abundance during the mid- to post-bloom period, which seemed to be associated with a particle-attached lifestyle. Alternatively, genes in carbon metabolism pathways were generally more abundant in the low chlorophyll a period than the bloom period. These results indicate that changes in trophic condition associated with seasonal phytoplankton succession altered the community function of prokaryotes. Our findings on seasonal changes of predicted function provide fundamental information for future research on the mechanisms that shape marine microbial communities.

Functional characterization of arginine metabolic pathway enzymes in the antibacterial immune response of penaeid shrimp

Arginine metabolism pathway enzymes and products are important modulators of several physiological processes in animals, including immune response. Although some components of the arginine metabolic pathway have been reported in penaeid shrimps, no systematic study has explored all the key pathway enzymes involved in shrimp antimicrobial response. Here, we explored the role of the three key arginine metabolism enzymes (nitric-oxide synthase (NOS), arginase (ARG), agmatinase (AGM)) in Penaeus vannamei antimicrobial immunity. First, P. vannamei homologs of ARG and AGM (PvARG and PvAGM) were cloned and found to be evolutionally conserved with invertebrate counterparts. Transcript levels of PvARG, PvAGM, and PvNOS were ubiquitously expressed in healthy shrimp tissues and induced in hemocytes and hepatopancreas upon challenge with Gram-negative (Vibrio parahaemolyticus) and Gram-positive (Streptoccocus iniae) bacteria, suggesting their involvement in shrimp antimicrobial immune response. Besides, RNA interference knockdown and enzyme activity assay revealed an antagonistic relationship between PvARG/PvAGM and PvNOS, while this relationship was broken upon pathogen stimulation. Interestingly, knockdown of PvNOS increased Vibrio abundance in shrimp hemolymph, whereas knockdown of PvAGR reduced Vibrio abundance. Taken together, our present data shows that homologs of the key arginine metabolism pathway enzymes in penaeid shrimp (PvARG, PvAGM, and PvNOS) work synergistically and/or antagonistically to modulate antibacterial immune response.

Protective effects of all-trans retinoic acid against gastric premalignant lesions by repressing exosomal LncHOXA10-pyruvate carboxylase axis.

Long noncoding RNAs (LncRNAs) play a pivotal role in gastric tumorigenesis, while exosomes facilitate the LncRNAs transferring to recipient cells. However, the roles of exosomal LncRNAs in gastric premalignant lesions (GPL) remain unclear. We analyzed the expression of LncHOXA10 and its role in GPL progression. The protective effect of all-trans retinoic acid (ATRA) on GPL was explored in vitro and in vivo. Here, we found that LncHOXA10 expression was obviously increased in serum exosomes and gastric tissues from individuals with GPL, and exosomal LncHOXA10 from patients with GPL markedly promoted the malignant progression of human gastric epithelial cell line GES-1. Furthermore, RNA-pulldown assay revealed that LncHOXA10 mainly interacted with pyruvate carboxylase (PC), an essential enzyme in various cellular metabolic pathways. In gastric tissues from patients with GPL and gastric cancer (GC), PC was also upregulated and positively correlated with LncHOXA10 expression, which predicted a poor prognosis as well. Moreover, PC silencing attenuated the malignant effects of exosomal LncHOXA10 on GES-1 cells. ATRA also ameliorated the deterioration of GPL and prevented the malignant progression of GPL by reducing exosomal LncHOXA10 and PC expression. Collectively, the LncHOXA10-PC axis participated in the early stage of GC tumorigenesis, and ATRA might be useful to prevent GPL from developing into GC because it targets this axis.

