bims-mascan 2020-05-24 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2020–05–24
thirty papers selected by
Giovanny Rodriguez Blanco, The Beatson Institute for Cancer Research

2,4-dienoyl-CoA reductase regulates lipid homeostasis in treatment-resistant prostate cancer.
Anion-exchange chromatography mass spectrometry provides extensive coverage of primary metabolic pathways revealing altered metabolism in IDH1 mutant cells.
The lipid homeostasis regulation study of arenobufagin in zebrafish HepG2 xenograft model and HepG2 cells using integrated lipidomics-proteomics approach.
Regulation of lipid droplet homeostasis by hypoxia inducible lipid droplet associated HILPDA.
Profiling the 'deamidome' of complex biosamples using mixed-mode chromatography-coupled tandem mass spectrometry.
An accurate and sensitive analytical method for the simultaneous determination of glycine, methionine and homocysteine in biological matrices by matrix matching strategy and LC-quadrupole-time-of-flight-MS/MS.
Role of the HIF oxygen sensing pathway in cell defense and proliferation through the control of amino acid metabolism.
Development and validation of a rapid, robust and sensitive UPLC-QQQ-MS/MS method for simultaneous quantification of GSH metabolism in lung cancer cells.
A review of strategies for untargeted urinary metabolomic analysis using gas chromatography-mass spectrometry.
PolySTest: Robust statistical testing of proteomics data with missing values improves detection of biologically relevant features.
Double bond characterization of free fatty acids directly from biological tissues by ultraviolet photodissociation.
Enhanced single-cell metabolomics by capillary electrophoresis electrospray ionization-mass spectrometry with field amplified sample injection.
Chemical Proteomic Profiling of the Interacting Proteins of Isoprenoid Pyrophosphates.
Stress-induced TRAILR2 expression overcomes TRAIL resistance in cancer cell spheroids.
Metabolic Adaptation during nab-Paclitaxel Resistance in Pancreatic Cancer Cell Lines.
Multiplatform Investigation of Plasma and Tissue Lipid Signatures of Breast Cancer Using Mass Spectrometry Tools.
Improved discrimination of asymmetric and symmetric arginine dimethylation by optimization of the normalized collision energy in LC-MS proteomics.
Reprogramming of serine, glycine and one-carbon metabolism in cancer.
A New Pathway Promotes Adaptation of Human Glioblastoma Cells to Glucose Starvation.
Metabolic Plasticity in Ovarian Cancer Stem Cells.
Quantitative Proteomics Using Formalin-fixed, Paraffin-embedded Biopsy Tissues in Inflammatory Disease.
Comprehensive metabolomics analysis of prostate cancer tissue in relation to tumor aggressiveness and TMPRSS2-ERG fusion status.
HILIC-ESI-FTMS with All Ion Fragmentation (AIF) Scans as a Tool for Fast Lipidome Investigations.
Small intestine proteomics coupled with serum metabolomics reveal disruption of amino acid metabolism in Chinese hamsters with type 2 diabetes mellitus.
Development of a Novel High-Performance Thin Layer Chromatography-Based Method for the Simultaneous Quantification of Clinically Relevant Lipids from Cells and Tissue Extracts.
ACLY is the novel signaling target of PIP2/PIP3 and Lyn in acute myeloid leukemia.
Sodium-glucose cotransporter 2 inhibition suppresses HIF-1α-mediated metabolic switch from lipid oxidation to glycolysis in kidney tubule cells of diabetic mice.
β-Oxidation and autophagy are critical energy providers during acute glucose depletion in Saccharomyces cerevisiae.
Ferroptosis is involved in alcohol-induced cell death in vivo and in vitro.
PPARδ-mediated mitochondrial rewiring of osteoblasts determines bone mass.

Nat Commun. 2020 May 19. 11(1): 2508

2,4-dienoyl-CoA reductase regulates lipid homeostasis in treatment-resistant prostate cancer.

Arnaud Blomme, Catriona A Ford, Ernest Mui, Rachana Patel, Chara Ntala, Lauren E Jamieson, Mélanie Planque, Grace H McGregor, Paul Peixoto, Eric Hervouet, Colin Nixon, Mark Salji, Luke Gaughan, Elke Markert, Peter Repiscak, David Sumpton, Giovanny Rodriguez Blanco, Sergio Lilla, Jurre J Kamphorst, Duncan Graham, Karen Faulds, Gillian M MacKay, Sarah-Maria Fendt, Sara Zanivan, Hing Y Leung.

Despite the clinical success of Androgen Receptor (AR)-targeted therapies, reactivation of AR signalling remains the main driver of castration-resistant prostate cancer (CRPC) progression. In this study, we perform a comprehensive unbiased characterisation of LNCaP cells chronically exposed to multiple AR inhibitors (ARI). Combined proteomics and metabolomics analyses implicate an acquired metabolic phenotype common in ARI-resistant cells and associated with perturbed glucose and lipid metabolism. To exploit this phenotype, we delineate a subset of proteins consistently associated with ARI resistance and highlight mitochondrial 2,4-dienoyl-CoA reductase (DECR1), an auxiliary enzyme of beta-oxidation, as a clinically relevant biomarker for CRPC. Mechanistically, DECR1 participates in redox homeostasis by controlling the balance between saturated and unsaturated phospholipids. DECR1 knockout induces ER stress and sensitises CRPC cells to ferroptosis. In vivo, DECR1 deletion impairs lipid metabolism and reduces CRPC tumour growth, emphasizing the importance of DECR1 in the development of treatment resistance.

DOI: https://doi.org/10.1038/s41467-020-16126-7
Commun Biol. 2020 May 20. 3(1): 247

Anion-exchange chromatography mass spectrometry provides extensive coverage of primary metabolic pathways revealing altered metabolism in IDH1 mutant cells.

John Walsby-Tickle, Joan Gannon, Ingvild Hvinden, Chiara Bardella, Martine I Abboud, Areesha Nazeer, David Hauton, Elisabete Pires, Tom Cadoux-Hudson, Christopher J Schofield, James S O McCullagh.

Altered central carbon metabolism is a hallmark of many diseases including diabetes, obesity, heart disease and cancer. Identifying metabolic changes will open opportunities for better understanding aetiological processes and identifying new diagnostic, prognostic, and therapeutic targets. Comprehensive and robust analysis of primary metabolic pathways in cells, tissues and bio-fluids, remains technically challenging. We report on the development and validation of a highly reproducible and robust untargeted method using anion-exchange tandem mass spectrometry (IC-MS) that enables analysis of 431 metabolites, providing detailed coverage of central carbon metabolism. We apply the method in an untargeted, discovery-driven workflow to investigate the metabolic effects of isocitrate dehydrogenase 1 (IDH1) mutations in glioblastoma cells. IC-MS provides comprehensive coverage of central metabolic pathways revealing significant elevation of 2-hydroxyglutarate and depletion of 2-oxoglutarate. Further analysis of the data reveals depletion in additional metabolites including previously unrecognised changes in lysine and tryptophan metabolism.

