bims-mascan 2019-06-02 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2019–06–02
thirty-six papers selected by
Giovanny Rodríguez Blanco, The Beatson Institute for Cancer Research

Cancer cell lipid class homeostasis is altered under nutrient-deprivation but stable under hypoxia.
Lactate Dehydrogenases as Metabolic Links between Tumor and Stroma in the Tumor Microenvironment.
Effect of cold atmospheric plasma treatment on the metabolites of human leukemia cells.
Dietary Walnut Supplementation Alters Mucosal Metabolite Profiles During DSS-Induced Colonic Ulceration.
Characterization of Hydroxy and Hydroperoxy Polyunsaturated Fatty Acids by Mass Spectrometry.
High-quality MS/MS spectrum prediction for data-dependent and data-independent acquisition data analysis.
CDK9 Inhibition Induces a Metabolic Switch that Renders Prostate Cancer Cells Dependent on Fatty Acid Oxidation.
Metabolomics Profiling before, during, and after the Beijing Olympics: A Panel Study of Within-Individual Differences during Periods of High and Low Air Pollution.
Lipid Metabolism and Endocrine Resistance in Prostate Cancer, and New Opportunities for Therapy.
Inflammation and Metabolism in Cancer Cell-Mitochondria Key Player.
Analysis of oxidised and glycated aminophospholipids: Complete structural characterisation by C30 liquid chromatography-high resolution tandem mass spectrometry.
PKM2 Knockdown Induces Autophagic Cell Death via AKT/mTOR Pathway in Human Prostate Cancer Cells.
Inhibition of nucleotide synthesis promotes replicative senescence of human mammary epithelial cells.
When fat goes down, prostate cancer is on the ropes.
Isocitrate dehydrogenase 1-mutated cancers are sensitive to the green tea polyphenol epigallocatechin-3-gallate.
Prosit: proteome-wide prediction of peptide tandem mass spectra by deep learning.
A systematic review of the effects of increasing arachidonic acid intake on PUFA status, metabolism and health-related outcomes in humans.
Mass spectrometry analysis of protein biomarkers for hepatocellular carcinoma.
Inhibition of glycogen catabolism induces intrinsic apoptosis and augments multikinase inhibitors in hepatocellular carcinoma cells.
Metformin inhibits PPARδ agonist-mediated tumor growth by reducing Glut1 and SLC1A5 expressions of cancer cells.
The Relevance of Mass Spectrometry Analysis for Personalized Medicine through Its Successful Application in Cancer "Omics".
Characterization of Lipid and Lipid Droplet Metabolism in Human HCC.
Human pancreatic cancer cell exosomes, but not human normal cell exosomes, act as an initiator in cell transformation.
Malignant manipulaTORs of metabolism: suppressing BCAA catabolism to enhance mTORC1 activity.
Mapping and Profiling Lipid Distribution in a 3D Model of Breast Cancer Progression.
Analysis of Proteasome-Generated Antigenic Peptides by Mass Spectrometry.
Metabolomics identifies serum and exosomes metabolite markers of pancreatic cancer.
Direct Analysis of Protein S-Acylation by Mass Spectrometry.
The metabolites NADP+ and NADPH are the targets of the circadian protein Nocturnin (Curled).
Fecal Metabolomics and Potential Biomarkers for Systemic Lupus Erythematosus.
Effects of the Extracellular Matrix on the Proteome of Primary Skin Fibroblasts.
Enrichment of S-Palmitoylated Proteins for Mass Spectrometry Analysis.
MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation.
Linoleic acid esters of hydroxy linoleic acids are anti-inflammatory lipids found in plants and mammals.
High glucose promotes breast cancer proliferation and metastasis by impairing angiotensinogen expression.
Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation.

BMC Cancer. 2019 May 28. 19(1): 501

Cancer cell lipid class homeostasis is altered under nutrient-deprivation but stable under hypoxia.

Jan Lisec, Carsten Jaeger, Rida Rashid, Rimsha Munir, Nousheen Zaidi.

   BACKGROUND: Cancer cells modify the balance between fatty acid (FA) synthesis and uptake under metabolic stress, induced by oxygen/nutrient deprivation. These modifications were shown to alter the levels of individual triglyceride (TG) or phospholipid sub-species. To attain a holistic overview of the lipidomic profiles of cancer cells under stress we performed a broad lipidomic assay, comprising 244 lipids from six major classes. This assay allowed us to perform robust analyses and assess the changes in averages of broader lipid-classes, stratified on the basis of saturation index of their fatty-acyl side chains.
METHODS: Global lipidomic profiling using Liquid Chromatography-Mass Spectrometry was performed to assess lipidomic profiles of biologically diverse cancer cell lines cultivated under metabolically stressed conditions.
RESULTS: Neutral lipid compositions were markedly modified under serum-deprived conditions and, strikingly, the cellular level of triglyceride subspecies decreased with increasing number of double bonds in their fatty acyl chains. In contrast and unexpectedly, no robust changes were observed in lipidomic profiles of hypoxic (2% O2) cancer cells despite concurrent changes in proliferation rates and metabolic gene expression.
CONCLUSIONS: Serum-deprivation significantly affects lipidomic profiles of cancer cells. Although, the levels of individual lipid moieties alter under hypoxia (2% O2), the robust averages of broader lipid classes remain unchanged.

Keywords:  Fatty acid metabolism; Lipidomic profile; Metabolic stress; Tumor metabolism

DOI:  https://doi.org/10.1186/s12885-019-5733-y
Cancers (Basel). 2019 May 29. pii: E750. [Epub ahead of print]11(6):

Lactate Dehydrogenases as Metabolic Links between Tumor and Stroma in the Tumor Microenvironment.

Deepshikha Mishra, Debabrata Banerjee.

  Cancer is a metabolic disease in which abnormally proliferating cancer cells rewire metabolic pathways in the tumor microenvironment (TME). Molecular reprogramming in the TME helps cancer cells to fulfill elevated metabolic demands for bioenergetics and cellular biosynthesis. One of the ways through which cancer cell achieve this is by regulating the expression of metabolic enzymes. Lactate dehydrogenase (LDH) is the primary metabolic enzyme that converts pyruvate to lactate and vice versa. LDH also plays a significant role in regulating nutrient exchange between tumor and stroma. Thus, targeting human lactate dehydrogenase for treating advanced carcinomas may be of benefit. LDHA and LDHB, two isoenzymes of LDH, participate in tumor stroma metabolic interaction and exchange of metabolic fuel and thus could serve as potential anticancer drug targets. This article reviews recent research discussing the roles of lactate dehydrogenase in cancer metabolism. As molecular regulation of LDHA and LDHB in different cancer remains obscure, we also review signaling pathways regulating LDHA and LDHB expression. We highlight on the role of small molecule inhibitors in targeting LDH activity and we emphasize the development of safer and more effective LDH inhibitors. We trust that this review will also generate interest in designing combination therapies based on LDH inhibition, with LDHA being targeted in tumors and LDHB in stromal cells for better treatment outcome.

Keywords:  LDHA; LDHB; combination therapy; isoenzymes; lactate; lactate dehydrogenase; metabolic cooperation; metabolism; tumor microenvironment; tumor stroma

DOI:  https://doi.org/10.3390/cancers11060750
Cancer Cell Int. 2019 ;19 135

Effect of cold atmospheric plasma treatment on the metabolites of human leukemia cells.

Dehui Xu, Ning Ning, Yujing Xu, Bingchuan Wang, Qingjie Cui, Zhijie Liu, Xiaohua Wang, Dingxin Liu, Hailan Chen, Michael G Kong.

