Conversion Of Serine Research Articles

Inhibition of signaling protein ERN1 increases the sensitivity of serine synthesis gene expressions to glucose and glutamine deprivations in U87MG glioblastoma cells.

Objective. Glucose and glutamine supply as well as serine synthesis and endoplasmic reticulum (ER) stress are important factors of glioblastoma growth. Previous studies showed that the knockdown of ERN1 (ER to nucleus signaling 1) suppressed glioblastoma cell proliferation and modified the sensitivity of numerous gene expressions to nutrient deprivations. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of serine synthesis genes in U87MG glioblastoma cells in relation to ERN1 knockdown with the intent to reveal the role of ERN1 signaling pathway on the ER stress-dependent regulation of these gene expressions. Clarification of the regulatory mechanisms of serine synthesis is a great significance for glioblastoma therapy. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed under glucose and glutamine deprivation conditions for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine amino-transferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that the expression level of genes responsible for serine synthesis such as PHGDH, PSAT1, PSPH, and transcription factor ATF4 was up-regulated in U87MG glioblastoma cells under glucose and glutamine deprivations. Furthermore, inhibition of ERN1 significantly enhances the impact of glucose and especially glutamine deprivations on these gene expressions. At the same time, the expression of the SHMT1 gene, which is responsible for serine conversion to glycine, was down-regulated in both nutrient deprivation conditions with more significant changes in ERN1 knockdown glioblastoma cells. Conclusion. Taken together, the results of present study indicate that the expression of genes responsible for serine synthesis is sensitive to glucose and glutamine deprivations in gene-specific manner and that suppression of ERN1 signaling significantly modifies the impact of both glucose and glutamine deprivations on PHGDH, PSAT1, PSPH, ATF4, and SHMT1 gene expressions and reflects the ERN1-mediated genome reprograming introduced by nutrient deprivation condition.

Structural and functional analysis of two SHMT8 variants associated with soybean cyst nematode resistance.

Two amino acid variants in soybean serine hydroxymethyltransferase 8 (SHMT8) are associated with resistance to the soybean cyst nematode (SCN), a devastating agricultural pathogen with worldwide economic impacts on soybean production. SHMT8 is a cytoplasmic enzyme that catalyzes the pyridoxal 5-phosphate-dependent conversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate. A previous study of the P130R/N358Y double variant of SHMT8, identified in the SCN-resistant soybean cultivar (cv.) Forrest, showed profound impairment of folate binding affinity and reduced THF-dependent enzyme activity, relative to the highly active SHMT8 in cv. Essex, which is susceptible to SCN. Given the importance of SCN-resistance in soybean agriculture, we report here the biochemical and structural characterization of the P130R and N358Y single variants to elucidate their individual effects on soybean SHMT8. We find that both single variants have reduced THF-dependent catalytic activity relative to Essex SHMT8 (10- to 50-fold decrease in kcat /Km ) but are significantly more active than the P130R/N368Y double variant. The kinetic data also show that the single variants lack THF-substrate inhibition as found in Essex SHMT8, an observation with implications for regulation of the folate cycle. Five crystal structures of the P130R and N358Y variants in complex with various ligands (resolutions from 1.49 to 2.30 Å) reveal distinct structural impacts of the mutations and provide new insights into allosterism. Our results support the notion that the P130R/N358Y double variant in Forrest SHMT8 produces unique and unexpected effects on the enzyme, which cannot be easily predicted from the behavior of the individual variants.

HOXD8 suppresses renal cell carcinoma growth by upregulating SHMT1 expression.

Amplification of amino acids synthesis is reported to promote tumorigenesis. The serine/glycine biosynthesis pathway is a reversible conversion of serine and glycine catalyzed by cytoplasmic serine hydroxymethyltransferase (SHMT)1 and mitochondrial SHMT2; however, the role of SHTM1 in renal cell carcinoma (RCC) is still unclear. We found that low SHMT1 expression is correlated with poor survival of RCC patients. The in vitro study showed that overexpression of SHMT1 suppressed RCC proliferation and migration. In the mouse tumor model, SHMT1 significantly retarded RCC tumor growth. Furthermore, by gene network analysis, we found several SHMT1-related genes, among which homeobox D8 (HOXD8) was identified as the SHMT1 regulator. Knockdown of HOXD8 decreased SHMT1 expression, resulting in faster RCC growth, and rescued the SHMT1 overexpression-induced cell migration defects. Additionally, ChIP assay found the binding site of HOXD8 to SHMT1 promoter was at the -456~-254 bp region. Taken together, SHMT1 functions as a tumor suppressor in RCC. The transcription factor HOXD8 can promote SHMT1 expression and suppress RCC cell proliferation and migration, which provides new mechanisms of SHMT1 in RCC tumor growth and might be used as a potential therapeutic target candidate for clinical treatment.