DOI: https://doi.org/10.1038/s42003-020-0957-6
J Ethnopharmacol. 2020 May 15. pii: S0378-8741(20)30047-7. [Epub ahead of print] 112943

The lipid homeostasis regulation study of arenobufagin in zebrafish HepG2 xenograft model and HepG2 cells using integrated lipidomics-proteomics approach.

Li-Juan Zhao, Hai-Yu Zhao, Xiao-Lu Wei, Fei-Fei Guo, Jun-Ying Wei, Hong-Jie Wang, Jian Yang, Zhi-Gang Yang, Nan Si, Bao-Lin Bian.

   ETHNOPHARMACOLOGICAL RELEVANCE: Arenobufagin (ArBu) is an important anti-tumor ingredient of Chan'su which has long been used as traditional Chinese medicine in clinic for tumor therapy in China.
AIM OF THE STUDY: The purpose of our study is to investigate the lipid homeostasis regulation effects of ArBu on zebrafish model of liver cancer and hepatoma cells, and to provide a reference for further clarifying its active mechanisms.
MATERIALS AND METHODS: The zebrafish xenograft model was established by injecting HepG2 cells stained with CM-Dil red fluorescent dye. Both the xenograft model and HepG2 cells were used to evaluate the anti-hepatoma activity of ArBu. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was the main method to study lipidomics, proteomics and the semiquantification of endogenous metabolites. Bioinformatics was used as an assistant tool to further explore the antitumor mechanism of ArBu.
RESULTS: The lipidomics analysis revealed that ArBu caused differential lipids changes in a dose-dependent manner, including PCs, PEs, TGs, SMs, DGs, Cer and PA. PCs, PEs, SMs and TGs were markedly altered in both two models. The influence of glycerophospholipid metabolism was the major and commonly affected pathway. Notably, DGs and Cer were significantly changed only in HepG2 cells. Furthermore, the proteomics research in HepG2 cells fished the target proteins related to lipid homeostasis abnormalities and tumor suppression. ArBu reduced the expression of 65 differential proteins associated with the lipid metabolism, apoptosis and autophagy, such as LCLAT1, STAT3, TSPO and RPS27. Meanwhile, 7 amino acids of 29 determined metabolites were significantly changed, including tyrosine, glutamate, glutamine, leucine, threonine, arginine and isoleucine.
CONCLUSION: ArBu has a significant anti-hepatoma effect in vitro and a therapeutic effect on zebrafish xenograft model. It regulated the lipid homeostasis. Activated SM synthase and arginine deiminase, inhibited sphingomyelinase, amino acid supply and JAK-STAT3 signaling pathway, and the affected glycerophospholipid metabolism might explain these results.

Keywords:  Amino acid; Anti-hepatoma; Arenobufagin; Lipid homeostasis; Lipidomics; Proteomics

DOI:  https://doi.org/10.1016/j.jep.2020.112943
Biochim Biophys Acta Mol Cell Biol Lipids. 2020 May 08. pii: S1388-1981(20)30130-X. [Epub ahead of print] 158738

Regulation of lipid droplet homeostasis by hypoxia inducible lipid droplet associated HILPDA.

Montserrat A de la Rosa Rodriguez, Sander Kersten.

  Nearly all cell types have the ability to store excess energy as triglycerides in specialized organelles called lipid droplets. The formation and degradation of lipid droplets is governed by a diverse set of enzymes and lipid droplet-associated proteins. One of the lipid droplet-associated proteins is Hypoxia Inducible Lipid Droplet Associated (HILPDA). HILPDA was originally discovered in a screen to identify novel hypoxia-inducible proteins. Apart from hypoxia, levels of HILPDA are induced by fatty acids and adrenergic agonists. HILPDA is a small protein of 63 amino acids in humans and 64 amino acids in mice. Inside cells, HILPDA is located in the endoplasmic reticulum and around lipid droplets. Gain- and loss-of-function experiments have demonstrated that HILPDA promotes lipid storage in hepatocytes, macrophages and cancer cells. HILPDA increases lipid droplet accumulation at least partly by inhibiting triglyceride hydrolysis via ATGL and stimulating triglyceride synthesis via DGAT1. Overall, HILPDA is a novel regulatory signal that adjusts triglyceride storage and the intracellular availability of fatty acids to the external fatty acid supply and the capacity for oxidation.

Keywords:  ATGL; Fatty acids; Hypoxia; Lipid droplets; Lipolysis; Triglycerides

DOI:  https://doi.org/10.1016/j.bbalip.2020.158738
Methods. 2020 May 08. pii: S1046-2023(19)30285-3. [Epub ahead of print]

Profiling the 'deamidome' of complex biosamples using mixed-mode chromatography-coupled tandem mass spectrometry.

Siu Kwan Sze, Gnanasekaran JebaMercy, SoFong Cam Ngan.

  Deamidation is a spontaneous degenerative protein modification (DPM) that disrupts the structure and function of both endogenous proteins and various therapeutic agents. While deamidation has long been recognized as a critical event in human aging and multiple degenerative diseases, research progress in this field has been restricted by the technical challenges associated with studying this DPM in complex biological samples. Asparagine (Asn) deamidation generates L-aspartic acid (L-Asp), D-aspartic acid (D-Asp), L-isoaspartic acid (L-isoAsp) or D-isoaspartic acid (D-isoAsp) residues at the same position of Asn in the affected protein, but each of these amino acids displays similar hydrophobicity and cannot be effectively separated by reverse phase liquid chromatography. The Asp and isoAsp isoforms are also difficult to resolve using mass spectrometry since they have the same mass and fragmentation pattern in MS/MS. Moreover, the 13C peaks of the amidated peptide are often misassigned as monoisotopic peaks of the corresponding deamidated peptides in protein database searches. Furthermore, typical protein isolation and proteomic sample preparation methods induce artificial deamidation that cannot be distinguished from the physiological forms. To better understand the role of deamidation in biological aging and degenerative pathologies, new technologies are now being developed to address these analytical challenges, including mixed mode electrostatic-interaction modified hydrophilic interaction liquid chromatography (emHILIC). When coupled to high resolution, high accuracy tandem mass spectrometry this technology enables unprecedented, proteome-wide study of the 'deamidome' of complex samples. The current article therefore reviews recent advances in sample preparation methods, emHILIC-MS/MS technology, and MS instrumentation / data processing approaches to achieving accurate and reliable characterization of protein deamidation in complex biological and clinical samples.

Keywords:  Asparagine; DPMs; Deamidation; Deamidome; ERLIC-MS/MS; PTMs

DOI:  https://doi.org/10.1016/j.ymeth.2020.05.005
Spectrochim Acta A Mol Biomol Spectrosc. 2020 May 05. pii: S1386-1425(20)30372-3. [Epub ahead of print]239 118394

An accurate and sensitive analytical method for the simultaneous determination of glycine, methionine and homocysteine in biological matrices by matrix matching strategy and LC-quadrupole-time-of-flight-MS/MS.

Meltem Şaylan, Elif Öztürk Er, Zeynep Tekin, Sezgin Bakırdere.