   Background: Acute myeloid leukemia (AML) is a typically fatal malignancy and new drug and treatment need to be developed for a better survival outcome. Cold atmospheric plasma (CAP) is a novel technology, which has been widely applied in biomedicine, especially in various of cancer treatment. However, the changes in cell metabolism after CAP treatment of leukemia cells have been rarely studied.
Methods: In this study, we investigated the metabolite profiling of plasma treatment on leukemia cells based on Gas Chromatography Tandem Time-of-Flight Mass Spectrometry (GC-TOFMS). Simultaneously, we conducted a series of bioinformatics analysis of metabolites and metabolic pathways with significant differences after basic data analysis.
Results: 800 signals were detected by GC-TOF mass-spectrometry and then evaluated using PCA and OPLS-DA. All the differential metabolites were listed and the related metabolic pathways were analyzed by KEGG pathway. The results showed that alanine, aspartate and glutamate metabolism had a significant change after plasma treatment. Meanwhile, d-glutamine and d-glutamate metabolism were significantly changed by CAP. Glutaminase activity was decreased after plasma treatment, which might lead to glutamine accumulation and leukemia cells death.
Conclusions: We found the above two metabolic pathways vulnerable to plasma treatment, which might result in leukemia cells death and might be the cornerstone of further exploration of plasma treatment targets.

Keywords:  Acute myeloid leukemia; Alanine, aspartate and glutamate metabolism; Cold atmospheric plasma; Metabolite profiling; d-Glutamine and d-glutamate metabolism

DOI:  https://doi.org/10.1186/s12935-019-0856-4
Nutrients. 2019 May 20. pii: E1118. [Epub ahead of print]11(5):

Dietary Walnut Supplementation Alters Mucosal Metabolite Profiles During DSS-Induced Colonic Ulceration.

Masako Nakanishi, Alyssa Matz, Cory Klemashevich, Daniel W Rosenberg.

  Walnuts contain a complex array of natural compounds and phytochemicals that exhibit a wide range of health benefits, including protection against inflammation and colon cancer. In this study, we assess the effects of dietary supplementation with walnuts on colonic mucosal injury induced in mice by the ulcerogenic agent, dextran sodium sulfate (DSS). C57Bl/6J mice were started on the Total Western Diet supplemented with freshly-ground whole walnuts (0, 3.5, 7 and 14% g/kg) 2 weeks prior to a 5-day DSS treatment and walnut diets were continued throughout the entire experimental period. Mice were examined at 2 days or 10 days after withdrawal of DSS. In a separate study, a discovery-based metabolite profiling analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on fecal samples and colonic mucosa following two weeks of walnut supplementation. Dietary walnut supplementation showed significant effects in the 10-day post-DSS recovery-phase study, in which the extent of ulceration was significantly reduced (7.5% vs. 0.3%, p < 0.05) with 14% walnuts. In the metabolite-profiling analysis, walnuts caused a significant increase in several polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) and 9-oxo-10(E),12(E)-octadecadienoic acid (9-oxoODA), as well as kynurenic acid. In colon tissue samples, walnuts caused a significant increase in the levels of S-adenosylhomocysteine (SAH) and betaine, important components of fatty acid β-oxidation. These metabolite changes may contribute in part to the observed protection against DSS-induced inflammatory tissue injury.

Keywords:  Inflammatory Bowel Disease; inflammation; lipid metabolites; omega-3 fatty acids; ulcerative colitis; walnuts

DOI:  https://doi.org/10.3390/nu11051118
Adv Exp Med Biol. 2019 ;1127 21-35

Characterization of Hydroxy and Hydroperoxy Polyunsaturated Fatty Acids by Mass Spectrometry.

Priscilla Bento Matos Cruz Derogis, Adriano B Chaves-Fillho, Sayuri Miyamoto.

  Lipids containing unsaturated doubles bonds are oxidized by enzymatic and non-enzymatic mechanisms yielding hydroperoxides and hydroxides as primary products.This process has been implicated in physiological and pathological mechanisms. Thus, precise characterization and quantification of lipid oxidation products in biological samples can provide important mechanistic insights. In this context, the use of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to characterize the primary products of polyunsaturated fatty acids, like hydroxides and hydroperoxides, is a sensitive, specific and versatile tool. Here we will focus on the characterization and specific analysis of hydroxy and hydroperoxy regioisomers of linoleic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid by LC-MS/MS.

Keywords:  Fatty acid hydroperoxides; Fatty acid hydroxides; Mass spectrometry; Polyunsaturated fatty acids

DOI:  https://doi.org/10.1007/978-3-030-11488-6_2
Nat Methods. 2019 Jun;16(6): 519-525

High-quality MS/MS spectrum prediction for data-dependent and data-independent acquisition data analysis.

Shivani Tiwary, Roie Levy, Petra Gutenbrunner, Favio Salinas Soto, Krishnan K Palaniappan, Laura Deming, Marc Berndl, Arthur Brant, Peter Cimermancic, Jürgen Cox.

Peptide fragmentation spectra are routinely predicted in the interpretation of mass-spectrometry-based proteomics data. However, the generation of fragment ions has not been understood well enough for scientists to estimate fragment ion intensities accurately. Here, we demonstrate that machine learning can predict peptide fragmentation patterns in mass spectrometers with accuracy within the uncertainty of measurement. Moreover, analysis of our models reveals that peptide fragmentation depends on long-range interactions within a peptide sequence. We illustrate the utility of our models by applying them to the analysis of both data-dependent and data-independent acquisition datasets. In the former case, we observe a q-value-dependent increase in the total number of peptide identifications. In the latter case, we confirm that the use of predicted tandem mass spectrometry spectra is nearly equivalent to the use of spectra from experimental libraries.

DOI: https://doi.org/10.1038/s41592-019-0427-6
Neoplasia. 2019 May 28. pii: S1476-5586(19)30108-3. [Epub ahead of print]21(7): 713-720

CDK9 Inhibition Induces a Metabolic Switch that Renders Prostate Cancer Cells Dependent on Fatty Acid Oxidation.

Harri M Itkonen, Ninu Poulose, Suzanne Walker, Ian G Mills.

Cyclin-dependent kinase 9 (CDK9), a key regulator of RNA-polymeraseII, is a candidate drug target for cancers driven by transcriptional deregulation. Here we report a multi-omics-profiling of prostate cancer cell responses to CDK9 inhibition to identify synthetic lethal interactions. These interactions were validated using live-cell imaging, mitochondrial flux-, viability- and cell death activation assays. We show that CDK9 inhibition induces acute metabolic stress in prostate cancer cells. This is manifested by a drastic down-regulation of mitochondrial oxidative phosphorylation, ATP depletion and induction of a rapid and sustained phosphorylation of AMP-activated protein kinase (AMPK), the key sensor of cellular energy homeostasis. We used metabolomics to demonstrate that inhibition of CDK9 leads to accumulation of acyl-carnitines, metabolic intermediates in fatty acid oxidation (FAO). Acyl-carnitines are produced by carnitine palmitoyltransferase enzymes 1 and 2 (CPT), and we used both genetic and pharmacological tools to show that inhibition of CPT-activity is synthetically lethal with CDK9 inhibition. To our knowledge this is the first report to show that CDK9 inhibition dramatically alters cancer cell metabolism.

DOI: https://doi.org/10.1016/j.neo.2019.05.001
Environ Health Perspect. 2019 May;127(5): 57010

Metabolomics Profiling before, during, and after the Beijing Olympics: A Panel Study of Within-Individual Differences during Periods of High and Low Air Pollution.

Lina Mu, Zhongzheng Niu, Rachael Hageman Blair, Han Yu, Richard W Browne, Matthew R Bonner, Tiffany Fanter, Furong Deng, Mya Swanson.