Storage of carbon reserves in spruce trees is prioritized over growth in the face of carbon limitation

Climate change is expected to pose a global threat to forest health by intensifying extreme events like drought and insect attacks. Carbon allocation is a fundamental process that determines the adaptive responses of long-lived late-maturing organisms like trees to such stresses. However, our mechanistic understanding of how trees coordinate and set allocation priorities among different sinks (e.g., growth and storage) under severe source limitation remains limited. Using flux measurements, isotopic tracing, targeted metabolomics, and transcriptomics, we investigated how limitation of source supply influences sink activity, particularly growth and carbon storage, and their relative regulation in Norway spruce (Picea abies) clones. During photosynthetic deprivation, absolute rates of respiration, growth, and allocation to storage all decline. When trees approach neutral carbon balance, i.e., daytime net carbon gain equals nighttime carbon loss, genes encoding major enzymes of metabolic pathways remain relatively unaffected. However, under negative carbon balance, photosynthesis and growth are down-regulated while sucrose and starch biosynthesis pathways are up-regulated, indicating that trees prioritize carbon allocation to storage over growth. Moreover, trees under negative carbon balance actively increase the turnover rate of starch, lipids, and amino acids, most likely to support respiration and mitigate stress. Our study provides molecular evidence that trees faced with severe photosynthetic limitation strategically regulate storage allocation and consumption at the expense of growth. Understanding such allocation strategies is crucial for predicting how trees may respond to extreme events involving steep declines in photosynthesis, like severe drought, or defoliation by heat waves, late frost, or insect attack.

Tryptophan and Its Metabolites in Lung Cancer: Basic Functions and Clinical Significance.

Lung cancer is the most lethal malignancy worldwide. Recently, it has been recognized that metabolic reprogramming is a complex and multifaceted factor, contributing to the process of lung cancer. Tryptophan (Try) is an essential amino acid, and Try and its metabolites can regulate the progression of lung cancer. Here, we review the pleiotropic functions of the Try metabolic pathway, its metabolites, and key enzymes in the pathogenic process of lung cancer, including modulating the tumor environment, promoting immune suppression, and drug resistance. We summarize the recent advance in therapeutic drugs targeting the Try metabolism and kynurenine pathway and their clinical trials.

A high extra-virgin olive oil diet induces changes in metabolic pathways of experimental mammary tumors

Breast cancer is the most common malignancy in women worldwide, and environmental factors, especially diet, have a role in the etiology of this disease. This work aimed to investigate the influence of high fat diets (rich in corn oil or extra virgin olive oil -EVOO-) and the timing of dietary intervention (from weaning or after induction) on tumor metabolism in a seven,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer model in rat. The effects of lipids (oils and fatty acids) have also been investigated in MCF-7 cells. The results have confirmed different effects on tumor progression depending on the type of lipid. Molecular analysis at mRNA, protein and activity level of enzymes of the main metabolic pathways have also shown differences among groups. Thus, the animals fed with the EVOO-enriched diet developed tumors with less degree of clinical and morphological malignancy and showed modified glucose and mitochondrial metabolism when compared to the animals fed with the corn oil-enriched diet. Paradoxically, no clear influence on lipid metabolism by the high fat diets was observed. Considering previous studies on proliferation and apoptosis in the same samples, the results suggest that metabolic changes have a role in the molecular context that results in the modulation of different signaling pathways. Moreover, metabolic characteristics, without the context of other pathways, may not reflect tumor malignancy. The time of dietary intervention plays also a role, suggesting the importance of metabolic plasticity and the relation with mammary gland status when the tumor is induced.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath.

ABSTRACTThe ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO2 was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth’s biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2.IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.

CETSA interaction proteomics define specific RNA-modification pathways as key components of fluorouracil-based cancer drug cytotoxicity

The optimal use of many cancer drugs is hampered by a lack of detailed understanding of their mechanism of action (MoA). Here, we apply a high-resolution implementation of the proteome-wide cellular thermal shift assay (CETSA) to follow protein interaction changes induced by the antimetabolite 5-fluorouracil (5-FU) and related nucleosides. We confirm anticipated effects on the known main target, thymidylate synthase (TYMS), and enzymes in pyrimidine metabolism and DNA damage pathways. However, most interaction changes we see are for proteins previously not associated with the MoA of 5-FU, including wide-ranging effects on RNA-modification and -processing pathways. Attenuated responses of specific proteins in a resistant cell model identify key components of the 5-FU MoA, where intriguingly the abrogation of TYMS inhibition is not required for cell proliferation.