Abstract 5870: T-cell factor-4 variants regulate PD-L1 expression in liver cancer cells

Abstract Background: We previously identified 14 isoforms of T-cell factor (TCF)-4, a key transcriptional factor in the Wnt signaling pathway, from human hepatocellular carcinoma (HCC) cell lines (Exp Cell Res 2011). The TCF-4J and K pair has been characterized based on the presence (K) or absence (J) of a conserved SxxSS motif at the head of exon 9. TCF-4J-overexpressing HCC cells (J cells) exhibited high tumorigenic potential in contrast to TCF-4K-overexpressing cells (K cells) (PLoS ONE 2012); however, resistance to anti-cancer agents was more prominent in K cells than J cells. Another analysis demonstrated that loss of exon 4 increased resistance to chemotherapeutic agents and induced epithelial-mesenchymal transition (EMT) (Liver Int 2013). EMT and the expression of programmed cell death 1-ligand 1 (PD-L1) is closely associated with each other, and our preliminary study revealed that EMT was regulated by TCF-4 variants, suggesting possible involvement of the variants in the regulation of PD-L1 expression. In general, PD-L1 is known to be upregulated by hypoxia-inducible factor-1α (HIF-1α) under hypoxic conditions. Therefore, if the TCF-4 variants regulate PD-L1 expression under normoxia, it would be a unique mechanism bridging the Wnt signaling pathway and the tumor immune microenvironment (TIME). Aim: To assess whether or not the TCF-4 variants regulate the expression of PD-L1 in cancer cells under normoxia. Methods: HAK-1A, a well-differentiated HCC cell line (Hepatology 1993), was used in this study. TCF-4K mutants (S269A, S272A, and S273A) were prepared with the conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. HAK-1A-derived stable clones overexpressing TCF-4J, K, and K-mutants (S269A, S272A, and S273A cells, respectively) were established. Empty vector-transfected mock cells (EV cells) were used as a control. Western blot analysis and real-time RT-PCR were employed to evaluate protein and mRNA expression levels, respectively. Results: J cells and K cells expressed PD-L1 mRNA 2-fold and 4-fold of that in the EV cells, respectively. Of note, the PD-L1 expression mRNA was enhanced up to 11-fold in S269A cells, 11-fold in S272A cells, and 17-fold in S273A cells. The increased mRNA expressions were verified in protein levels. Conclusion: PD-L1 expression was regulated by TCF-4 variants in liver cancer cells under normoxic conditions. The finding suggests that the Wnt signaling pathway fine-tunes the TIME by regulating the expression levels of PD-L1 in a HIF-1α-independent manner. Citation Format: Hironori Koga, Yasuko Imamura, Toshimitsu Tanaka, Hideki Iwamoto, Toru Nakamura, Atsutaka Masuda, Takahiko Sakaue, Hiroyuki Suzuki, Hirohisa Yano, Takumi Kawaguchi. T-cell factor-4 variants regulate PD-L1 expression in liver cancer cells. [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 5870.

Abstract 4903: Levels of folate transporters impact the compartmentalization of one-carbon metabolism in the mitochondria vs cytosol providing a unique vulnerability to SHMT inhibition