BACKGROUND: The metabolome is a collection of exogenous chemicals and metabolites from cellular processes that may reflect the body's response to environmental exposures. Studies of air pollution and metabolomics are limited.
OBJECTIVES: To explore changes in the human metabolome before, during, and after the 2008 Beijing Olympics Games, when air pollution was high, low, and high, respectively.
METHODS: Serum samples were collected before, during, and after the Olympics from 26 participants in an existing panel study. Gas and ultra-high performance liquid chromatography/mass spectrometry were used in metabolomics analysis. Repeated measures ANOVA, network analysis, and enrichment analysis methods were employed to identify metabolites and classes associated with air pollution changes.
RESULTS: A total of 886 molecules were measured in our metabolomics analysis. Network partitioning identified four modules with 65 known metabolites that significantly changed across the three time points. All known molecules in the first module ([Formula: see text]) were lipids (e.g., eicosapentaenoic acid, stearic acid). The second module consisted primarily of dipeptides ([Formula: see text], e.g., isoleucylglycine) plus 8 metabolites from four other classes (e.g., hypoxanthine, 12-hydroxyeicosatetraenoic acid). Most of the metabolites in Modules 3 (19 of 23) and 4 (5 of 5) were unknown. Enrichment analysis of module-identified metabolites indicted significantly overrepresented pathways, including long- and medium-chain fatty acids, polyunsaturated fatty acids (n3 and n6), eicosanoids, lysolipid, dipeptides, fatty acid metabolism, and purine metabolism [(hypo) xanthine/inosine-containing pathways].
CONCLUSIONS: We identified two major metabolic signatures: one consisting of lipids, and a second that included dipeptides, polyunsaturated fatty acids, taurine, and xanthine. Metabolites in both groups decreased during the 2008 Beijing Olympics, when air pollution was low, and increased after the Olympics, when air pollution returned to normal (high) levels. https://doi.org/10.1289/EHP3705.

DOI: https://doi.org/10.1289/EHP3705
Int J Mol Sci. 2019 May 28. pii: E2626. [Epub ahead of print]20(11):

Lipid Metabolism and Endocrine Resistance in Prostate Cancer, and New Opportunities for Therapy.

Gergana E Stoykova, Isabel R Schlaepfer.

  Prostate cancer (PCa) is the most common cancer in men, and more than 10% of men will be diagnosed with PCa during their lifetime. Patients that are not cured with surgery or radiation are largely treated with endocrine therapies that target androgens or the androgen receptor (AR), a major driver of PCa. In response to androgen deprivation, most PCas progress to castrate resistant PCa, which is treated with anti-androgens like enzalutamide, but tumors still progress and become incurable. Thus, there is a critical need to identify cellular pathways that allow tumors to escape anti-androgen therapies. Epidemiological studies suggest that high-fat diets play important roles in PCa progression. Lipid metabolism rewires the PCa metabolome to support growth and resistance to endocrine therapies, although the exact mechanisms remain obscure. Therapeutic effects have been observed inhibiting several aspects of PCa lipid metabolism: Synthesis, uptake, and oxidation. Since AR remains a driver of PCa in advanced disease, strategies targeting both lipid metabolism and AR are starting to emerge, providing new opportunities to re-sensitize tumors to endocrine therapies with lipid metabolic approaches.

Keywords:  AR; CPT1A; FASN; anti-androgens; combination therapy; dietary lipids; endocrine resistance; lipid oxidation; lipid synthesis; prostate cancer

DOI:  https://doi.org/10.3390/ijms20112626
Front Oncol. 2019 ;9 348

Inflammation and Metabolism in Cancer Cell-Mitochondria Key Player.

Monica Neagu, Carolina Constantin, Iulia Dana Popescu, Donato Zipeto, George Tzanakakis, Dragana Nikitovic, Concettina Fenga, Constantine A Stratakis, Demetrios A Spandidos, Aristidis M Tsatsakis.

  Cancer metabolism is an essential aspect of tumorigenesis, as cancer cells have increased energy requirements in comparison to normal cells. Thus, an enhanced metabolism is needed in order to accommodate tumor cells' accelerated biological functions, including increased proliferation, vigorous migration during metastasis, and adaptation to different tissues from the primary invasion site. In this context, the assessment of tumor cell metabolic pathways generates crucial data pertaining to the mechanisms through which tumor cells survive and grow in a milieu of host defense mechanisms. Indeed, various studies have demonstrated that the metabolic signature of tumors is heterogeneous. Furthermore, these metabolic changes induce the exacerbated production of several molecules, which result in alterations that aid an inflammatory milieu. The therapeutic armentarium for oncology should thus include metabolic and inflammation regulators. Our expanding knowledge of the metabolic behavior of tumor cells, whether from solid tumors or hematologic malignancies, may provide the basis for the development of tailor-made cancer therapies.

Keywords:  cancer cell; inflammation; metabolic pathways; therapy targets; tumorigenesis

DOI:  https://doi.org/10.3389/fonc.2019.00348
Free Radic Biol Med. 2019 May 28. pii: S0891-5849(19)30356-9. [Epub ahead of print]

Analysis of oxidised and glycated aminophospholipids: Complete structural characterisation by C30 liquid chromatography-high resolution tandem mass spectrometry.

Simone Colombo, Angela Criscuolo, Martin Zeller, Maria Fedorova, M Rosário Domingues, Pedro Domingues.

  The aminophospholipids (APL), phosphatidylethanolamine (PE) and phosphatidylserine (PS) are widely present in cell membranes and lipoproteins. Glucose and reactive oxygen species (ROS), such as the hydroxyl radical (•OH), can react with APL leading to an array of oxidised, glycated and glycoxidised derivatives. Modified APL have been implicated in inflammatory diseases and diabetes, and were identified as signalling molecules in regulating cell death. However, the biological relevance of these molecules has not been completely established, since they are present in very low amounts, and new sensitive methodologies are needed to detect them in biological systems. Few studies have focused on the characterisation of APL modifications using liquid chromatography-tandem mass spectrometry (LC-MS/MS), mainly using C5 or C18 reversed phase (RP) columns. In the present study, we propose a new analytical approach for the characterisation of complex mixtures of oxidised, glycated and glycoxidised PE and PS. This LC approach was based on a reversed-phase C30 column combined with high-resolution MS, and higher energy C-trap dissociation (HCD) MS/MS. C30 RP-LC separated short and long fatty acyl oxidation products, along with glycoxidised APL bearing oxidative modifications on the glucose moiety and the fatty acyl chains. Functional isomers (e.g. hydroxy-hydroperoxy-APL and tri-hydroxy-APL) and positional isomers (e.g. 9-hydroxy-APL and 13-hydroxy-APL) were also discriminated by the method. HCD fragmentation patterns allowed unequivocal structural characterisation of the modified APL, and are translatable into targeted MS/MS fingerprinting of the modified derivatives in biological samples.

Keywords:  Glycation; Lipidomics; Mass spectrometry; Oxidation; Phosphatidylethanolamine; Phosphatidylserine

DOI:  https://doi.org/10.1016/j.freeradbiomed.2019.05.025
Cell Physiol Biochem. 2019 ;52(6): 1535-1552

PKM2 Knockdown Induces Autophagic Cell Death via AKT/mTOR Pathway in Human Prostate Cancer Cells.

Prasanta Dey, Amit Kundu, Richa Sachan, Jae Hyeon Park, Mee Young Ahn, Kyungsil Yoon, Jaewon Lee, Nam Deuk Kim, In Su Kim, Byung Mu Lee, Hyung Sik Kim.