  Nowadays, it is well known that early diagnosis directly affects the success of treatment. Biomarkers play a crucial role in early diagnosis of diseases or explanation of pathological condition. The investigation of new biomarkers depends on the reliable quantification of analytes in biological matrices. Regarding to the critical roles of amino acids in metabolism, functions and nutrition of human body, the careful monitoring of their levels in biological samples is required to evaluate their potential in biomarker studies for clinical research. In this study, a reliable and accurate analytical strategy was developed for the simultaneous determination of glycine, methionine and homocysteine using LC-quadrupole-time of flight-tandem MS system. The method detection limit was found to be 0.73 μg/mL, 0.017 μg/mL and 0.019 μg/mL for glycine, methionine and homocysteine, respectively. The calibration curves were obtained with great linearity (R2 ≥ 0.9993) and low relative standard deviation values showed the repeatability of proposed method. The method applicability was determined using human plasma and urine samples, and high percent recoveries demonstrated the accuracy of method developed. Each measurement was taken less than 4.0 min indicating a promising strategy for the fast and reliable quantification of target amino acids in clinical laboratories.

Keywords:  Amino acids; Biomarkers; Human plasma; Human urine; Tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.saa.2020.118394
Biochim Biophys Acta Mol Cell Res. 2020 May 13. pii: S0167-4889(20)30091-4. [Epub ahead of print] 118733

Role of the HIF oxygen sensing pathway in cell defense and proliferation through the control of amino acid metabolism.

Antonio Bouthelier, Julián Aragonés.

  Cell responses to reduced oxygen supply (hypoxia) are largely mediated by hypoxia-inducible transcription factors (HIFs). The pathophysiological role of the HIF pathway is driven by its ability to potentiate key biological processes as part of the adaptation to hypoxia, such as erythropoiesis and angiogenesis. Moreover, the role of HIF signaling in the reprogramming of cell metabolism is also critical to understand the role of these transcription factors in health and disease. In this regard, HIFs reprogram oxidative metabolism of glucose and fatty acids, offering a molecular mechanism by which the HIF pathway can help cells become more tolerant of redox stress during hypoxic/ischemic episodes. However, the way in which HIFs influence amino acid metabolism and the pathophysiology consequences of these interactions have been less well explored. Here we review recent studies into the role of the HIF1α and HIF2α isoforms in amino acid metabolism, which provides insight to better understand how these factors can influence cell autonomous proliferation and cellular tolerance to hypoxia.

Keywords:  Amino acid metabolism; HIF; Hypoxia-inducible factors; Proliferation; Redox stress; mTORC1

DOI:  https://doi.org/10.1016/j.bbamcr.2020.118733
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 May 05. pii: S1570-0232(19)31685-X. [Epub ahead of print]1148 122145

Development and validation of a rapid, robust and sensitive UPLC-QQQ-MS/MS method for simultaneous quantification of GSH metabolism in lung cancer cells.

Yu-Fei Zhang, Yang Wang, Ke-Ren Zhang, Hui-Min Lei, Ya-Bin Tang, Liang Zhu.

  Changes in cellular metabolism accompany tumor therapeutic resistance. Metabolite concentrations specifically reflect the cellular state. Glutathione (GSH) metabolism maintains the redox homeostasis while also confers therapeutic resistance to cancer cells. However, analytical methods for studying GSH metabolism rely on high-resolution-based untargeted metabolomics. Since the aim of untargeted metabolomics studies is covering as much metabolites as possible, these methods lack sensitivity for simultaneous analysis of intracellular GSH-related metabolites with different polarities and structures. In this study, based on cultured lung cancer cells, we described a rapid, robust and sensitive ultra-performance liquid chromatography-triple quadrupole tandem mass spectrographic method (UPLC-QQQ-MS/MS) to simultaneously quantify a repertoire of GSH-related metabolites, including GSH, GSSG, glycine, cysteine, glutamine, glutamate, cystine, γ-glutamyl-cysteine and cysteinyl-glycine. This method avoided the use of derivatization and/or ion-pairing reagents and was validated according to United States Food and Drug Administration (US FDA) criteria. The lower limit of quantification was determined to be 0.5-100 ng/mL with lower limits of detection at 0.14-10.07 ng/mL. The intra- and inter-day precision values for all the analytes were <15% CV, and the accuracy ranged from 85.4% to 114% at three levels of quality control. This method combined simple preparation with rapid analytical procedure (8 min), allowed for high-throughput analysis of GSH metabolism in basic and therapeutic treatment conditions within cultured cells. Our data showed a significant alteration of GSH metabolism in two independent resistant cells compared to sensitive cells. This method monitored the impact of molecularly targeted drugs on GSH metabolism within lung cancer cells and therefore helped identifying potential metabolic vulnerability for the therapeutic resistance in lung cancer.

Keywords:  EGFR-TKI resistance; Glutathione metabolism; LC-MS/MS; Lung cancer; Metabolic change

DOI:  https://doi.org/10.1016/j.jchromb.2020.122145
Metabolomics. 2020 May 18. 16(6): 66

A review of strategies for untargeted urinary metabolomic analysis using gas chromatography-mass spectrometry.

Mohammad Khodadadi, Morteza Pourfarzam.

   BACKGROUND: Human urine gives evidence of the metabolism in the body and contains different metabolites at various concentrations. A number of analytical techniques including mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been used to obtain metabolites levels in urine samples. However, gas chromatography-mass spectrometry (GC-MS) is one of the most widely used techniques for urinary metabolomics studies due to its higher sensitivity, resolution, reproducibility, reliability, relatively low cost and ease of operation compared to liquid chromatography-mass spectrometry and NMR.
AIM OF REVIEW: This review looks at various aspects of urine preparation prior to analysis by GC-MS including sample storage, urease pretreatment, derivatization, use of internal standard and quality control samples for data correction. In addition, most common types of inlet liners, ionization techniques and columns are discussed and a summary of mass analyzers are also highlighted. Lastly, the role of retention index in metabolite identification and data normalization methods are presented.
KEY SCIENTIFIC CONCEPTS OF REVIEW: The purpose of this review is summarizing methods of sample storage, pretreatment, and GC-MS analysis that are mostly used in urine metabolomics studies. Specific emphasis is given to the critical steps within the GC-MS urine metabolomics that those new to this field need to be aware of and the remaining challenges that require further attention and studies.

Keywords:  Derivatization; Gas chromatography; Mass spectrometry; Metabolomics; Urine

DOI:  https://doi.org/10.1007/s11306-020-01687-x
Mol Cell Proteomics. 2020 May 18. pii: mcp.RA119.001777. [Epub ahead of print]

PolySTest: Robust statistical testing of proteomics data with missing values improves detection of biologically relevant features.

Veit Schwämmle, Christina E Hagensen, Adelina Rogowska-Wrzesinska, Ole N Jensen.