Conserved Metabolic and Evolutionary Themes in Microbial Degradation of Carbamate Pesticides.

Carbamate pesticides are widely used as insecticides, nematicides, acaricides, herbicides and fungicides in the agriculture, food and public health sector. However, only a minor fraction of the applied quantity reaches the target organisms. The majority of it persists in the environment, impacting the non-target biota, leading to ecological disturbance. The toxicity of these compounds to biota is mediated through cholinergic and non-cholinergic routes, thereby making their clean-up cardinal. Microbes, specifically bacteria, have adapted to the presence of these compounds by evolving degradation pathways and thus play a major role in their removal from the biosphere. Over the past few decades, various genetic, metabolic and biochemical analyses exploring carbamate degradation in bacteria have revealed certain conserved themes in metabolic pathways like the enzymatic hydrolysis of the carbamate ester or amide linkage, funnelling of aryl carbamates into respective dihydroxy aromatic intermediates, C1 metabolism and nitrogen assimilation. Further, genomic and functional analyses have provided insights on mechanisms like horizontal gene transfer and enzyme promiscuity, which drive the evolution of degradation phenotype. Compartmentalisation of metabolic pathway enzymes serves as an additional strategy that further aids in optimising the degradation efficiency. This review highlights and discusses the conclusions drawn from various analyses over the past few decades; and provides a comprehensive view of the environmental fate, toxicity, metabolic routes, related genes and enzymes as well as evolutionary mechanisms associated with the degradation of widely employed carbamate pesticides. Additionally, various strategies like application of consortia for efficient degradation, metabolic engineering and adaptive laboratory evolution, which aid in improvising remediation efficiency and overcoming the challenges associated with in situ bioremediation are discussed.

Abstract 2495: SPARC: a bladder cancer tumor-suppressor via targeting metabolic programming

Abstract Bladder cancer (BC) is the sixth most common cancer in the United States. Approximately, 75% of BC patients present with non-muscle invasive bladder cancer (NMIBC), which is responsive to local treatment and surveillance. However, recurrence is common with progression to muscle-invasive bladder cancer (MIBC). Forty percent of MIBC patients develop distant metastases, with few treatment options and poor survival. Limited advances have been made in BC therapy with only a few FDA-approved new treatments for advanced disease since 2016 with response rates hovering around 15%. Thus, there is an unmet need for better understanding of the pathobiology of the disease to develop more effective therapeutics. Metabolic re-programming of cancer is increasingly recognized as a hallmark of cancer as cancer cells take advantage of metabolic products to meet the increasing demands of proliferating cells. Our lab has identified the Secreted Protein Acidic Rich in Cysteine (SPARC) as a multi-faceted tumor suppressor within the BC tumor microenvironment. Herein, we sought to elucidate the role of SPARC in regulating metabolic programming in BC and identify pathways that may serve as diagnostic and therapeutic targets. We used three BC cell lines UMUC3, T24 (SPARChigh) and its isogenic SPARClow T24T. We treated BC cells with recombinant SPARC protein and genetically manipulated SPARC expression in these cells. We performed real-time monitoring of the effect of SPARC on bioenergetics using Seahorse assays. In tandem, we profiled transcriptomes of T24, T24T, and T24shSP-cell lines to identify aggressive phenotype signatures using GSEA. We then compared signatures with survival data from BC cohorts from the GEO and TCGA. We found that SPARC inhibited ATP production, basal and maximal respiration, and spare respiratory capacity in BC cell lines. This effect is more pronounced in more aggressive SPARClow T24T cells compared to its isogenic SPARChigh T24 cells. Knockdown of SPARC in BC cell lines not only induced an aggressive phenotype, but also increased ATP production and mitochondrial respiration. Integrated transcriptomic profiling indicated that SPARC-loss is associated with enriched glycolytic, lipidomic, and mitochondrial metabolic pathways. Importantly, in patient samples, SPARC expression negatively correlated with rate limiting enzymes in metabolic pathways associated with lower survival. SPARC loss also associated with oncogenic signatures of EMT, PI3K-AKT-mTOR signaling, and G1/S cell cycle arrest. Our data reveals a novel function of SPARC inhibiting mitochondrial bioenergetics and ATP production that fuel growth and invasion in BC cells. The SPARC-regulated metabolic profile highlights potential vulnerabilities that can be exploited as therapeutic targets. Citation Format: Sameh Waleed Almousa, Alia Ghoneum, Hesham Afify, Neveen Said. SPARC: a bladder cancer tumor-suppressor via targeting metabolic programming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2495.