Abstract These studies characterize the critical role of the Reduced Folate Carrier (RFC) and the Proton Coupled Folate Transporter (PCFT) as determinants of cancer cell reliance on mitochondrial vs cytosolic one carbon (C1) metabolism, to identify a unique susceptibility towards SHMT1 and SHMT2 inhibition in cancer cells. C1 metabolism is frequently reprogrammed in cancer cells to provide nucleotides, amino acids, and glutathione, and to maintain redox homeostasis for proliferation. The mitochondrial C1 converting enzymes serine hydroxymethyltransferase (SHMT) 2 and methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) are among the top overexpressed metabolic enzymes in human cancers, suggesting these are important cancer-specific targets. We previously discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates (with AGF347 being the lead) that potently inhibit mitochondrial SHMT2, as well as SHMT1. Folates are essential cofactors for C1 converting reactions; folates and antifolates primarily rely on the major facilitative folate transporters RFC and PCFT for internalization by tumor cells. In these studies, we explore the effects of folate transporter expression on the compartmentalization of C1 metabolism in the mitochondria and cytosol and the potential impact on SHMT1 and SHMT2 inhibition. We used a tetracycline inducible system for RFC expression in RFC/PCFT-null HeLa cells, with and without constitutive PCFT, and characterized the impact on transport and accumulation of folates and AGF347 in response to folate transporter expression. Increased accumulation of folates was observed with increasing RFC in the absence of PCFT. In the presence of PCFT, baseline folate accumulation was substantial and independent of RFC expression, indicating a major role of PCFT as a facilitative folate transporter. SHMT2 catalyzes the conversion of serine and THF to glycine and 5, 10-methylene THF in the mitochondria, while SHMT1 catalyzes the reverse reaction in the cytosol, producing THF and serine. To determine how folate transporter expression affects the C1 flux through SHMT1 vs SHMT2, [2,3,3-2H]serine tracer experiments were performed; increased mitochondrial C1 flux through SHMT2 was observed with increasing RFC in the absence of PCFT, whereas the mitochondrial flux through SHMT2 predominated in the presence of PCFT. By cell proliferation assays it was discovered that cells with lower RFC expression exhibit a hypersensitive phenotype towards AGF347 in the absence of PCFT, indicating a unique therapeutic opportunity for targeting SHMT forms in cancer cells with low folate transport activity. Here we identify RFC and PCFT as key determinants for the compartmentalization of C1 flux in the mitochondria vs the cytosol, in turn affecting the sensitivity of cancer cells towards SHMT inhibition. Citation Format: Mathew Joseph Schneider, Carrie O'connor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry H. Matherly. Levels of folate transporters impact the compartmentalization of one-carbon metabolism in the mitochondria vs cytosol providing a unique vulnerability to SHMT inhibition. [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 4903.

Hypoxia controls the expression of genes responsible for serine synthesis in U87MG cells on ERN1-dependent manner.

Objective. Serine synthesis as well as endoplasmic reticulum stress and hypoxia are important factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of ERN1 (endoplasmic reticulum to nucleus signaling) significantly suppressed the glioblastoma cell proliferation and modified the hypoxia regulation. The present study is aimed to investigate the impact of hypoxia on the expression of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine aminotransferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) in U87MG glioblastoma cells in relation to knockdown of ERN1 with the intent to reveal the role of ERN1 signaling pathway on the endoplasmic reticulum stress-dependent regulation of expression of these genes. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed to hypoxia introduced by dimethyloxalylglycine for 4 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH, PSAT1, PDPH, SHMT1, and ATF4 genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that hypoxia up-regulated the expression level of PHGDH, PSAT1, and ATF4 genes in control U87MG cells, but PSPH and SHMT1 genes expression was down-regulated. The expression of PHGDH, PSAT1, and ATF4 genes in glioblastoma cells with knockdown of ERN1 signaling protein was more sensitive to hypoxia, especially PSAT1 gene. At the same time, the expression of PSPH gene in ERN1 knockdown cells was resistant to hypoxia. The expression of SHMT1 gene, encoding the enzyme responsible for conversion of serine to glycine, showed similar negative sensitivity to hypoxia in both control and ERN1 knockdown glioblastoma cells. Conclusion. The results of the present study demonstrate that the expression of genes responsible for serine synthesis is sensitive to hypoxia in gene-specific manner and that ERN1 knockdown significantly modifies the impact of hypoxia on the expression of PHGDH, PSAT1, PSPH, and ATF4 genes in glioblastoma cells and reflects the ERN1-mediated reprograming of hypoxic regulation at gene expression level.

A mutation in SlCHLH encoding a magnesium chelatase H subunit is involved in the formation of yellow stigma in tomato (Solanum lycopersicum L.)