  Statistical testing remains one of the main challenges for high-confidence detection of differentially regulated proteins or peptides in large-scale quantitative proteomics experiments by mass spectrometry. Statistical tests need to be sufficiently robust to deal with experiment intrinsic data structures and variations and often also reduced feature coverage across different biological samples due to ubiquitous missing values. A robust statistical test provides accurate confidence scores of large-scale proteomics results, regardless of instrument platform, experimental protocol and software tools. However, the multitude of different combinations of experimental strategies, mass spectrometry techniques and informatics methods complicate the decision of choosing appropriate statistical approaches. We address this challenge by introducing PolySTest, a user-friendly web service for statistical testing, data browsing and data visualization. We introduce a new method, Miss test, that simultaneously tests for missingness and feature abundance, thereby complementing common statistical tests by rescuing otherwise discarded data features. We demonstrate that PolySTest with integrated Miss test achieves higher confidence and higher sensitivity for artificial and experimental proteomics data sets with known ground truth. Application of PolySTest to mass spectrometry based large-scale proteomics data obtained from differentiating muscle cells resulted in the rescue of 10%-20% additional proteins in the identified molecular networks relevant to muscle differentiation. We conclude that PolySTest is a valuable addition to existing tools and instrument enhancements that improve coverage and depth of large-scale proteomics experiments. A fully functional demo version of PolySTest and Miss test is available via http://computproteomics.bmb.sdu.dk/Apps/PolySTest.

Keywords:  Bioinformatics software; Biostatistics; Computational Biology; Quantification; Statistics

DOI:  https://doi.org/10.1074/mcp.RA119.001777
Anal Chem. 2020 May 18.

Double bond characterization of free fatty acids directly from biological tissues by ultraviolet photodissociation.

Clara Leigh Feider, Luis A Macias, Jennifer S Brodbelt, Livia S Eberlin.

Free fatty acids (FA) are a vital component of cells and are critical to cellular structure and function, so much so that alterations in FA are often associated with cell malfunction and disease. Analysis of FA from biological samples can be achieved by mass spectrometry (MS), but these analyses are often not capable of distinguishing the fine structural alterations within FA isomers, and often limited to global profiling of lipids without spatial resolution. Here, we present the use of ultraviolet photodissociation (UVPD) for the characterization of double bond positional isomers of charge inverted dication●FA complexes and the subsequent implementation of this method for online desorption electrospray ionization (DESI) MS imaging of FA isomers from human tissue sections. This method allows relative quantification of FA isomers from heterogenous biological tissue sections, yielding spatially resolved information about alterations in double bond isomers within these samples. Applying this method to the analysis of the monounsaturated FA 18:1 within breast cancer subtypes uncovered a correlation between double bond positional isomer abundance and the hormone receptor status of the tissue sample, an important factor in the prognosis and treatment of breast cancer patients. This result further validates similar studies that suggest FA synthase activity and FA isomer abundances are significantly altered within breast cancer tissue.

DOI: https://doi.org/10.1021/acs.analchem.0c00970
Anal Chim Acta. 2020 Jun 29. pii: S0003-2670(20)30425-6. [Epub ahead of print]1118 36-43

Enhanced single-cell metabolomics by capillary electrophoresis electrospray ionization-mass spectrometry with field amplified sample injection.

Hsiao-Wei Liao, Stanislav S Rubakhin, Marina C Philip, Jonathan V Sweedler.

  Single-cell metabolomics provides information on the biochemical state of an individual cell and its relationship with the surrounding environment. Characterization of metabolic cellular heterogeneity is challenging, in part due to the small amounts of analytes and their wide dynamic concentration ranges within individual cells. CE-ESI-MS is well suited to single-cell assays because of its low sample-volume requirements and low detection limits. While the volume of a cell is in the picoliter range, after isolation, the typical volume of the lysed cell sample is on the order of a microliter; however, only nanoliters are injected into the CE system, with the volume mismatch limiting analytical performance. Here we developed an approach for the detection of intracellular metabolites from a single neuron using field amplified sample injection (FASI) CE-ESI-MS. Through the application of FASI, we achieved 100- to 300-fold detection limit enhancement compared to hydrodynamic injections. We further enhanced the analyte identification and quantification accuracy via introduction of two internal standards. As a result, the relative standard deviations of migration times were reduced to <5%, aiding identification. Finally, we successfully applied FASI CE-ESI-MS to the untargeted profiling of metabolites of Aplysia californica pleural sensory neurons with <50 μm diameter cell somata. As a result, twenty one neurotransmitters and metabolites have been quantified in these neurons.

Keywords:  Capillary electrophoresis electrospray ionization-mass spectrometry; Field amplified sample injection; Single-cell metabolomics

DOI:  https://doi.org/10.1016/j.aca.2020.04.028
Anal Chem. 2020 May 18.

Chemical Proteomic Profiling of the Interacting Proteins of Isoprenoid Pyrophosphates.

Rong Cai, Xuejiao Dong, Kailin Yu, Xiaomei He, Xiaochuan Liu, Yinsheng Wang.

Isoprenoid pyrophosphates are involved in protein prenylation and assume regulatory roles in cells; however, little is known about the cellular proteins that can interact with isoprenoid pyrophosphates. Here, we devised a chemical proteomic strategy, capitalizing on the use of desthiobiotin-geranyl pyrophosphate (GPP) acyl phosphate probe, for the enrichment and subsequent identification of GPP-binding proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By combining stable isotope labeling by amino acids in cell culture (SILAC) and competitive labeling with low vs. high concentrations of GPP probe, with ATP vs. GPP acyl phosphate probes, or with the GPP probe in the presence of different concentrations of free GPP, we uncovered a number of candidate GPP-binding proteins. We also discovered, for the first time, histone deacetylase 1 (HDAC1) as a GPP-binding protein. Furthermore, we found that the enzymatic activity of HDAC1 could be modulated by isoprenoid pyrophosphates. Together, we developed a novel chemical proteomic method for the proteome-wide discovery of GPP-binding proteins, which sets the stage for a better understanding about the biological functions of isoprenoids.

DOI: https://doi.org/10.1021/acs.analchem.0c01676
Cell Death Differ. 2020 May 20.

Stress-induced TRAILR2 expression overcomes TRAIL resistance in cancer cell spheroids.

Daniela Stöhr, Jens O Schmid, Tobias B Beigl, Alexandra Mack, Daniela S Maichl, Kai Cao, Beate Budai, Gavin Fullstone, Roland E Kontermann, Thomas E Mürdter, Stephen W G Tait, Cathrin Hagenlocher, Nadine Pollak, Peter Scheurich, Markus Rehm.

The influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated.

DOI: https://doi.org/10.1038/s41418-020-0559-3
Cells. 2020 May 19. pii: E1251. [Epub ahead of print]9(5):

Metabolic Adaptation during nab-Paclitaxel Resistance in Pancreatic Cancer Cell Lines.

Lukas M Braun, Simon Lagies, Jessica Guenzle, Stefan Fichtner-Feigl, Uwe A Wittel, Bernd Kammerer.

  Pancreatic ductal adenocarcinoma (PDAC) correlates with high mortality and is about to become one of the major reasons for cancer-related mortality in the next decades. One reason for that high mortality is the limited availability of effective chemotherapy as well as the intrinsic or acquired resistance against it. Here, we report the impact of nab-paclitaxel on the cellular metabolome of PDAC cell lines. After establishment of nab-paclitaxel resistant cell lines, comparison of parental and resistant PDAC cell lines by metabolomics and biochemical assessments revealed altered metabolism, enhanced viability and reduced apoptosis. The results unveiled that acute nab-paclitaxel treatment affected primary metabolism to a minor extent. However, acquisition of resistance led to altered metabolites in both cell lines tested. Specifically, aspartic acid and carbamoyl-aspartic acid were differentially abundant, which might indicate an increased de novo pyrimidine synthesis. This pathway has already shown a similar behavior in other cancerous entities and thus might serve in the future as vulnerable target fighting resistance acquisition occurring in common malignancies.