Identification of Metabolic Enzymes ENO1 and KMO As Novel Immunotherapeutic Targets in Myeloma

Introduction. Our previous studies showed an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. pDCs are present in increased numbers in MM BM, promote tumor growth and survival, protect tumor from therapy-induced cytotoxicity; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323) . Aberrant pDC function is evidenced from their interactions with MM tumor and T/NK effector cells in the BM milieu (Ray et al, Leukemia 2015, 29:1441-1444) . Direct interruption of pDC-MM and pDC-T cells interactions and their sequelae utilizing novel targeted agents is necessary to further enhance both anti-tumor immunity and cytotoxicity. Based on DNA microarray analysis, we identified several novel targets on MM cells that are upregulated after coculture with pDCs, and focused on metabolic pathway enzymes that are upregulated both in pDC and MM cells. We identified alpha-Enolase (ENO1) and Kynurenine 3-monooxygenase (KMO) as the two most promising therapeutic targets, based on their roles in normal and cancer metabolism. ENO1 is a glycolytic enzyme, and its surface localization is a unique feature of the tumor cells. Moreover, ENO1 promotes tumor metastasis. KMO is one of the two rate-limiting enzymes involved in eukaryotic tryptophan catabolic kynurenine pathway; and importantly, its overexpression has been reported to inhibit functional immune responses in cancer. Here, we utilized our pDC/MM or pDC/T cells co-culture model to show that targeting ENO1 or KMO generates MM-specific cytotoxic T lymphocyte activity.MethodsDNA MicroArray Analysis: MM.1S cells were cocultured with pDCs for 72h, separated using CD138 Microbeads, and harvested. Poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM.1S cells cultured in the presence vs absence of pDCs were compared, and heat map was generated (>1.5-fold change in transcript was considered significant, CI > 95%). pDC-induced overexpression of target proteins : MM cells were cocultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in target protein expression. CD138+ tumor cells were gated out, and KMO-FITC or ENO1-AlexaFluor488-stained cells were analyzed. C TL activity assays : MM patient BM CD8+ T cells were cocultured with autologous pDCs at 1:10 (pDC:T) ratio in the presence or absence of KMO inhibitor Ro 61-8048 (10 nM) or ENO1 inhibitor ENOblock (0.1-0.2 µM) or anti-ENO1 Ab for 5 days; pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T:MM), followed by quantification of viable MM cells by FACS. Statistical parameters were calculated using GraphPad Prism. Pharmacological inhibitors were obtained from Selleck Chemicals.Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of both ENO1 and KMO transcripts in MM cells (1.772 and 2.153-fold, respectively, vs MM.1S alone; n=3; CI > 95%). 2) Both pDC and MM cells showed high KMO and surface ENO1 expression. Coculture of MM cells with pDCs further increased ENO1 surface expression levels in MM cells (5-6-fold increase; n=3; p = 0.003), as well as the ENO1-positive MM cell population (3-4-fold versus MM alone; mean ± SD; n=3; p = 0.005). 3) Like ENO1, pDCs also increased both KMO expression on MM cells and KMO-positive MM cells (MFI: 2-3-fold in the presence vs absence of pDCs; p = 0.006). 4) Functional studies showed that treatment of pDC-T cell cocultures with ENO1 antagonists triggers a robust MM-specific CD8+ CTL activity against autologous CD138-positive MM cells (2 fold in treatment vs control; p = 0.005). 5) pDC-mediated MM-specific CD8+ CTL activity was also confirmed using allogeneic HLA-A2+ U266 MM cells (1.8-2-fold vs control; p = 0.008). Finally, 6) KMO inhibitor Ro 61-8048 activates patient BM-CD8+T cells in autologous pDCs/T cell cocultures, and triggers robust allogeneic and autologous MM-specific CD8+ CTL activity (~2.0-fold vs control; p = 0.009).Conclusions Our study shows that pDC-MM interactions upregulate tumor promoting and immunosuppressive metabolic enzymes in MM. Blockade of ENO1 or KMO enzymes tiggers MM-specific CD8-positive CTL activity. These data therefore identify novel immune-based therapeutic strategies targeting ENO1 and KMO to enhance MM cytotoxicity and restore anti-MM immunity. DisclosuresAnderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Study on relationship between bacterial diversity and quality of Huangjiu (Chinese Rice Wine) fermentation.