Chlorophylls are ubiquitous pigments responsible for the green color in plants. Changes in the chlorophyll content have a significant impact on photosynthesis, plant growth and development. In this study, we used a yellow stigma mutant (ys) generated from a green stigma tomato WT by using ethylmethylsulfone (EMS)-induced mutagenesis. Compared with WT, the stigma of ys shows low chlorophyll content and impaired chloroplast ultrastructure. Through map-based cloning, the ys gene is localized to a 100 kb region on chromosome 4 between dCAPS596 and dCAPS606. Gene expression analysis and nonsynonymous SNP determination identified the Solyc04g015750, as the potential candidate gene, which encodes a magnesium chelatase H subunit (CHLH). In ys mutant, a single base C to T substitution in the SlCHLH gene results in the conversion of Serine into Leucine (Ser92Leu) at the N-terminal region. The functional complementation test shows that the SlCHLH from WT can rescue the green stigma phenotype of ys. In contrast, knockdown of SlCHLH in green stigma tomato AC, observed the yellow stigma phenotype at the stigma development stage. Overexpression of the mutant gene Slys in green stigma tomato AC results in the light green stigma. These results indicate that the mutation of the N-terminal S92 to Leu in SlCHLH is the main reason for the formation of the yellow stigma phenotype. Characterization of the ys mutant enriches the current knowledge of the tomato chlorophyll mutant library and provides a novel and effective tool for understanding the function of CHLH in tomato.

Sublethal concentrations of heavy metals Cu2+ and Zn2+ can induce the emergence of bacterial multidrug resistance

The emergence of multidrug resistance has become an environmental safety issue of worldwide concern. Numerous studies have confirmed that heavy metals can promote the spread of bacterial resistance. However, in addition to co-resistance plasmids, the role and mechanism of metals at sublethal levels (< minimum inhibitory concentration [MIC]) on antibiotic resistance remain poorly defined. Here we investigated the alteration of resistance phenotype under Cu 2 + and Zn 2 + exposure and explored the molecular mechanism by which heavy metals affect antibiotic resistance. The results showed that the MIC of Escherichia coli and Staphylococcus aureus on antibiotics, especially norfloxacin and tetracyclines, was significantly increased after seven consecutive days of exposure to Cu 2 + and Zn 2 + and exhibited a higher cross-resistance rate, which meant that heavy metals can induce bacteria to develop antibiotic resistances that were not previously available. It was observed the downregulation of outer membrane protein gene omp C after heavy metal exposure, while the expression of other efflux pump and resistance genes in E. coli was upregulated, especially tet B, tol C and arc AB genes, suggesting that heavy metals might enhance antibiotic resistance by altering the expression of efflux pump genes. Moreover, many point mutations were found in the efflux system of resistant mutant strains. In particular, quinolone-resistant mutant strains showed a point mutation in the conversion of serine (Ser) to phenylalanine (Phe) at residue 464 of the gyr B gene, which may also be an important cause of increased quinolone resistance. This study provides an explanation for the effect of heavy metals on bacterial resistance. • The MIC of E. coli and S. aureus on antibiotics increased after Cu 2+ and Zn 2+ exposure. • After exposure to heavy metals, the cross resistance rates also showed an increase. • Heavy metals may enhance antibiotic resistance by altering efflux pump expression. • Many point mutations were found in the efflux system of resistant mutant strains.

Targeting HOTAIRM1 ameliorates glioblastoma by disrupting mitochondrial oxidative phosphorylation and serine metabolism

SummarySerine hydroxymethyltransferase 2 (SHMT2), which catalyzes the conversion of serine to glycine and one-carbon transfer reactions in mitochondria, is significantly upregulated in glioblastoma (GBM). However, the mechanism by which the stability of SHMT2 gene expression is maintained to drive GBM tumorigenesis has not been clarified. Herein, through microarray screening, we identified that HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) modulates the SHMT2 level in various GBM cell lines. Serine catabolism and mitochondrial oxidative phosphorylation activities were decreased by HOTAIRM1 inhibition. Mechanistically, according to our mass spectrometry and eCLIP-seq results, HOTAIRM1 can bind to PTBP1 and IGF2BP2. Furthermore, HOTAIRM1 maintains the stability of SHMT2 by promoting the recognition of an m6A site and the interaction of PTBP1/IGF2BP2 with SHMT2 mRNA. The stability of HOTAIRM1 can also be enhanced and results in positive feedback regulation to support the progression of GBM. Thus, targeting HOTAIRM1 could be a promising metabolic therapy for GBM.