Keywords:  GC/MS; PDAC; chemotherapy resistance; metabolic reprogramming; metabolomics; nab-Paclitaxel; pancreatic cancer; pancreatic ductal adenocarcinoma

DOI:  https://doi.org/10.3390/cells9051251
Int J Mol Sci. 2020 May 20. pii: E3611. [Epub ahead of print]21(10):

Multiplatform Investigation of Plasma and Tissue Lipid Signatures of Breast Cancer Using Mass Spectrometry Tools.

Alex Ap Rosini Silva, Marcella R Cardoso, Luciana Montes Rezende, John Q Lin, Fernando Guimaraes, Geisilene R Paiva Silva, Michael Murgu, Denise Gonçalves Priolli, Marcos N Eberlin, Alessandra Tata, Livia S Eberlin, Sophie F M Derchain, Andreia M Porcari.

  Plasma and tissue from breast cancer patients are valuable for diagnostic/prognostic purposes and are accessible by multiple mass spectrometry (MS) tools. Liquid chromatography-mass spectrometry (LC-MS) and ambient mass spectrometry imaging (MSI) were shown to be robust and reproducible technologies for breast cancer diagnosis. Here, we investigated whether there is a correspondence between lipid cancer features observed by desorption electrospray ionization (DESI)-MSI in tissue and those detected by LC-MS in plasma samples. The study included 28 tissues and 20 plasma samples from 24 women with ductal breast carcinomas of both special and no special type (NST) along with 22 plasma samples from healthy women. The comparison of plasma and tissue lipid signatures revealed that each one of the studied matrices (i.e., blood or tumor) has its own specific molecular signature and the full interposition of their discriminant ions is not possible. This comparison also revealed that the molecular indicators of tissue injury, characteristic of the breast cancer tissue profile obtained by DESI-MSI, do not persist as cancer discriminators in peripheral blood even though some of them could be found in plasma samples.

Keywords:  breast cancer; desorption-electrospray-ionization—mass spectrometry imaging; lipidomics; liquid chromatography-mass spectrometry; plasma; tumor tissue

DOI:  https://doi.org/10.3390/ijms21103611
J Proteome Res. 2020 May 19.

Improved discrimination of asymmetric and symmetric arginine dimethylation by optimization of the normalized collision energy in LC-MS proteomics.

Nicolas G Hartel, Christopher Z Liu, Nicholas A Graham.

Protein arginine methylation regulates diverse biological processes including signaling, metabolism, splicing, and transcription. Despite its important biological roles, arginine dimethylation remains an understudied post-translational modification. Partly, this is because the two forms of arginine dimethylation, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), are isobaric and therefore indistinguishable by traditional mass spectrometry techniques. Thus, there exists a need for methods that can differentiate these two modifications. Recently, it has been shown that the ADMA and SDMA can be distin-guished by the characteristic neutral loss (NL) of dimethylamine and methylamine, respectively. However, the utility of this meth-od is limited because the vast majority of dimethylarginine peptides do not generate measurable NL ions. Here, we report that increasing the normalized collision energy (NCE) in a higher-energy collisional dissociation (HCD) cell increases the generation of the characteristic NL that distinguish ADMA and SDMA. By analyzing both synthetic and endogenous methyl-peptides, we iden-tify an optimal NCE value that maximizes NL generation and simultaneously improves methyl-peptide identification. Using two orthogonal methyl peptide enrichment strategies, high pH strong cation exchange (SCX) and immunoaffinity purification (IAP), we demonstrate that the optimal NCE increases improves NL-based ADMA and SDMA annotation and dimethyl peptide identi-fications by 125% and 17%, respectively, compared to the standard NCE. This simple parameter change will greatly facilitate the identification and annotation of ADMA and SDMA in mass spectrometry-based methyl-proteomics to improve our understanding of how these modifications differentially regulate protein function. All raw data has been deposited in the PRIDE database with accession number PXD017193.

DOI: https://doi.org/10.1021/acs.jproteome.0c00116
Biochim Biophys Acta Mol Basis Dis. 2020 May 18. pii: S0925-4439(20)30188-5. [Epub ahead of print] 165841

Reprogramming of serine, glycine and one-carbon metabolism in cancer.

Albert M Li, Jiangbin Ye.

  Metabolic pathways leading to the synthesis, uptake, and usage of the nonessential amino acid serine are frequently amplified in cancer. Serine encounters diverse fates in cancer cells, including being charged onto tRNAs for protein synthesis, providing head groups for sphingolipid and phospholipid synthesis, and serving as a precursor for cellular glycine and one-carbon units, which are necessary for nucleotide synthesis and methionine cycle reloading. This review will focus on the participation of serine and glycine in the mitochondrial one-carbon (SGOC) pathway during cancer progression, with an emphasis on the genetic and epigenetic determinants that drive SGOC gene expression. We will discuss recently elucidated roles for SGOC metabolism in nucleotide synthesis, redox balance, mitochondrial function, and epigenetic modifications. Finally, therapeutic considerations for targeting SGOC metabolism in the clinic will be discussed.

Keywords:  Cancer; Glycine; Metabolism; Mitochondria; One-carbon; Serine

DOI:  https://doi.org/10.1016/j.bbadis.2020.165841
Cells. 2020 May 18. pii: E1249. [Epub ahead of print]9(5):

A New Pathway Promotes Adaptation of Human Glioblastoma Cells to Glucose Starvation.

Alberto Azzalin, Francesca Brambilla, Eloisa Arbustini, Katia Basello, Attilio Speciani, Pierluigi Mauri, Paola Bezzi, Lorenzo Magrassi.

  Adaptation of glioblastoma to caloric restriction induces compensatory changes in tumor metabolism that are incompletely known. Here we show that in human glioblastoma cells maintained in exhausted medium, SHC adaptor protein 3 (SHC3) increases due to down-regulation of SHC3 protein degradation. This effect is reversed by glucose addition and is not present in normal astrocytes. Increased SHC3 levels are associated to increased glucose uptake mediated by changes in membrane trafficking of glucose transporters of the solute carrier 2A superfamily (GLUT/SLC2A). We found that the effects on vesicle trafficking are mediated by SHC3 interactions with adaptor protein complex 1 and 2 (AP), BMP-2-inducible protein kinase and a fraction of poly ADP-ribose polymerase 1 (PARP1) associated to vesicles containing GLUT/SLC2As. In glioblastoma cells, PARP1 inhibitor veliparib mimics glucose starvation in enhancing glucose uptake. Furthermore, cytosol extracted from glioblastoma cells inhibits PARP1 enzymatic activity in vitro while immunodepletion of SHC3 from the cytosol significantly relieves this inhibition. The identification of a new pathway controlling glucose uptake in high grade gliomas represents an opportunity for repositioning existing drugs and designing new ones.