   BACKGROUND/AIMS: Pyruvate kinase M2 (PKM2) is essential for aerobic glycolysis. Although high PKM2 expression is observed in various cancer tissues, its functional role in cancer metabolism is unclear. Here, we investigated the role of PKM2 in regulating autophagy and its associated pathways in prostate cancer cells.
METHODS: Immunohistochemistry was performed to compare the expression level of PKM2 in prostate cancer patients and normal human, whereas expression of PKM2 in several cell lines was also examined by using western blot. PKM2 expression was silenced using various small interfering RNAs (siRNAs). Cell viability was examined using IncuCyte ZOOM™ live cell imaging system. Western blotting and immunofluorescence were performed to investigate the PKM2 knockdown on other cellular signaling molecules. Acridine orange and Monodansylcadaverine staining was performed to check effect of PKM2 knockdown on autophagy induction. High performance thin layer chromatography was carried out to quantify the level of different cellular metabolites (pyruvate and lactate). Colony formation assay was performed to determine the ability of a cells to form large colonies.
RESULTS: PKM2 was highly expressed in prostate cancer patients as compared to normal human. PKM2 siRNA-transfected prostate cancer cells showed significantly reduced viability. Acridine orange, Monodansylcadaverine staining and western blotting analysis showed that PKM2 downregulation markedly increased autophagic cell death. Results of western blotting analysis showed that PKM2 knockdown affected protein kinase B/mechanistic target of rapamycin 1 pathway, which consequently downregulated the expression of glycolytic enzymes lactate dehydrogenase A and glucose transporter 1. Knockdown of PKM2 also reduced the colony formation ability of human prostate cancer cell DU145.
CONCLUSION: To the best of our knowledge, this is the first study to show that PKM2 inhibition alters prostate cancer cell metabolism and induces autophagy, thus providing new perspectives for developing PKM2-targeting anticancer therapies for treating prostate cancer.

Keywords:  Autophagy; Cancer metabolism; Prostate cancer; Pyruvate kinase M2; mTOR

DOI:  https://doi.org/10.33594/000000107
J Biol Chem. 2019 May 28. pii: jbc.RA118.005806. [Epub ahead of print]

Inhibition of nucleotide synthesis promotes replicative senescence of human mammary epithelial cells.

Alireza Delfarah, Sydney Parrish, Jason A Junge, Jesse Yang, Frances Seo, Si Li, John Mac, Pin Wang, Scott E Fraser, Nicholas A Graham.

  Cellular senescence is a mechanism by which cells permanently withdraw from the cell cycle in response to stresses including telomere shortening, DNA damage, or oncogenic signaling. Senescent cells contribute to both age-related degeneration and hyperplastic pathologies, including cancer. In culture, normal human epithelial cells enter senescence after a limited number of cell divisions, known as replicative senescence. Here, to investigate how metabolic pathways regulate replicative senescence, we used LC-MS-based metabolomics to analyze senescent primary human mammary epithelial cells (HMECs). We did not observe significant changes in glucose uptake or lactate secretion in senescent HMECs. However, analysis of intracellular metabolite pool sizes indicated that senescent cells exhibit depletion of metabolites from nucleotide synthesis pathways. Furthermore, stable isotope tracing with 13C-labeled glucose or glutamine revealed a dramatic blockage of flux of these two metabolites into nucleotide synthesis pathways in senescent HMECs. To test whether cellular immortalization would reverse these observations, we expressed telomerase in HMECs. In addition to preventing senescence, telomerase expression maintained metabolic flux from glucose into nucleotide synthesis pathways. Finally, we investigated whether inhibition of nucleotide synthesis in proliferating HMECs is sufficient to induce senescence. In proliferating HMECs, both pharmacological and genetic inhibition of ribonucleotide reductase regulatory subunit M2 (RRM2), a rate-limiting enzyme in dNTP synthesis, induced premature senescence with concomitantly decreased metabolic flux from glucose into nucleotide synthesis. Taken together, our results suggest that nucleotide synthesis inhibition plays a causative role in the establishment of replicative senescence in HMECs.

Keywords:  aging; cell stress; cellular senescence; epithelial cell; growth arrest; metabolomics; nucleoside/nucleotide biosynthesis; ribonucleotide reductase regulatory subunit M2 (RRM2); systems biology

DOI:  https://doi.org/10.1074/jbc.RA118.005806
Mol Cell Oncol. 2019 ;6(3): 1595308

When fat goes down, prostate cancer is on the ropes.

Giorgia Zadra, Massimo Loda.

  Reprogrammed lipid metabolism and persistent androgen receptor signaling commonly mark aggressive prostate cancer. We describe that targeting de-novo lipogenesis deprives prostate cancer cells of substrates and fuel, while inhibiting androgen receptor signaling. Our study uncovers the interplay between lipogenesis and androgen receptor and proposes novel combinatorial therapeutic approaches.

Keywords:  Metastatic prostate cancer; androgen receptor; fatty acid synthesis; lipid metabolism; reticulum endoplasmic stress response

DOI:  https://doi.org/10.1080/23723556.2019.1595308
Cancer Metab. 2019 ;7 4

Isocitrate dehydrogenase 1-mutated cancers are sensitive to the green tea polyphenol epigallocatechin-3-gallate.

Tom H Peeters, Krissie Lenting, Vincent Breukels, Sanne A M van Lith, Corina N A M van den Heuvel, Remco Molenaar, Arno van Rooij, Ron Wevers, Paul N Span, Arend Heerschap, William P J Leenders.

   Background: Mutations in isocitrate dehydrogenase 1 (IDH1) occur in various types of cancer and induce metabolic alterations resulting from the neomorphic activity that causes production of D-2-hydroxyglutarate (D-2-HG) at the expense of α-ketoglutarate (α-KG) and NADPH. To overcome metabolic stress induced by these alterations, IDH-mutated (IDH mut ) cancers utilize rescue mechanisms comprising pathways in which glutaminase and glutamate dehydrogenase (GLUD) are involved. We hypothesized that inhibition of glutamate processing with the pleiotropic GLUD-inhibitor epigallocatechin-3-gallate (EGCG) would not only hamper D-2-HG production, but also decrease NAD(P)H and α-KG synthesis in IDH mut cancers, resulting in increased metabolic stress and increased sensitivity to radiotherapy.
Methods: We performed 13C-tracing studies to show that HCT116 colorectal cancer cells with an IDH1 R132H knock-in allele depend more on glutaminolysis than on glycolysis for the production of D-2-HG. We treated HCT116 cells, HCT116-IDH1 R132H cells, and HT1080 cells (carrying an IDH1 R132C mutation) with EGCG and evaluated D-2-HG production, cell proliferation rates, and sensitivity to radiotherapy.
Results: Significant amounts of 13C from glutamate accumulate in D-2-HG in HCT116-IDH1 wt/R132H but not in HCT116-IDH1 wt/wt . Preventing glutamate processing in HCT116-IDH1 wt/R132H cells with EGCG resulted in reduction of D-2-HG production. In addition, EGCG treatment decreased proliferation rates of IDH1 mut cells and at high doses sensitized cancer cells to ionizing radiation. Effects of EGCG in IDH-mutated cell lines were diminished by treatment with the IDH1mut inhibitor AGI-5198.
Conclusions: This work shows that glutamate can be directly processed into D-2-HG and that reduction of glutamatolysis may be an effective and promising new treatment option for IDH mut cancers.

Keywords:  EGCG; Glutamate; IDH mutations; Metabolism; Radiotherapy

DOI:  https://doi.org/10.1186/s40170-019-0198-7
Nat Methods. 2019 Jun;16(6): 509-518

Prosit: proteome-wide prediction of peptide tandem mass spectra by deep learning.