Red light regulates metabolic pathways of soybean hypocotyl elongation and thickening

Plant stem growth and development are regulated by red light, which is an important environmental signal. Using proteomic and metabolomic approaches, differentially expressed proteins (DEPs) and differentially expressed metabolites (DEMs) in soybean hypocotyls were examined between soybean plants subjected to darkness and red light conditions, elucidating the pathways underlying the regulatory effects of red light on soybean hypocotyl growth. The results revealed that red light inhibits hypocotyl elongation and increases the hypocotyl diameter in soybeans. Red light inhibited hypocotyl elongation mainly by enhancing the firmness of the cell wall. First, red light promoted the expression of enzymes related to the conversion of serine and threonine to glycine, decreased the level of intracellular reactive oxygen species (ROS), and reduced the degradation of cell wall polysaccharides. Furthermore, by enhancing the expression of enzymes involved in the lignin monomer synthetic pathway, red light promoted the biosynthesis of lignin to strengthen the cell wall. Moreover, red light promoted hypocotyl thickening mainly by upregulating the expression of aminocyclopropanecarboxylate oxidase, thereby promoting the accumulation of ethylene precursors. This study provides a theoretical basis for using light to regulate soybean stem growth.

SHMT2 promotes cell viability and inhibits ROS-dependent, mitochondrial-mediated apoptosis via the intrinsic signaling pathway in bladder cancer cells.

Mitochondrial serine hydroxymethyltransferase (SHMT2) catalyzes the conversion of serine to glycine and concomitantly produces one-carbon units to support cell growth and is upregulated in various cancer cells. SHMT2 knockdown triggers cell apoptosis; however, the detailed mechanism of apoptosis induced by SHMT2 inactivation remains unknown. Here, we demonstrate that SHMT2 supports the proliferation of bladder cancer (BC) cells by maintaining redox homeostasis. SHMT2 knockout decreased the pools of purine and one-carbon units and delayed cell cycle progression in a manner that was rescued by formate, demonstrating that SHMT2-mediated one-carbon units are essential for BC cell proliferation. SHMT2 deficiency promoted the accumulation of intracellular reactive oxygen species (ROS) by decreasing the NADH/NAD+, NADPH/NADP+, and GSH/GSSG ratios, leading to a loss in mitochondrial membrane potential, release of cytochrome c, translocation of Bcl-2 family protein and activation of caspase-3. Notably, blocking ROS production with the one-carbon donor formate and the ROS scavenger N-acetyl-cysteine (NAC) effectively rescued SHMT2 deficiency-induced cell apoptosis via the intrinsic signaling pathway. Treatment with the SHMT inhibitor SHIN1 resulted in a significant inhibitory effect on cell proliferation and induced cell apoptosis. Formate and NAC rescued SHIN1-induced cell apoptosis. Our findings reveal an important mechanism by which the loss of SHMT2 triggers ROS-dependent, mitochondrial-mediated apoptosis, which gives insight into the link between serine metabolism and cell apoptosis and provides a promising target for BC treatment and drug discovery.

Virtual high-throughput screening: potential inhibitors for the mycobacterial α-subunit of tryptophan synthase

ABSTRACT Mycobacterium tuberculosis (Mtb), which causes tuberculosis (TB), is the world’s most successful pathogen. If it is to survive in phagosomes, the tryptophan biosynthetic pathway is essential. The α-subunit of tryptophan synthase (TRPS) executes the irreversible catalysis of indole-3-glyceral-3-phosphate to generate indole. The β-subunit catalyses the conversion of indole and serine to tryptophan. Targeting the TRPS α-subunit may, therefore, render the enzyme inactive. The crystal structure of α-subunit downloaded from the protein data bank (PDB: 5OCW) has gaps at the amino acid sequence positions in three places. We, therefore, retrieved the entire amino acid sequence of the protein from the UniProt and built a complete protein structure by adding the missing amino acids using a Modeller. The 3D model of the α-subunit was then used as the target to develop inhibitors for Mtb. We downloaded 58,699 natural products from the Maybridge database. After space filtering, molecular docking, molecular dynamic simulation and binding free energy calculation were done to develop inhibitors for the TRPS α-subunit. Out of the 18 compounds selected, five virtual hit compounds, namely MFCD00103482, MFCD00118123, MFCD04110719, MFCD01005315 and MFCD04112426, bind well to the TRPS α-subunit. These five potential inhibitors for Mtb will be taken for preclinical testing.