Keywords:  GLUT/SLC2A; PARP1; SHC3; aerobic glycolysis; glioblastoma cells

DOI:  https://doi.org/10.3390/cells9051249
Cancers (Basel). 2020 May 17. pii: E1267. [Epub ahead of print]12(5):

Metabolic Plasticity in Ovarian Cancer Stem Cells.

Alia Ghoneum, Daniela Gonzalez, Ammar Yasser Abdulfattah, Neveen Said.

  Ovarian Cancer is the fifth most common cancer in females and remains the most lethal gynecological malignancy as most patients are diagnosed at late stages of the disease. Despite initial responses to therapy, recurrence of chemo-resistant disease is common. The presence of residual cancer stem cells (CSCs) with the unique ability to adapt to several metabolic and signaling pathways represents a major challenge in developing novel targeted therapies. The objective of this study is to investigate the transcripts of putative ovarian cancer stem cell (OCSC) markers in correlation with transcripts of receptors, transporters, and enzymes of the energy generating metabolic pathways involved in high grade serous ovarian cancer (HGSOC). We conducted correlative analysis in data downloaded from The Cancer Genome Atlas (TCGA), studies of experimental OCSCs and their parental lines from Gene Expression Omnibus (GEO), and Cancer Cell Line Encyclopedia (CCLE). We found positive correlations between the transcripts of OCSC markers, specifically CD44, and glycolytic markers. TCGA datasets revealed that NOTCH1, CD133, CD44, CD24, and ALDH1A1, positively and significantly correlated with tricarboxylic acid cycle (TCA) enzymes. OVCAR3-OCSCs (cancer stem cells derived from a well-established epithelial ovarian cancer cell line) exhibited enrichment of the electron transport chain (ETC) mainly in complexes I, III, IV, and V, further supporting reliance on the oxidative phosphorylation (OXPHOS) phenotype. OVCAR3-OCSCs also exhibited significant increase in CD36, ACACA, SCD, and CPT1A, with CD44, CD133, and ALDH1A1 exhibiting positive correlations with lipid metabolic enzymes. TCGA data show positive correlations between OCSC markers and glutamine metabolism enzymes, whereas in OCSC experimental models of GSE64999, GSE28799, and CCLE, the number of positive and negative correlations observed was significantly lower and was different between model systems. Appropriate integration and validation of data model systems with those in patients' specimens is needed not only to bridge our knowledge gap of metabolic programing of OCSCs, but also in designing novel strategies to target the metabolic plasticity of dormant, resistant, and CSCs.

Keywords:  chemo-resistance; metabolic plasticity; ovarian cancer; stem cells

DOI:  https://doi.org/10.3390/cancers12051267
J Proteomics Bioinform. 2019 ;12(7): 104-112

Quantitative Proteomics Using Formalin-fixed, Paraffin-embedded Biopsy Tissues in Inflammatory Disease.

Abhimanyu Amarnani, Joseph R Capri, Puneet Souda, David A Elashoff, Ivan A Lopez, Julian P Whitelegge, Ram R Singh.

   Background: Investigations in human disease pathogenesis have been hampered due to paucity of access to fresh-frozen tissues (FFT) for use in global, data-driven methodologies. As an alternative, formalin-fixed, paraffin-embedded (FFPE) tissues are readily available in pathology banks. However, the use of formalin for fixation can lead to the loss of proteins that appear during inflammation, thus introducing an inherent sample bias. To address this, we compared FF and FFPE tissue proteomics to determine whether FFPE-tissue can be used effectively in inflammatory diseases.
Methods: Adjacent kidney slices from lupus nephritic mice were processed as FFPE or FFTs. Their tissue lysates were run together using proteomics workflow involving filter-aided sample preparation, in-solution dimethyl isotope labeling, StageTip fractionation, and nano-LC MS/MS through an Orbitrap XL MS.
Results: We report a >97% concordance in protein identification between adjacent FFPE and FFTs in murine lupus nephritic kidneys. Specifically, proteins representing pathways, namely, 'systemic lupus erythematosus', 'interferon-α', 'TGF-β', and 'extracellular matrix', were reproducibly quantified between FFPE and FFTs. However, 12%-29% proteins were quantified differently in FFPE compared to FFTs, but the differences were consistent across experiments. In particular, certain proteins represented in pathways, including 'inflammatory response' and 'innate immune system' were quantified less in FFPE than in FFTs. In a pilot study of human FFPE tissues, we identified proteins relevant to pathogenesis in lupus nephritic kidney biopsies compared to control kidneys.
Conclusion: This is the first report of lupus nephritis kidney proteomics using FFPE tissue. We concluded that archived FFPE tissues can be reliably used for proteomic analyses in inflammatory diseases, with a caveat that certain proteins related to immunity and inflammation may be quantified less in FFPE than in FFTs.

Keywords:  Formalin-fixed paraffin-embedded; Fresh frozen; Inflammatory disease; Kidney biopsy; Lupus nephritis; Proteomics

DOI:  https://doi.org/10.35248/0974-276X.12.19.503
BMC Cancer. 2020 May 18. 20(1): 437

Comprehensive metabolomics analysis of prostate cancer tissue in relation to tumor aggressiveness and TMPRSS2-ERG fusion status.

Ilona Dudka, Elin Thysell, Kristina Lundquist, Henrik Antti, Diego Iglesias-Gato, Amilcar Flores-Morales, Anders Bergh, Pernilla Wikström, Gerhard Gröbner.

   BACKGROUND: Prostate cancer (PC) can display very heterogeneous phenotypes ranging from indolent asymptomatic to aggressive lethal forms. Understanding how these PC subtypes vary in their striving for energy and anabolic molecules is of fundamental importance for developing more effective therapies and diagnostics. Here, we carried out an extensive analysis of prostate tissue samples to reveal metabolic alterations during PC development and disease progression and furthermore between TMPRSS2-ERG rearrangement-positive and -negative PC subclasses.
METHODS: Comprehensive metabolomics analysis of prostate tissue samples was performed by non-destructive high-resolution magic angle spinning nuclear magnetic resonance (1H HR MAS NMR). Subsequently, samples underwent moderate extraction, leaving tissue morphology intact for histopathological characterization. Metabolites in tissue extracts were identified by 1H/31P NMR and liquid chromatography-mass spectrometry (LC-MS). These metabolomics profiles were analyzed by chemometric tools and the outcome was further validated using proteomic data from a separate sample cohort.
RESULTS: The obtained metabolite patterns significantly differed between PC and benign tissue and between samples with high and low Gleason score (GS). Five key metabolites (phosphocholine, glutamate, hypoxanthine, arginine and α-glucose) were identified, who were sufficient to differentiate between cancer and benign tissue and between high to low GS. In ERG-positive PC, the analysis revealed several acylcarnitines among the increased metabolites together with decreased levels of proteins involved in β-oxidation; indicating decreased acyl-CoAs oxidation in ERG-positive tumors. The ERG-positive group also showed increased levels of metabolites and proteins involved in purine catabolism; a potential sign of increased DNA damage and oxidative stress.
CONCLUSIONS: Our comprehensive metabolomic analysis strongly indicates that ERG-positive PC and ERG-negative PC should be considered as different subtypes of PC; a fact requiring different, sub-type specific treatment strategies for affected patients.