Siegfried Gessulat, Tobias Schmidt, Daniel Paul Zolg, Patroklos Samaras, Karsten Schnatbaum, Johannes Zerweck, Tobias Knaute, Julia Rechenberger, Bernard Delanghe, Andreas Huhmer, Ulf Reimer, Hans-Christian Ehrlich, Stephan Aiche, Bernhard Kuster, Mathias Wilhelm.

In mass-spectrometry-based proteomics, the identification and quantification of peptides and proteins heavily rely on sequence database searching or spectral library matching. The lack of accurate predictive models for fragment ion intensities impairs the realization of the full potential of these approaches. Here, we extended the ProteomeTools synthetic peptide library to 550,000 tryptic peptides and 21 million high-quality tandem mass spectra. We trained a deep neural network, termed Prosit, resulting in chromatographic retention time and fragment ion intensity predictions that exceed the quality of the experimental data. Integrating Prosit into database search pipelines led to more identifications at >10× lower false discovery rates. We show the general applicability of Prosit by predicting spectra for proteases other than trypsin, generating spectral libraries for data-independent acquisition and improving the analysis of metaproteomes. Prosit is integrated into ProteomicsDB, allowing search result re-scoring and custom spectral library generation for any organism on the basis of peptide sequence alone.

DOI: https://doi.org/10.1038/s41592-019-0426-7
Br J Nutr. 2019 May 27. 1-14

A systematic review of the effects of increasing arachidonic acid intake on PUFA status, metabolism and health-related outcomes in humans.

Philip C Calder, Cristina Campoy, Ans Eilander, Mathilde Fleith, Stewart Forsyth, Per-Olof Larsson, Bettina Schelkle, Szimonetta Lohner, Aliz Szommer, Bert J M van de Heijning, Ronald P Mensink.

  We conducted a systematic review of randomised controlled trials (RCT) of increased intake of arachidonic acid (ARA) on fatty acid status and health outcomes in humans. We identified twenty-two articles from fourteen RCT. Most studies were conducted in adults. These used between 80 and 2000 mg ARA per d and were of 1-12 weeks duration. Supplementation with ARA doses as low as 80 mg/d increased the content of ARA in different blood fractions. Overall there seem to be few marked benefits for adults of increasing ARA intake from the typical usual intake of 100-200 mg/d to as much as 1000 mg/d; the few studies using higher doses (1500 or 2000 mg/d) also report little benefit. However, there may be an impact of ARA on cognitive and muscle function which could be particularly relevant in the ageing population. The studies reviewed here suggest no adverse effects in adults of increased ARA intake up to at least 1000-1500 mg/d on blood lipids, platelet aggregation and blood clotting, immune function, inflammation or urinary excretion of ARA metabolites. However, in many areas there are insufficient studies to make firm conclusions, and higher intakes of ARA are deserving of further study. Based on the RCT reviewed, there are not enough data to make any recommendations for specific health effects of ARA intake.

Keywords:   ARA arachidonic acid; CE cholesteryl ester; DGLA dihomo-γ-linolenic acid; FA fatty acid; LA linoleic acid; LC-PUFA long-chain PUFA; PL phospholipid; RCT randomised controlled trial; Arachidonic acid; Fatty acids; Human health; Inflammation; n-6 Fatty acids

DOI:  https://doi.org/10.1017/S0007114519000692
Expert Rev Proteomics. 2019 May 30.

Mass spectrometry analysis of protein biomarkers for hepatocellular carcinoma.

Kwang Hoe Kim, Jin Young Kim, Jong Shin Yoo.

  Introduction: Diagnosis of hepatocellular carcinoma (HCC) is important for improving the survival rate and selecting the optimum therapeutic option. However, some patients with HCC are not diagnosed until after symptoms appear, when the tumor is already advanced. Thus, biomarkers associated with HCC and novel diagnostic methods are required to improve the diagnosis of HCC. Mass spectrometry (MS) is one of the most widely used analytical tools in proteomic research. Furthermore, tandem MS (MS/MS) has been applied for the discovery and verification of protein biomarkers for clinical use. Areas covered: We review candidate glycoprotein biomarkers, including their aberrant glycosylation discovered by MS-based proteomics techniques and their diagnostic strategies using human blood samples. Finally, we discuss the limitations and prospects of MS-based approaches for clinical applications. Expert commentary: The development of biomarkers with high sensitivity and specificity is essential for optimizing the management of HCC. Various glycoprotein biomarkers of HCC have been identified using MS-based techniques. MS-based assays will continue to play an important role in clinical applications for discovery and verification of biomarkers. Furthermore, combination of multibiomarker, improvements in sample enrichment and the development of highly sensitive MS methods will facilitate more rapid adoption of MS for the diagnosis of HCC.

Keywords:  biomarker; fucosylation; glycosylation; hepatocellular carcinoma; mass spectrometry; proteomics

DOI:  https://doi.org/10.1080/14789450.2019.1626235
Exp Cell Res. 2019 May 22. pii: S0014-4827(19)30258-7. [Epub ahead of print]

Inhibition of glycogen catabolism induces intrinsic apoptosis and augments multikinase inhibitors in hepatocellular carcinoma cells.

Shrikant Barot, Ehab M Abo-Ali, Daisy L Zhou, Christian Palaguachi, Vikas V Dukhande.

  Hepatocellular carcinoma (HCC) is one of the leading cancers in the world in incidence and mortality. Current pharmacotherapy of HCC is limited in the number and efficacy of anticancer agents. Metabolic reprogramming is a prominent feature of many cancers and has rekindled interest in targeting metabolic proteins for cancer therapy. Glycogen is a storage form of glucose, and the levels of glycogen have been found to correlate with biological processes in reprogrammed cancer cells. However, the contribution of glycogen metabolism to carcinogenesis, cancer cell growth, metastasis, and chemoresistance is poorly understood. Thus, we studied the processes involved in the inhibition of glycogen metabolism in HCC cells. Pharmacological inhibition of glycogen phosphorylase (GP), a rate-limiting enzyme in glycogen catabolism, by CP-91149 led to a decrease in HCC cell viability. GP inhibition induced cancer cell death through the intrinsic apoptotic pathway. Mitochondrial dysfunction and autophagic adaptations accompanied this apoptosis process whereas endoplasmic reticulum stress, necrosis, and necroptosis were not major components of the cell death. In addition, GP inhibition potentiated the effects of multikinase inhibitors sorafenib and regorafenib, which are key drugs in advanced-stage HCC therapy. Our study provides mechanistic insights into cell death by perturbation of glycogen metabolism and identifies GP inhibition as a potential HCC pharmacotherapy target.

Keywords:  Apoptosis; Autophagy; Glycogen phosphorylase; Hepatocellular carcinoma; Metabolic reprogramming; Sorafenib

DOI:  https://doi.org/10.1016/j.yexcr.2019.05.017
Eur J Pharmacol. 2019 May 28. pii: S0014-2999(19)30376-0. [Epub ahead of print] 172425

Metformin inhibits PPARδ agonist-mediated tumor growth by reducing Glut1 and SLC1A5 expressions of cancer cells.

Jiajun Ding, Qian Gou, Jianhua Jin, Juanjuan Shi, Qian Liu, Yongzhong Hou.

  As a nuclear receptor, ligand binding and activated PPARδ (peroxisome-proliferator-activated receptor δ) plays an important role in regulation of inflammation, metabolism and cancer, while it is unclear the effect of metformin on PPARδ-mediated cancer cell metabolism. Here we found that PPARδ agonist GW501516 significantly increased Glut1 (Glucose transporter 1) and SLC1A5 (solutecarrier family 1 member 5) gene and protein expressions in HCT-116, SW480, HeLa, and MCF-7 cancer cell lines, while metformin inhibited this event, which was associated with metformin-mediated inhibition of PPARδ activity in response to GW501516. Importantly, GW501516 inhibited the binding of PPARδ to AMPK, while metformin reversed this process. Metformin inhibited Glut1 and SLC1A5 expressions leading to reduced influx of glucose and glutamine in cancer cells, which is associated with reduced tumor growth. These findings suggest that metformin inhibited PPARδ agonist GW501516-induced cancer cell metabolism and tumor growth.