Gene mutations in resistant strains of Mycobacterium tuberculosis isolated from pulmonary tuberculosis patients in Mazandaran and Golestan provinces of Iran - A cross-sectional study

Due to the significant prevalence of tuberculosis (TB) in Mazandaran and Golestan provinces in northern Iran, we aimed to investigate the gene mutations in resistant Mycobacterium tuberculosis isolated from pulmonary TB patients in these provinces of Iran. In this cross-sectional study, sputum of TB suspected patients were collected and cultured in a period of 3 months. The antibiotic-resistant cases identified using the resistance ratio method. Then, the DNAs of the resistant isolates were extracted manually by the CTAB-Proteinase K method and investigated for gene mutations using PCR and sequencing. Among 26 isolated M. tuberculosis, 4 isolates (15.38%) were resistant to antibiotics, while one (3.84%), 2 (7.69%), and one (3.84%) isolates were resistant to rifampin, isoniazid, and multidrug-resistant (MDR), respectively. The MDR isolate had a mutation in codon 315 of the KatG gene resulting in the conversion of serine to threonine, and a mutation in the codon 450 of the rpoB gene, causing the alteration of serine to leucine. In the rifampin-resistant isolate, a mutation occurred at the same locus converting serine to leucine. The low prevalence of antibiotic resistance in this study indicates the appropriate treatment of the patients, however, the single MDR isolate was identified in Mazandaran province.

Novel transaminases from thermophiles: from discovery to application.

SummaryTransaminases (TAs) are promising biocatalysts for chiral amine synthesis; however, only few thermophilic TAs have been described to date. In this work, a genome mining approach was taken to seek novel TAs from nine thermophilic microorganisms. TA sequences were identified from their respective genome sequences and their Pfam were predicted confirming that TAs class I–II are the most abundant (50%), followed by class III (26%), V (16%), IV (8%) and VI (1%). The percentage of open reading frames (ORFs) that are TAs ranges from 0.689% in Thermococcus litoralis to 0.424% in Sulfolobus solfataricus. A total of 94 putative TAs were successfully cloned and expressed into E. coli, showing mostly good expression levels when using a chemical chaperone media containing d‐sorbitol. Kinetic and end‐point colorimetric assays with different amino donors–acceptors confirmed TAs activity allowing for initial exploration of the substrate scope. Stereoselective and non‐stereoselective serine‐TAs were selected for the synthesis of hydroxypyruvate (HPA). Low HPA reaction yields were observed with four non‐stereoselective serine‐TAs, whilst two stereoselective serine‐TAs showed significantly higher yields. Coupling serine‐TA reactions to a transketolase to yield l‐erythrulose (Ery) substantially increased serine conversion into HPA. Combining both stereoselective serine‐TAs and transketolase using the inexpensive racemic D/L‐serine led to high Ery yield (82%). Thermal characterization of stereoselective serine‐TAs confirmed they have excellent thermostability up to 60°C and high optimum temperatures.

Synthesis of Enantiopure Unnatural Amino Acids by Metallaphotoredox Catalysis.

We describe herein a two-step process for the conversion of serine to a wide array of optically pure unnatural amino acids. This method utilizes a photocatalytic cross-electrophile coupling between a bromoalkyl intermediate and a diverse set of aryl halides to produce artificial analogues of phenylalanine, tryptophan, and histidine. The reaction is tolerant of a broad range of functionalities and can be leveraged toward the scalable synthesis of valuable pharmaceutical scaffolds via flow technology.