Huangjiu (Chinese rice wine) is brewed in an open environment, where bacteria play an important role during the fermentation process. In this study, bacterial community structure and composition changes in the fermented mash liquid of mechanized Huangjiu, well‐fermented manual Huangjiu (wines of good qualities), and poorly fermented manual Huangjiu (wines of poor qualities: spoilage, high acidity, low alcohol content) in different fermentation stages from Guyuelongshan Shaoxing Huangjiu company were analyzed via metagenomic sequencing. And bacterial metabolic difference was analyzed via gene prediction of metabolic pathway enzymes. The results showed that the bacterial diversity degree was abundant, and the number of bacterial species in every sample was approximately 200–400. Lactic acid bacteria (LAB) dominated the bacterial community of Huangjiu fermentation, and lactobacillus was predominant species in well‐fermented Huangjiu while Lactobacillus brevis had an absolute dominance in spoilage Huangjiu. Further, gene prediction revealed that transformation of malate to pyruvate and lactate anabolism was more active in mash liquid of well‐fermented manual Huangjiu, while acetate accumulation was stronger in mash liquid of poorly fermented manual Huangjiu, which explained acidity excess reason in poorly fermented Huangjiu at gene level.

Cloning of <italic>Ta4CL1 </italic>and its function in promoting plant growth and lignin deposition in transgenic <italic>Arabidopsis</italic> plants

<p id="C3">4-Coumarate:coenzyme A ligase (4CL; EC 6.2.1.12) acts upstream of the branch point of phenylpropanoid, which is a key enzyme in the phenylpropanoid metabolic pathways for monolignol and flavonoid biosynthesis, and these compounds play important roles in plant growth and development as well as stress adaptability. Many 4CLs had been extensively studied in dicotyledons, but their function in monocotyledons, especially in crops, was relatively poorly understood. In this study, <italic>Ta4CL1, </italic>which encoded 4-Coumarate:coenzyme A ligase, was isolated from common wheat by RACE technique. Polygenetic analysis revealed that Ta4CL1 could be clustered to the same group of 4CLs from rice, maize, and sorghum, which was mainly involved in lignin biosynthesis. <italic>Ta4CL1-</italic>overexpressed <italic>Arabidopsis</italic> lines, <italic>at4cl1</italic>, <italic>at4cl3</italic>, and <italic>at4cl14cl3</italic> as well as their corresponding functional recovery lines were used to elucidate the function of<italic> Ta4CL1 </italic>in the phenylpropanoid metabolic pathway. The results suggested that Ta4CL1 had similar function with At4CL1 in regulating lignin biosynthesis but it had no effect on flavonoid biosynthesis. Ta4CL was the major contributor of 4CL enzyme activity in transgenic <italic>Arabidopsis</italic> plants. Overexpression of <italic>Ta4CL</italic> led to enlarged leaves and thickened stems in transgenic <italic>Arabidopsis</italic> seedlings, the expression of <italic>Ta4CL </italic>was also affected by MeJA, GA, and IAA treatments. These results provide the theoretical basis for improving the utilizing efficiency of plant straws using <italic>Ta4CL1 </italic>by genetic engineering.