Keywords:  1H HRMAS NMR; Gleason score; Metabolomics; Prostate cancer; TMPRSS2-ERG

DOI:  https://doi.org/10.1186/s12885-020-06908-z
Molecules. 2020 May 14. pii: E2310. [Epub ahead of print]25(10):

HILIC-ESI-FTMS with All Ion Fragmentation (AIF) Scans as a Tool for Fast Lipidome Investigations.

Giovanni Ventura, Mariachiara Bianco, Cosima Damiana Calvano, Ilario Losito, Tommaso R I Cataldi.

  Lipidomics suffers from the lack of fast and reproducible tools to obtain both structural information on intact phospholipids (PL) and fatty acyl chain composition. Hydrophilic interaction liquid chromatography with electrospray ionization coupled to an orbital-trap Fourier-transform analyzer operating using all ion fragmentation mode (HILIC-ESI-FTMS-AIF MS) is seemingly a valuable resource in this respect. Here, accurate m/z values, HILIC retention times and AIF MS scan data were combined for PL assignment in standard mixtures or real lipid extracts. AIF scans in both positive and negative ESI mode, achieved using collisional induced dissociation for fragmentation, were applied to identify both the head-group of each PL class and the fatty acyl chains, respectively. An advantage of the AIF approach was the concurrent collection of tandem MS-like data, enabling the identification of linked fatty acyl chains of precursor phospholipids through the corresponding carboxylate anions. To illustrate the ability of AIF in the field of lipidomics, two different types of real samples, i.e., the lipid extracts obtained from human plasma and dermal fibroblasts, were examined. Using AIF scans, a total of 253 intact lipid species and 18 fatty acids across 4 lipid classes were recognized in plasma samples, while FA C20:3 was confirmed as the fatty acyl chain belonging to phosphatidylinositol, PI 38:3, which was found to be down-regulated in fibroblast samples of Parkinson's disease patients.

Keywords:  AIF scan MS; HILIC separation; collisional induced dissociation; fatty acids; phospholipids

DOI:  https://doi.org/10.3390/molecules25102310
J Proteomics. 2020 May 16. pii: S1874-3919(20)30191-3. [Epub ahead of print] 103823

Small intestine proteomics coupled with serum metabolomics reveal disruption of amino acid metabolism in Chinese hamsters with type 2 diabetes mellitus.

Chenyang Wang, Jingjing Yu, Ruihu Zhang, Wentao Wang, Zeya Shi, Yu Liu, Guohua Song, Hailong Wang, Naijian Han, Liqun Huang, Yuze An, Shouyuan Tian, Zhaoyang Chen.

  Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by hyperglycemia, with metabolic disturbances resulting from defects in insulin secretion, insulin resistance (IR), or both. Chinese hamsters have potential value as non-obese animal models of spontaneous T2DM for studying the pathogenesis and molecular characteristics of diabetes. In this study, the molecular characteristics of the Chinese hamster diabetes animal model were investigated through small intestine proteomics and serum metabolomics. A total of 213 differentially abundant proteins and 14 differentially abundant metabolites were identified through liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-time of flight mass spectrometry (GC-TOF/MS) analysis, respectively. Annotation by bioinformatics analysis revealed that these differentially abundant proteins in the small intestine were commonly associated with abnormal glucose and lipid metabolism, IR, impaired insulin secretion, amino acid metabolism disorders, and inflammatory dysregulation. Moreover, differentially abundant metabolites in the serum were amino acids and were related to diabetic IR. Through the analysis of small intestine proteomics and serum metabolomics in the Chinese hamster diabetes model, we provide a preliminary understanding of the diabetic characteristics of this model from a molecular perspective. This study provides data incentivizing the popularization and application of Chinese hamsters in T2DM research. SIGNIFICANCE: Spontaneous rodent models of diabetes, such as Chinese hamsters, effectively summarizes the clinical characteristics of type 2 diabetes and has high applicative value for studying the pathophysiology of diabetes. In order to explore the potential value of the Chinese hamster diabetes animal model in the study of the T2DM molecular mechanism, we performed small intestine proteomic analysis and serum metabolomic analysis in Chinese hamsters for the first time. After an integrated analysis of proteomics and metabolomics, we have a preliminary understanding of the diabetic characteristics of this model from a molecular perspective. Further, we found that in the occurrence and development of T2DM, the metabolic abnormalities of this model are particularly prominent, especially the metabolism of amino acids. These findings not only provide basic data in support of the popularization and application of the current model in T2DM research, but also provide a new perspective for the exploration of mechanisms related to T2DM.

Keywords:  Chinese hamster; Metabolomics; Proteomics; Type 2 diabetes mellitus

DOI:  https://doi.org/10.1016/j.jprot.2020.103823
Lipids. 2020 May 18.

Development of a Novel High-Performance Thin Layer Chromatography-Based Method for the Simultaneous Quantification of Clinically Relevant Lipids from Cells and Tissue Extracts.

Michelle Pinault, Cyrille Guimaraes, Céline Ben Hassen, Jorge L Gutierrez-Pajares, Stéphan Chevalier, Caroline Goupille, Pierre Bernard-Savary, Philippe G Frank.

  Lipids such as cholesterol, triacylglycerols, and fatty acids play important roles in the regulation of cellular metabolism and cellular signaling pathways and, as a consequence, in the development of various diseases. It is therefore important to understand how their metabolism is regulated to better define the components involved in the development of various human diseases. In the present work, we describe the development and validation of a high-performance thin layer chromatography (HPTLC) method allowing the separation and quantification of free cholesterol, cholesteryl esters, nonesterified fatty acids, and triacylglycerols. This method will be of interest as the quantification of these lipids in one single assay is difficult to perform.

Keywords:  Cancer; Cholesterol; Fatty acids; Triacylglycerols

DOI:  https://doi.org/10.1002/lipd.12245
Heliyon. 2020 May;6(5): e03910

ACLY is the novel signaling target of PIP2/PIP3 and Lyn in acute myeloid leukemia.

Johnvesly Basappa, Mevlut Citir, Qian Zhang, Hong Y Wang, Xiaobin Liu, Olga Melnikov, Hafiz Yahya, Frank Stein, Rainer Muller, Alexis Traynor-Kaplan, Carsten Schultz, Mariusz A Wasik, Andrzej Ptasznik.

  A fundamental feature of tumor progression is reprogramming of metabolic pathways. ATP citrate lyase (ACLY) is a key metabolic enzyme that catalyzes the generation of Acetyl-CoA and is upregulated in cancer cells and required for their growth. The phosphoinositide 3-kinase (PI3K) and Src-family kinase (SFK) Lyn are constitutively activate in many cancers. We show here, for the first time, that both the substrate and product of PI3K, phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), respectively, bind to ACLY in Acute Myeloid Leukemia (AML) patient-derived, but not normal donor-derived cells. We demonstrate the binding of PIP2 to the CoA-binding domain of ACLY and identify the six tyrosine residues of ACLY that are phosphorylated by Lyn. Three of them (Y682, Y252, Y227) can be also phosphorylated by Src and they are located in catalytic, citrate binding and ATP binding domains, respectively. PI3K and Lyn inhibitors reduce the ACLY enzyme activity, ACLY-mediated Acetyl-CoA synthesis, phospholipid synthesis, histone acetylation and cell growth. Thus, PIP2/PIP3 binding and Src tyrosine kinases-mediated stimulation of ACLY links oncogenic pathways to Acetyl-CoA-dependent pro-growth and survival metabolic pathways in cancer cells. These results indicate a novel function for Lyn, as a regulator of Acetyl-CoA-mediated metabolic pathways.