Abstract 1992: Serine hydroxymethyltransferase-2 as regulator of oxidative stress and chemoradiation resistance in lung adenocarcinoma

Abstract Purpose/Objective(s): Lung cancer is the leading cause of cancer deaths in the United States and worldwide. There has been little improvement in survival over the past several decades and surgery is generally considered to be the only curative treatment. We, therefore, performed a genome-wide functional genomics screen on human lung adenocarcinoma cells to identify genes involved in chemo- and radiotherapy resistance. Based on these results, we hypothesized that serine hydroxymethyltransferase-2 (SHMT2), which catalyzes the conversion of serine to glycine, is a target for chemoradiotherapy resistance in preclinical models of lung adenocarcinoma. Materials/Methods: PC9 cells were treated three times with 1 uM cisplatin and 2 Gy radiation before being subjected to genomic, untargeted metabolomic, or proteomic analysis. For the genomic analysis, PC9 cells were transfected with an shRNA viral library containing ~60,000 individual shRNAs following treatment. shRNAs that were depleted were hypothesized to represent genes involved in chemo-/radioresistance. Proteomics analysis was conducted after treatment and quantified by tandem mass tags isobaric labeling, totaling ~2,100 quantifiable proteins. An untargeted metabolomics approach was conducted after treatment, using gas chromatography mass spectrometry to quantify ~95 metabolites. Protein expression following treatment was determined by Western blot. Potential drug targets were assessed via colony formation assays. The Cancer Genome Atlas (TCGA) was queried to confirm in vivo human clinical correlations. Results: Our genomic, proteomic, and metabolomics screens indicated glutathione detoxification and oxidative stress response were major compensatory pathways upregulated after cisplatin and radiation treatment. We validated three targets, SLC7A11, GPX4, and SHMT2, as genes involved in chemo-radioresistance in vitro. The respective small molecule inhibitors of these gene targets, IKE, RSL3, and SHIN1, were synergistic when combined with cisplatin and radiation. SHIN1 appeared to also induce oxidative stress through ferroptosis, although this mechanism of cell death must be interrogated further. Analysis of TCGA revealed SHMT2 expression is upregulated in human lung adenocarcinoma in vivo and that higher expression is associated with decreased overall and disease-free survival. Conclusion: SHMT2 was identified as a novel target to overcome treatment resistance in human lung adenocarcinoma, which may be regulated ferroptotic cell death. Citation Format: Kunal R. Chaudhary, Connor J. Kinslow, Ling F. Ye, Pavan S. Upadhyayula, Eun Y. Lee, Ramon C. Sun, Simon K. Cheng. Serine hydroxymethyltransferase-2 as regulator of oxidative stress and chemoradiation resistance in lung adenocarcinoma [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 1992.

Loss of SHMT2 and Folate Deficiency Impair Energy Metabolism in Mouse Embryonic Fibroblasts Cells

ObjectivesSerine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine producing THF-conjugated one-carbon units and glycine in the mitochondria. Recently biallelic SHMT2 variants were identified in humans and suggested to alter the protein’s active site, potentially disrupting enzymatic function. Indeed, patient fibroblasts exhibited impaired oxidative capacity, however, with no changes in oxidative phosphorylation protein levels. Furthermore, immortalized cells models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. We have developed a mouse model of reduced Shmt2 expression that provides an appropriate reference model to further study the deleterious human SHMT2 variants and their connection to folate metabolism. MethodsPrimary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6 dam crossed with a heterozygous Shmt2+/– (HET) male. MEF embryos were genotyped, and Shmt2+/+ (wild type) or Shmt2+/– MEF cells were cultured for 4 doubling in 2 nM (deficient) or 25 nM folate (sufficient) medium. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, and mitochondrial membrane potential and function. ResultsLoss of Shmt2 and folate deficiency significantly reduced cell proliferation and oxygen consumption rate. Total cellular folate levels and pyruvate kinase enzyme activity and protein levels were reduced with folate deficiency. While there were no changes in mitochondrial DNA content or complex 1 proteins NDUFA9 or ND3, mitochondrial membrane potential was impaired and PGC1α protein levels were elevated in Shmt2+/– MEF cells. ConclusionsOur results demonstrate that loss of SHMT2 impairs cellular proliferation and that SHMT2 expression and folate status impact cellular energy metabolism and mitochondrial health. Funding SourcesThis study was financially supported by a President’s Council of Cornell Women Award and Cornell University Division of Nutritional Sciences funds.