Keywords:  AMPK; GW501516; Glut1; Metformin; PPARδ; SLC1A5; Transcriptional activity

DOI:  https://doi.org/10.1016/j.ejphar.2019.172425
Int J Mol Sci. 2019 May 25. pii: E2576. [Epub ahead of print]20(10):

The Relevance of Mass Spectrometry Analysis for Personalized Medicine through Its Successful Application in Cancer "Omics".

Cristina Alexandra Ciocan-Cartita, Ancuța Jurj, Mihail Buse, Diana Gulei, Cornelia Braicu, Lajos Raduly, Roxana Cojocneanu, Lavinia Lorena Pruteanu, Cristina Adela Iuga, Ovidiu Coza, Ioana Berindan-Neagoe.

  Mass spectrometry (MS) is an essential analytical technology on which the emerging omics domains; such as genomics; transcriptomics; proteomics and metabolomics; are based. This quantifiable technique allows for the identification of thousands of proteins from cell culture; bodily fluids or tissue using either global or targeted strategies; or detection of biologically active metabolites in ultra amounts. The routine performance of MS technology in the oncological field provides a better understanding of human diseases in terms of pathophysiology; prevention; diagnosis and treatment; as well as development of new biomarkers; drugs targets and therapies. In this review; we argue that the recent; successful advances in MS technologies towards cancer omics studies provides a strong rationale for its implementation in biomedicine as a whole.

Keywords:  biomarkers; mass spectrometry; omics technology; personalized medicine

DOI:  https://doi.org/10.3390/ijms20102576
Cells. 2019 May 27. pii: E512. [Epub ahead of print]8(5):

Characterization of Lipid and Lipid Droplet Metabolism in Human HCC.

Nikolaus Berndt, Johannes Eckstein, Niklas Heucke, Robert Gajowski, Martin Stockmann, David Meierhofer, Hermann-Georg Holzhütter.

  Human hepatocellular carcinoma (HCC) is the most common type of primary liver cancer in adults and the most common cause of death in people with cirrhosis. While previous metabolic studies of HCC have mainly focused on the glucose metabolism (Warburg effect), less attention has been paid to tumor-specific features of the lipid metabolism. Here, we applied a computational approach to analyze major pathways of fatty acid utilization in individual HCC. To this end, we used protein intensity profiles of eleven human HCCs to parameterize tumor-specific kinetic models of cellular lipid metabolism including formation, enlargement, and degradation of lipid droplets (LDs). Our analysis reveals significant inter-tumor differences in the lipid metabolism. The majority of HCCs show a reduced uptake of fatty acids and decreased rate of β-oxidation, however, some HCCs display a completely different metabolic phenotype characterized by high rates of β-oxidation. Despite reduced fatty acid uptake in the majority of HCCs, the content of triacylglycerol is significantly enlarged compared to the tumor-adjacent tissue. This is due to tumor-specific expression profiles of regulatory proteins decorating the surface of LDs and controlling their turnover. Our simulations suggest that HCCs characterized by a very high content of triglycerides comprise regulatory peculiarities that render them susceptible to selective drug targeting without affecting healthy tissue.

Keywords:  hepatocellular carcinoma; kinetic modeling; lipid droplet metabolism; liver; mathematical model; tumor metabolism

DOI:  https://doi.org/10.3390/cells8050512
Elife. 2019 May 28. pii: e40226. [Epub ahead of print]8

Human pancreatic cancer cell exosomes, but not human normal cell exosomes, act as an initiator in cell transformation.

Karoliina Stefanius, Kelly Servage, Marcela de Souza Santos, Hillery Fields Gray, Jason E Toombs, Suneeta Chimalapati, Min S Kim, Venkat S Malladi, Rolf Brekken, Kim Orth.

  Cancer evolves through a multistep process that occurs by the temporal accumulation of genetic mutations. Tumor-derived exosomes are emerging contributors to tumorigenesis. To understand how exosomes might contribute to cell transformation, we utilized the classic two-step NIH/3T3 cell transformation assay and observed that exosomes isolated from pancreatic cancer cells, but not normal human cells, can initiate malignant cell transformation and these transformed cells formed tumors in vivo. However, cancer cell exosomes are unable to transform cells alone or to act as a promoter of cell transformation. Utilizing proteomics and exome sequencing, we discovered cancer cell exosomes act as an initiator by inducing random mutations in recipient cells. Cells from the pool of randomly mutated cells are driven to transformation by a classic promoter resulting in foci, each of which encode a unique genetic profile. Our studies describe a novel molecular understanding of how cancer cell exosomes contribute to cell transformation.
Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that major issues remain unresolved (see decision letter).

Keywords:  cancer; cancer biology; cell biology; exosomes; mouse; pancreatic cancer; proteomics; transformation

DOI:  https://doi.org/10.7554/eLife.40226
Mol Cell Oncol. 2019 ;6(3): 1585171

Malignant manipulaTORs of metabolism: suppressing BCAA catabolism to enhance mTORC1 activity.

Russell E Ericksen, Weiping Han.

  The mammalian target of rapamycin complex 1 (mTORC1) plays an important role in the development and progression of multiple cancers. Its activity is regulated by both growth factor and nutrient signals, and the branched-chain amino acid (BCAA) leucine plays an important and unique role in this process. Recently we found that cancers of the liver and multiple other tissues suppress the catabolism of BCAAs, thereby facilitating the chronic activation of mTORC1. Our results unveil how mTORC1's nutrient-sensing arm can be manipulated by tumors, and suggest that restoring BCAA catabolism may help control mTORC1 activity in cancer cells.

Keywords:  branched-chain amino acids; cancer metabolism; dietary intake; liver cancer; mTOR

DOI:  https://doi.org/10.1080/23723556.2019.1585171
ACS Cent Sci. 2019 May 22. 5(5): 768-780

Mapping and Profiling Lipid Distribution in a 3D Model of Breast Cancer Progression.

Netta Vidavsky, Jennie A M R Kunitake, Maria Elena Diaz-Rubio, Aaron E Chiou, Hyun-Chae Loh, Sheng Zhang, Admir Masic, Claudia Fischbach, Lara A Estroff.

Aberrant lipid accumulation and marked changes in cellular lipid profiles are related to breast cancer metabolism and disease progression. In vitro, these phenomena are primarily studied using cells cultured in monolayers (2D). Here, we employ multicellular spheroids, generated using the MCF10A cell line series of increasing malignancy potential, to better recapitulate the 3D microenvironmental conditions that cells experience in vivo. Breast cancer cell lipid compositions were assessed in 2D and 3D culture models as a function of malignancy using liquid chromatography coupled with mass spectrometry. Further, the spatial distribution of lipids was examined using Raman chemical imaging and lipid staining. We show that with changes in the cellular microenvironment when moving from 2D to 3D cell cultures, total lipid amounts decrease significantly, while the ratio of acylglycerols to membrane lipids increases. This ratio increase could be associated with the formation of large lipid droplets (>10 μm) that are spatially evident throughout the spheroids but absent in 2D cultures. Additionally, we found a significant difference in lipid profiles between the more and less malignant spheroids, including changes that support de novo sphingolipid production and a reduction in ether-linked lipid fractions in the invasive spheroids. These differences in lipid profiles as a function of cell malignancy and microenvironment highlight the importance of coupled spatial and lipidomic studies to better understand the connections between lipid metabolism and cancer.