Keywords:  ACLY; Acetyl-CoA; Biochemistry; Biological sciences; Cancer; Cancer research; Health sciences; Lyn; Metabolism; PI3K; Src

DOI:  https://doi.org/10.1016/j.heliyon.2020.e03910
Cell Death Dis. 2020 May 22. 11(5): 390

Sodium-glucose cotransporter 2 inhibition suppresses HIF-1α-mediated metabolic switch from lipid oxidation to glycolysis in kidney tubule cells of diabetic mice.

Ting Cai, Qingqing Ke, Yi Fang, Ping Wen, Hanzhi Chen, Qi Yuan, Jing Luo, Yu Zhang, Qi Sun, Yunhui Lv, Ke Zen, Lei Jiang, Yang Zhou, Junwei Yang.

Inhibition of sodium-glucose cotransporter 2 (SGLT2) in the proximal tubule of the kidney has emerged as an effective antihyperglycemic treatment. The potential protective role of SGLT2 inhibition on diabetic kidney disease (DKD) and underlying mechanism, however, remains unknown. In this study, metabolic switch was examined using kidney samples from human with diabetes and streptozocin (STZ)-induced experimental mouse model of diabetes treated with or without SGLT2 inhibitor dapagliflozin. Results were further validated using primarily cultured proximal tubule epithelial cells. We found that DKD development and progression to renal fibrosis entailed profound changes in proximal tubule metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation, which is associated with the increased expression of HIF-1α. Diabetes-induced tubulointerstitial damage, such as macrophage infiltration and fibrosis, was significantly improved by dapagliflozin. Consistent with the effects of these beneficial interventions, the metabolic disorder was almost completely eliminated by dapagliflozin. The increased level of HIF-1α in renal proximal tubule was nearly nullified by dapagliflozin. Moreover, dapagliflozin protects against glucose-induced metabolic shift in PTCs via inhibiting HIF-1α. It suggests that SGLT2 inhibition is efficient in rectifying the metabolic disorder and may be a novel prevention and treatment strategy for kidney tubule in DKD.

DOI: https://doi.org/10.1038/s41419-020-2544-7
Proc Natl Acad Sci U S A. 2020 May 19. pii: 201913370. [Epub ahead of print]

β-Oxidation and autophagy are critical energy providers during acute glucose depletion in Saccharomyces cerevisiae.

Carmen A Weber, Karthik Sekar, Jeffrey H Tang, Philipp Warmer, Uwe Sauer, Karsten Weis.

  The ability to tolerate and thrive in diverse environments is paramount to all living organisms, and many organisms spend a large part of their lifetime in starvation. Upon acute glucose starvation, yeast cells undergo drastic physiological and metabolic changes and reestablish a constant-although lower-level of energy production within minutes. The molecules that are rapidly metabolized to fuel energy production under these conditions are unknown. Here, we combine metabolomics and genetics to characterize the cells' response to acute glucose depletion and identify pathways that ensure survival during starvation. We show that the ability to respire is essential for maintaining the energy status and to ensure viability during starvation. Measuring the cells' immediate metabolic response, we find that central metabolites drastically deplete and that the intracellular AMP-to-ATP ratio strongly increases within 20 to 30 s. Furthermore, we detect changes in both amino acid and lipid metabolite levels. Consistent with this, both bulk autophagy, a process that frees amino acids, and lipid degradation via β-oxidation contribute in parallel to energy maintenance upon acute starvation. In addition, both these pathways ensure long-term survival during starvation. Thus, our results identify bulk autophagy and β-oxidation as important energy providers during acute glucose starvation.

Keywords:  Saccharomyces cerevisiae; acute glucose starvation; lipidomics; metabolomics; β-oxidation

DOI:  https://doi.org/10.1073/pnas.1913370117
Biosci Biotechnol Biochem. 2020 May 18. 1-8

Ferroptosis is involved in alcohol-induced cell death in vivo and in vitro.

Chun-Yu Liu, Min Wang, Hong-Min Yu, Fang-Xuan Han, Qiong-Shi Wu, Xing-Jun Cai, Hiroshi Kurihara, Yong-Xing Chen, Yi-Fang Li, Rong-Rong He.

  A critical pathogenic factor in the development of lethal liver failure is cell death induced by the accumulation of lipid reactive oxygen species. In this study, we discovered and illuminated a new mechanism that led to alcoholic liver disease via ferroptosis, an iron-dependent regulated cell death. Study in vitro showed that both necroptosis inhibitor and ferroptosis inhibitors performed significantly protective effect on alcohol-induced cell death, while apoptosis inhibitor and autophagy inhibitor had no such effect. Our data also indicated that alcohol caused the accumulation of lipid peroxides and the mRNA expression of prostaglandin-endoperoxide synthase 2, reduced the protein expression of the specific light-chain subunit of the cystine/glutamate antiporter and glutathione peroxidase 4. Importantly, ferrostatin-1 significantly ameliorated liver injury that was induced by overdosed alcohol both in vitro and in vivo. These findings highlight that targeting ferroptosis serves as a hepatoprotective strategy for alcoholic liver disease treatment.

Keywords:  Alcohol; ferroptosis; hepatotoxicity; lipid peroxidation

DOI:  https://doi.org/10.1080/09168451.2020.1763155
Sci Rep. 2020 May 21. 10(1): 8428

PPARδ-mediated mitochondrial rewiring of osteoblasts determines bone mass.

Dorothea I H Müller, Cornelia Stoll, Katrin Palumbo-Zerr, Christina Böhm, Brenda Krishnacoumar, Natacha Ipseiz, Jule Taubmann, Max Zimmermann, Martin Böttcher, Dimitrios Mougiakakos, Jan Tuckermann, Farida Djouad, Georg Schett, Carina Scholtysek, Gerhard Krönke.

Bone turnover, which is determined by osteoclast-mediated bone resorption and osteoblast-mediated bone formation, represents a highly energy consuming process. The metabolic requirements of osteoblast differentiation and mineralization, both essential for regular bone formation, however, remain incompletely understood. Here we identify the nuclear receptor peroxisome proliferator-activated receptor (PPAR) δ as key regulator of osteoblast metabolism. Induction of PPARδ was essential for the metabolic adaption and increased rate in mitochondrial respiration necessary for the differentiation and mineralization of osteoblasts. Osteoblast-specific deletion of PPARδ in mice, in turn, resulted in an altered energy homeostasis of osteoblasts, impaired mineralization and reduced bone mass. These data show that PPARδ acts as key regulator of osteoblast metabolism and highlight the relevance of cellular metabolic rewiring during osteoblast-mediated bone formation and bone-turnover.

DOI: https://doi.org/10.1038/s41598-020-65305-5