Silencing SHMT2 inhibits the progression of tongue squamous cell carcinoma through cell cycle regulation

BackgroundSerine hydroxymethyltransferase 2 (SHMT2) is a vital metabolic enzyme in one carbon metabolism catalyzing the conversion of serine to glycine, which has been reported to play a crucial role in the progression of tumors. However, its function in tongue squamous cell carcinoma (TSCC) remains unclear.MethodsSHMT2 expression was analyzed using samples in online databases, and was assessed through immunohistochemistry staining of collected clinical specimens. The correlation between SHMT2 expression and the cell cycle was predicted through bioinformatic analysis, including weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA). After transfection with siRNA, CCK8 assay, Edu staining, flow cytometry, trans-well assay, and wound healing experiments were performed to verify the functional role of SHMT2 in vitro. A stable cell line with SHMT2 silencing was established to detect the oncogenic function of SHMT2 in vivo.ResultsThe expression of SHMT2 was up-regulated in TSCC tissues and cell lines compared with normal groups, and highly expressed SHMT2 significantly indicated a poorer clinical outcome for TSCC patients. Bioinformatic analysis found that high expression of SHMT2 was closely related with biologic process including cell cycle and cell cycle G1/S transition. Down regulating of SHMT2 significantly suppressed the proliferation, invasive and migrative ability of TSCC cells, and induced the prolongation of the G1 phase of the cell cycle in vitro. Furthermore, western blot showed that cell cycle-related regulators such as cyclin-dependent kinase 4 (CDK4) and cyclinD1 expression levels were decreased, while the expression levels of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 were increased after SHMT2 knockdown. Silencing SHMT2 in the HN6 cell line using short hairpin RNA also impeded tumor growth in vivo.ConclusionsOverexpression of SHMT2 in TSCC indicated low survival rates, and was associated with aggressive behaviors of TSCC. It was also found to be involved in cell cycle regulation of TSCC cells. SHMT2 may serve as a novel prognostic indicator of TSCC.

Revealing the Metabolic Alterations during Biofilm Development of Burkholderia cenocepacia Based on Genome-Scale Metabolic Modeling.

Burkholderia cenocepacia is among the important pathogens isolated from cystic fibrosis (CF) patients. It has attracted considerable attention because of its capacity to evade host immune defenses during chronic infection. Advances in systems biology methodologies have led to the emergence of methods that integrate experimental transcriptomics data and genome-scale metabolic models (GEMs). Here, we integrated transcriptomics data of bacterial cells grown on exponential and biofilm conditions into a manually curated GEM of B. cenocepacia. We observed substantial differences in pathway response to different growth conditions and alternative pathway susceptibility to extracellular nutrient availability. For instance, we found that blockage of the reactions was vital through the lipid biosynthesis pathways in the exponential phase and the absence of microenvironmental lysine and tryptophan are essential for survival. During biofilm development, bacteria mostly had conserved lipid metabolism but altered pathway activities associated with several amino acids and pentose phosphate pathways. Furthermore, conversion of serine to pyruvate and 2,5-dioxopentanoate synthesis are also identified as potential targets for metabolic remodeling during biofilm development. Altogether, our integrative systems biology analysis revealed the interactions between the bacteria and its microenvironment and enabled the discovery of antimicrobial targets for biofilm-related diseases.

The serine hydroxymethyltransferase-2 (SHMT2) initiates lymphoma development through epigenetic tumor suppressor silencing.

Cancer cells adapt their metabolic activities to support growth and proliferation. However, increased activity of metabolic enzymes is not usually considered an initiating event in the malignant process. Here, we investigate the possible role of the enzyme serine hydroxymethyltransferase-2 (SHMT2) in lymphoma initiation. SHMT2 localizes to the most frequent region of copy number gains at chromosome 12q14.1 in lymphoma. Elevated expression of SHMT2 cooperates with BCL2 in lymphoma development; loss or inhibition of SHMT2 impairs lymphoma cell survival. SHMT2 catalyzes the conversion of serine to glycine and produces an activated one-carbon unit that can be used to support S-adenosyl methionine synthesis. SHMT2 induces changes in DNA and histone methylation patterns leading to promoter silencing of previously uncharacterized mutational genes, such as SASH1 and PTPRM. Together, our findings reveal that amplification of SHMT2 in cooperation with BCL2 is sufficient in the initiation of lymphomagenesis through epigenetic tumor suppressor silencing.