DOI: https://doi.org/10.1021/acscentsci.8b00932
Methods Mol Biol. 2019 ;1988 15-29

Analysis of Proteasome-Generated Antigenic Peptides by Mass Spectrometry.

Kathrin Textoris-Taube, Ulrike Kuckelkorn, Christin Beier, Peter M Kloetzel.

  Mass spectrometry (MS) is today one of the most important analytical techniques in biosciences. The development of electro spray ionization (ESI) as a gentle method, in which molecules are not destroyed, has revolutionized the analytic of peptides. MS is an ideal technique for detection and analysis of peptides generated by purified 20S proteasomes in in vitro experiments. This approach also provides a convenient and sensitive way to monitor the different processing characteristics of proteasome isoforms. The combination of high performance liquid chromatography (HPLC) with ESI-MS allows for the analysis of complex samples with separation in their specific constituents by LC and their subsequent detection by MS.

Keywords:  Antigen processing; Antigenic peptides; Epitope; Fragment generation; HPLC; Mass spectrometry; Polypeptides; Proteasome; Substrate degradation

DOI:  https://doi.org/10.1007/978-1-4939-9450-2_2
Metabolomics. 2019 May 30. 15(6): 86

Metabolomics identifies serum and exosomes metabolite markers of pancreatic cancer.

Lianyuan Tao, Juntuo Zhou, Chunhui Yuan, Lingfu Zhang, Deyu Li, Dandan Si, Dianrong Xiu, Lijun Zhong.

   INTRODUCTION: Pancreatic cancer (PC) is one of the most aggressive malignancies, and it's difficult to diagnosis PC at an early stage, which leads to the poor prognosis of PC.
OBJECTIVES: To identifiy the possible prognosis or dignosis metabolite biomarkers in the serum exosome of PC patients.
METHODS: We employed LC-DDA-MS based untargeted lipidomic analysis to search for potential candidate biomarkers in the serum exosome of PC patients. Then LC-MRM-MS based targeted lipid quantification was used to validate the trends of the candidate biomarkers in larger sample cohorts.
RESULTS: About 270 lipids belonging to 20 lipid species were found significantly dysregulated between the serum exosome of PC patients and healthy controls. 61 of them were validated in larger samples size. We further analysis the correlation between these dysregulated lipids and other PC related factors, and results show that LysoPC 22:0, PC (P-14:0/22:2) and PE (16:0/18:1) are all associated with tumor stage, CA19-9, CA242 and tumor diameter. What's more, PE (16:0/18:1) is also found to be significantly correlated with the patient's overall survival.
CONCLUSION: These data reveal dysregulated lipids in serum exosome of PC patients, which have potential to be biomarkers for diagnosis, or unveil pathological relationship between exosome and PC progress.

Keywords:  Exosome biomarker; Lipidomics; Pancreatic cancer; Serum exosome

DOI:  https://doi.org/10.1007/s11306-019-1550-1
Methods Mol Biol. 2019 ;2009 59-70

Direct Analysis of Protein S-Acylation by Mass Spectrometry.

Yuhuan Ji, Cheng Lin.

  Dynamic and reversible protein S-acylation, most commonly occurring as S-palmitoylation, plays an important role in protein/membrane association and the regulation of intracellular signaling via cycles of palmitoylation and depalmitoylation. Direct analysis of protein S-acylation by mass spectrometry (MS) offers several benefits over indirect detection methods in that it can definitively determine the location and nature of the acyl modification, and is not prone to false discoveries. However, characterization of acyl proteins is challenging because of the tendency of acyl loss during sample preparation and tandem MS analysis. In this chapter, we present a sample preparation protocol that preserves labile acyl modifications and an LC-MS/MS workflow for detection of S-acylation with high confidence and sensitivity.

Keywords:  Reversed phase liquid chromatography–mass spectrometry (RPLC-MS); S-palmitoylation; Tandem mass spectrometry (MS/MS)

DOI:  https://doi.org/10.1007/978-1-4939-9532-5_5
Nat Commun. 2019 May 30. 10(1): 2367

The metabolites NADP+ and NADPH are the targets of the circadian protein Nocturnin (Curled).

Michael A Estrella, Jin Du, Li Chen, Sneha Rath, Eliza Prangley, Alisha Chitrakar, Tsutomu Aoki, Paul Schedl, Joshua Rabinowitz, Alexei Korennykh.

Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. However, in contrast to other deadenylases, purified NOCT lacks the deadenylase activity. To identify the substrate of NOCT, we conducted a mass spectrometry screen and report that NOCT specifically and directly converts the dinucleotide NADP+ into NAD+ and NADPH into NADH. Further, we demonstrate that the Drosophila NOCT ortholog, Curled, has the same enzymatic activity. We obtained the 2.7 Å crystal structure of the human NOCT•NADPH complex, which revealed that NOCT recognizes the chemically unique ribose-phosphate backbone of the metabolite, placing the 2'-terminal phosphate productively for removal. We provide evidence for NOCT targeting to mitochondria and propose that NADP(H) regulation, which takes place at least in part in mitochondria, establishes the molecular link between circadian clock and metabolism.

DOI: https://doi.org/10.1038/s41467-019-10125-z
Front Immunol. 2019 ;10 976

Fecal Metabolomics and Potential Biomarkers for Systemic Lupus Erythematosus.

Qiong Zhang, Xiaofeng Yin, Haifang Wang, Xing Wu, Xin Li, Yao Li, Xiaohe Zhang, Chen Fu, Haixia Li, Yurong Qiu.

  The role of metabolomics in autoimmune diseases has been a rapidly expanding area in researches over the last decade, while its pathophysiologic impact on systemic lupus erythematosus (SLE) remains poorly elucidated. In this study, we analyzed the metabolic profiling of fecal samples from SLE patients and healthy controls based on ultra-high-performance liquid chromatography equipped with mass spectrometry for exploring the potential biomarkers of SLE. The results showed that 23 differential metabolites and 5 perturbed pathways were identified between the two groups, including aminoacyl-tRNA biosynthesis, thiamine metabolism, nitrogen metabolism, tryptophan metabolism, and cyanoamino acid metabolism. In addition, logistic regression and ROC analysis were used to establish a diagnostic model for distinguishing SLE patients from healthy controls. The combined model of fecal PG 27:2 and proline achieved an area under the ROC curve of 0.846, and had a good diagnostic efficacy. In the present study, we analyzed the correlations between fecal metabolic perturbations and SLE pathogenesis. In summary, we firstly illustrate the comprehensive metabolic profiles of feces in SLE patients, suggesting that the fecal metabolites could be used as the potential non-invasive biomarkers for SLE.

Keywords:  biomarker; feces; liquid chromatography; mass spectrometry; metabolomics; systemic lupus erythematosus

DOI:  https://doi.org/10.3389/fimmu.2019.00976
Methods Mol Biol. 2019 ;1993 193-204

Effects of the Extracellular Matrix on the Proteome of Primary Skin Fibroblasts.

Regine C Tölle, Jörn Dengjel.

  The cellular microenvironment often plays a crucial role in disease development and progression. In recessive dystrophic epidermolysis bullosa (RDEB), biallelic mutations of the gene COL7A1, encoding for collagen VII, the main component of anchoring fibrils, lead to a loss of collagen VII in the extracellular matrix (ECM). Loss of collagen VII in skin is linked to a destabilization of the dermal-epidermal junction zone, blister formation, chronic wounds, fibrosis, and aggressive skin cancer. Thus, RDEB cells can serve as a model system to study the effects of a perturbed ECM on the cellular proteome. In this chapter, we describe in detail the combination of stable isotope labeling by amino acids in cell culture (SILAC) of primary skin fibroblasts with reseeding of fibroblasts on decellularized collagen VII-positive and -negative ECM to study the consequences of collagen VII loss on the cellular proteome. This approach allows the quantitative, time-resolved analysis of cellular protein dynamics in response to ECM perturbation by liquid chromatography-mass spectrometry.