FRET-Based Genetically Encoded Sensor to Monitor Silver Ions.

Silver is commonly used in wound dressing, photography, health care products, laboratories, pharmacy, biomedical devices, and several industrial purposes. Silver (Ag+) ions are more toxic pollutants widely scattered in the open environment by natural processes and dispersed in soil, air, and water bodies. Ag+ binds with metallothionein, macroglobulins, and albumins, which may lead to the alteration of various enzymatic metabolic pathways. To analyze the uptake and metabolism of silver ions in vitro as well as in cells, a range of high-affinity fluorescence-based nanosensors has been constructed using a periplasmic protein CusF, a part of the CusCFBA efflux complex, which is involved in providing resistance against copper and silver ions in Escherichia coli. This nanosensor was constructed by combining of two fluorescent proteins (donor and acceptor) at the N- and C-terminus of the silver-binding protein (CusF), respectively. SenSil (WT) with a binding constant (Kd) of 5.171 μM was more efficient than its mutant variants (H36D and F71W). This nanosensor allows monitoring the level of silver ions in real time in prokaryotes and eukaryotes without any disruption of cells or tissues.

Selection of a Gentamicin-Resistant Variant Following Polyhexamethylene Biguanide (PHMB) Exposure in Escherichia coli Biofilms.

Antibiotic resistance is one of the most important issues facing modern medicine. Some biocides have demonstrated the potential of selecting resistance to antibiotics in bacteria, but data are still very scarce and it is important to better identify the molecules concerned and the underlying mechanisms. This study aimed to assess the potential of polyhexamethylene biguanide (PHMB), a widely used biocide in a variety of sectors, to select antibiotic resistance in Escherichia coli grown in biofilms. Biofilms were grown on inox coupons and then exposed daily to sublethal concentrations of PHMB over 10 days. Antibiotic-resistant variants were then isolated and characterized phenotypically and genotypically to identify the mechanisms of resistance. Repeated exposure to PHMB led to the selection of an E. coli variant (Ec04m1) with stable resistance to gentamycin (8-fold increase in minimum inhibitory concentration (MIC) compared to the parental strain. This was also associated with a significant decrease in the growth rate in the variant. Sequencing and comparison of the parental strain and Ec04m1 whole genomes revealed a nonsense mutation in the aceE gene in the variant. This gene encodes the pyruvate dehydrogenase E1 component of the pyruvate dehydrogenase (PDH) complex, which catalyzes the conversion of pyruvate to acetyl-CoA and CO2. A growth experiment in the presence of acetate confirmed the role of this mutation in a decreased susceptibility to both PHMB and gentamicin (GEN) in the variant. This work highlights the potential of PHMB to select resistance to antibiotics in bacteria, and that enzymes of central metabolic pathways should be considered as a potential target in adaptation strategies, leading to cross-resistance toward biocides and antibiotics in bacteria.

LXR and INSIG Act as Differentiators in the Regulation of the Gene Expression of G6PDH and FAS Under Insulin Resistant Conditions

Diabetes, a chronic metabolic disease that affects nearly 10% of the world's population can lead to very serious complications such as renal failure, liver cirrhosis, and heart attacks. The most common type is Type 2 diabetes and is diagnosed when a person has elevated amounts of blood glucose due to insulin resistance. This resistance to insulin leads to problems with glucose transport into tissues for subsequent metabolism. Over the years it has been shown that insulin regulates the expression of several key enzymes in both carbohydrate and fatty acid metabolic pathways via the phosphatidyl inositol 3-kinase (PI3K) pathway. Previously, using glucosamine, a precursor of the hexosamine biosynthetic pathway, we had established a model of insulin resistance in primary rat hepatocytes in culture. Using this primary cell culture model, we showed that under insulin resistant conditions, the expression of glucose 6 phosphate dehydrogenase (G6PDH), a key enzyme in carbohydrate metabolism and fatty acid synthase (FAS), a key enzyme in fat metabolism were differently regulated but the mechanism of this differentiation was unclear. Under this model of insulin resistance, we now show that this differential regulation is due to the liver X receptor (LXR) and insulin induced gene (INSIG).