Keywords:  Decellularization; Extracellular matrix; Fibroblasts; GASP; High-pH reversed-phase chromatography; Mass spectrometry; Protein kinetics; Proteomics; SILAC; Skin

DOI:  https://doi.org/10.1007/978-1-4939-9473-1_15
Methods Mol Biol. 2019 ;2009 71-79

Enrichment of S-Palmitoylated Proteins for Mass Spectrometry Analysis.

Melanie Cheung See Kit, Brent R Martin.

  As the 10-year anniversary of their first introduction approaches, alkynyl fatty acids have revolutionized the analysis of S-palmitoylation dynamics, acting as functional mimics incorporated into native modification sites in cultured cells. The alkyne functional group provides a robust handle for bioorthogonal Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to reporter-linked azides, forming a stable conjugate for enrichment for mass spectrometry analysis or in-gel fluorescence. Importantly, metabolic labeling enables time-dependent analysis of S-palmitoylation dynamics, which can be used to profile incorporation and turnover rates across the proteome. Here we present a protocol for cell labeling, click chemistry conjugation, enrichment, and isobaric tandem mass tag labeling for quantitative mass spectrometry analysis of protein S-palmitoylation.

Keywords:  Affinity purification; Click chemistry; Mass spectrometry; Metabolic labeling; Posttranslational modification; S-palmitoylation

DOI:  https://doi.org/10.1007/978-1-4939-9532-5_6
Nat Genet. 2019 Jun;51(6): 990-998

MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation.

Sara Sdelci, André F Rendeiro, Philipp Rathert, Wanhui You, Jung-Ming G Lin, Anna Ringler, Gerald Hofstätter, Herwig P Moll, Bettina Gürtl, Matthias Farlik, Sandra Schick, Freya Klepsch, Matthew Oldach, Pisanu Buphamalai, Fiorella Schischlik, Peter Májek, Katja Parapatics, Christian Schmidl, Michael Schuster, Thomas Penz, Dennis L Buckley, Otto Hudecz, Richard Imre, Shuang-Yan Wang, Hans Michael Maric, Robert Kralovics, Keiryn L Bennett, Andre C Müller, Karl Mechtler, Jörg Menche, James E Bradner, Georg E Winter, Kristaps Klavins, Emilio Casanova, Christoph Bock, Johannes Zuber, Stefan Kubicek.

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.

DOI: https://doi.org/10.1038/s41588-019-0413-z
J Biol Chem. 2019 May 31. pii: jbc.RA118.006956. [Epub ahead of print]

Linoleic acid esters of hydroxy linoleic acids are anti-inflammatory lipids found in plants and mammals.

Matthew J Kolar, Srihari Konduri, Tina Chang, Huijing Wang, Clare McNerlin, Lena Ohlsson, Magnus Härröd, Dionicio Siegel, Alan Saghatelian.

  Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids. Here, we identify the linoleic acid ester of 13-hydroxy linoleic acid (13-LAHLA) as an anti-inflammatory lipid. An oat oil fraction and FAHFA-enriched extract from this fraction showed anti-inflammatory activity in LPS-induced cytokine secretion assay. Structural studies identified three LAHLA isomers (15-, 13-, and 9-LAHLA) as being the most abundant FAHFAs in the oat oil fraction. Of these LAHLAs, 13-LAHLA is the most abundant LAHLA isomer in human serum after ingestion of liposomes made of fractionated oat oil, and it is also the most abundant endogenous LAHLA in mouse and human adipose tissue. As a result, we chemically synthesized 13-LAHLA for biological assays. 13-LAHLA suppresses LPS-stimulated secretion of cytokines and expression of pro-inflammatory genes. These studies identify LAHLAs as an evolutionarily conserved lipid with anti-inflammatory activity in mammalian cells.

Keywords:  FAHFAs; LAHLAs; inflammation; lipid; lipid signaling; lipid structure; lipid synthesis

DOI:  https://doi.org/10.1074/jbc.RA118.006956
Biosci Rep. 2019 May 29. pii: BSR20190436. [Epub ahead of print]

High glucose promotes breast cancer proliferation and metastasis by impairing angiotensinogen expression.

Shichao Sun, Yao Sun, Xiaoping Rong, Lei Bai.

  A number of investigations have addressed the importance of high glucose in breast cancer, however, the involvement of angiotensinogen (AGT) in this scenario is yet to be defined. Here we set out to analyze the potential pro-tumor effects of high glucose in breast cancer, and understand the underlying molecular mechanism. We demonstrated that high glucose promoted cell proliferation, viability and anchorage-independent growth of breast cancer cells. In addition, the migrative and invasive capacities were significantly enhanced by high glucose medium. Mechanistically, AGT expression was inhibited by high glucose at both transcriptional and translational levels. High AGT remarkably suppressed proliferation, inhibited viability and compromised migration/invasion of breast cancer cells. Most importantly, ectopic introduction of AGT almost completely abrogated pro-tumor effects of high glucose. Our study has characterized the pro-tumor properties of high glucose in breast cancer cells, which is predominantly attributed to the suppression of AGT.

Keywords:  angiotensinogen; breast cancer; high glucose; proliferation

DOI:  https://doi.org/10.1042/BSR20190436
Cell Metab. 2019 May 17. pii: S1550-4131(19)30243-8. [Epub ahead of print]

Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation.

Daniel J Puleston, Michael D Buck, Ramon I Klein Geltink, Ryan L Kyle, George Caputa, David O'Sullivan, Alanna M Cameron, Angela Castoldi, Yaarub Musa, Agnieszka M Kabat, Ying Zhang, Lea J Flachsmann, Cameron S Field, Annette E Patterson, Stefanie Scherer, Francesca Alfei, Francesc Baixauli, S Kyle Austin, Beth Kelly, Mai Matsushita, Jonathan D Curtis, Katarzyna M Grzes, Matteo Villa, Mauro Corrado, David E Sanin, Jing Qiu, Nora Pällman, Katelyn Paz, Maria Elena Maccari, Bruce R Blazar, Gerhard Mittler, Joerg M Buescher, Dietmar Zehn, Sabine Rospert, Edward J Pearce, Stefan Balabanov, Erika L Pearce.

  How cells adapt metabolism to meet demands is an active area of interest across biology. Among a broad range of functions, the polyamine spermidine is needed to hypusinate the translation factor eukaryotic initiation factor 5A (eIF5A). We show here that hypusinated eIF5A (eIF5AH) promotes the efficient expression of a subset of mitochondrial proteins involved in the TCA cycle and oxidative phosphorylation (OXPHOS). Several of these proteins have mitochondrial targeting sequences (MTSs) that in part confer an increased dependency on eIF5AH. In macrophages, metabolic switching between OXPHOS and glycolysis supports divergent functional fates stimulated by activation signals. In these cells, hypusination of eIF5A appears to be dynamically regulated after activation. Using in vivo and in vitro models, we show that acute inhibition of this pathway blunts OXPHOS-dependent alternative activation, while leaving aerobic glycolysis-dependent classical activation intact. These results might have implications for therapeutically controlling macrophage activation by targeting the polyamine-eIF5A-hypusine axis.

Keywords:  deoxyhypusine hydroxylase; deoxyhypusine synthase; eIF5A; hypusination; immunometabolism; macrophage activation; metabolism; polyamines

DOI:  https://doi.org/10.1016/j.cmet.2019.05.003