Integrating continuous hypermutation with high-throughput screening for optimization of cis,cis-muconic acid production in yeast.

SummaryDirected evolution is a powerful method to optimize proteins and metabolic reactions towards user‐defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection and amplification. While powerful, systematic searches through large sequence‐spaces is a labour‐intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here, we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generate enzyme variants using the recently developed orthogonal replication system, OrthoRep and screens for optimal performance in high‐throughput using a transcription factor‐based biosensor. We demonstrate the strengths of this workflow by evolving a rate‐limiting enzymatic reaction of the biosynthetic pathway for cis,cis‐muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high‐throughput sorting of best‐performing variants using a transcription factor‐based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers > 13‐fold compared with reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.

Phytochemicals Block Glucose Utilization and Lipid Synthesis to Counteract Metabolic Reprogramming in Cancer Cells

Aberrant metabolism is one of the hallmarks of cancers. The contributions of dysregulated metabolism to cancer development, such as tumor cell survival, metastasis and drug resistance, have been extensively characterized. “Reprogrammed” metabolic pathways in cancer cells are mainly represented by excessive glucose consumption and hyperactive de novo lipogenesis. Natural compounds with anticancer activities are constantly being demonstrated to target metabolic processes, such as glucose transport, aerobic glycolysis, fatty acid synthesis and desaturation. However, their molecular targets and underlying anticancer mechanisms remain largely unclear or controversial. Mounting evidence indicated that these natural compounds could modulate the expression of key regulatory enzymes in various metabolic pathways at transcriptional and translational levels. Meanwhile, natural compounds could also inhibit the activities of these enzymes by acting as substrate analogs or altering their protein conformations. The actions of natural compounds in the crosstalk between metabolism modulation and cancer cell destiny have become increasingly attractive. In this review, we summarize the activities of natural small molecules in inhibiting key enzymes of metabolic pathways. We illustrate the structural characteristics of these compounds at the molecular level as either inhibitor of various enzymes or regulators of metabolic pathways in cancer cells. Our ultimate goal is to both facilitate the clinical application of natural compounds in cancer therapies and promote the development of novel anticancer therapeutics.

Upregulated ethanolamine phospholipid synthesis via selenoprotein I is required for effective metabolic reprogramming during T cell activation

ObjectiveT cell activation triggers metabolic reprogramming to meet increased demands for energy and metabolites required for cellular proliferation. Ethanolamine phospholipid synthesis has emerged as a regulator of metabolic shifts in stem cells and cancer cells, which led us to investigate its potential role during T cell activation. MethodsAs selenoprotein I (SELENOI) is an enzyme participating in two metabolic pathways for the synthesis of phosphatidylethanolamine (PE) and plasmenyl PE, we generated SELENOI-deficient mouse models to determine loss-of-function effects on metabolic reprogramming during T cell activation. Ex vivo and in vivo assays were carried out along with metabolomic, transcriptomic, and protein analyses to determine the role of SELENOI and the ethanolamine phospholipids synthesized by this enzyme in cell signaling and metabolic pathways that promote T cell activation and proliferation. ResultsSELENOI knockout (KO) in mouse T cells led to reduced de novo synthesis of PE and plasmenyl PE during activation and impaired proliferation. SELENOI KO did not affect T cell receptor signaling, but reduced activation of the metabolic sensor AMPK. AMPK was inhibited by high [ATP], consistent with results showing SELENOI KO causing ATP accumulation, along with disrupted metabolic pathways and reduced glycosylphosphatidylinositol (GPI) anchor synthesis/attachment ConclusionsT cell activation upregulates SELENOI-dependent PE and plasmenyl PE synthesis as a key component of metabolic reprogramming and proliferation.