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1
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Ito-Harashima S, Miura N. Compartmentation of multiple metabolic enzymes and their preparation in vitro and in cellulo. Biochim Biophys Acta Gen Subj 2025; 1869:130787. [PMID: 40058614 DOI: 10.1016/j.bbagen.2025.130787] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2024] [Revised: 02/27/2025] [Accepted: 03/03/2025] [Indexed: 03/15/2025]
Abstract
Compartmentalization of multiple enzymes in cellulo and in vitro is a means of controlling the cascade reaction of metabolic enzymes. The compartmentation of enzymes through liquid-liquid phase separation may facilitate the reversible control of biocatalytic cascade reactions, thereby reducing the transcriptional and translational burden. This has attracted attention as a potential application in bioproduction. Recent research has demonstrated the existence and regulatory mechanisms of various enzyme compartments within cells. Mounting evidence suggests that enzyme compartmentation allows in vitro and in vivo regulation of cellular metabolism. However, the comprehensive regulatory mechanisms of enzyme condensates in cells and ideal organization of cellular systems remain unknown. This review provides an overview of the recent progress in multiple enzyme compartmentation in cells and summarizes strategies to reconstruct multiple enzyme assemblies in vitro and in cellulo. By examining parallel examples, we have evaluated the consensus and future perspectives of enzyme condensation.
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Affiliation(s)
- Sayoko Ito-Harashima
- Department of Applied Biological Chemistry, Graduate School of Agriculture, Osaka Metropolitan University, Sakai 599-8531, Japan
| | - Natsuko Miura
- Department of Applied Biological Chemistry, Graduate School of Agriculture, Osaka Metropolitan University, Sakai 599-8531, Japan.
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2
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André AA, Rehnberg N, Garg A, Kjærgaard M. Toward Design Principles for Biomolecular Condensates for Metabolic Pathways. Adv Biol (Weinh) 2025; 9:e2400672. [PMID: 40195042 PMCID: PMC12078866 DOI: 10.1002/adbi.202400672] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2024] [Revised: 01/14/2025] [Indexed: 04/09/2025]
Abstract
Biology uses membrane-less organelles or biomolecular condensates as dynamic reaction compartments that can form or dissolve to regulate biochemical pathways. This has led to a flurry of research aiming to design new synthetic organelles that function as reaction crucibles for enzymes and biomolecular cascades in biotechnology. The mechanisms by which a condensate can enhance multistep biochemical processes including mass action, tuning the chemical environment, scaffolding and metabolic channelling is reviewed. These mechanisms are not inherently beneficial for the rate of enzymatic processes but can also inhibit a reaction. Similarly, some aspects of condensates are likely intrinsically inhibitory including retardation of diffusion, where the net effect of a condensate will be a trade-off between inhibitory and stimulatory effects. It is discussed which generalizable conclusions can be drawn so far and how close it is to design principles for condensates for enzyme cascades in microbial cell factories including which reactions are likely to be enhanced by condensates and which type of condensate will be suited for which reaction.
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Affiliation(s)
- Alain A.M. André
- Department of Molecular Biology and GeneticsAarhus University, Denmar
- Interdisciplinary Nanoscience Center (iNANO)Aarhus University, Denmark
| | - Nikita Rehnberg
- Department of Molecular Biology and GeneticsAarhus University, Denmar
- Interdisciplinary Nanoscience Center (iNANO)Aarhus University, Denmark
| | - Ankush Garg
- Department of Molecular Biology and GeneticsAarhus University, Denmar
- Interdisciplinary Nanoscience Center (iNANO)Aarhus University, Denmark
| | - Magnus Kjærgaard
- Department of Molecular Biology and GeneticsAarhus University, Denmar
- Interdisciplinary Nanoscience Center (iNANO)Aarhus University, Denmark
- The Danish Research Institute for Translational Neuroscience (DANDRITE)Aarhus University, Denmark
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3
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Samuel Russell PP, Rickard MM, Pogorelov TV, Gruebele M. Enzymes in a human cytoplasm model organize into submetabolon complexes. Proc Natl Acad Sci U S A 2025; 122:e2414206122. [PMID: 39874290 PMCID: PMC11804712 DOI: 10.1073/pnas.2414206122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2024] [Accepted: 12/18/2024] [Indexed: 01/30/2025] Open
Abstract
Enzyme-enzyme interactions are fundamental to the function of cells. Their atomistic mechanisms remain elusive mainly due to limitations of in-cell measurements. We address this challenge by atomistically modeling, for a total of ≈80 μs, a slice of the human cell cytoplasm that includes three successive enzymes along the glycolytic pathway: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and phosphoglycerate mutase (PGM). We tested the model for nonspecific protein stickiness, an artifact of current atomistic force fields in crowded environments. The simulations reveal that the human enzymes co-organize in-cell into transient submetabolon complexes, consistent with previous experimental results. Our data both reiterate known specificity between GAPDH and PGK and reveal extensive direct interactions between GAPDH and PGM. Our simulations further reveal, through force field benchmarking, the critical role of protein solvation in facilitating these enzyme-enzyme interactions. Transient interenzyme interactions with μs lifetime occur repeatedly in our simulations via specific sticky protein surface patches, with interactions often mediated by charged patch residues. Some of the residues that interact frequently with one another lie in or near the active site of the enzymes. We show that some of these patches correspond to a general mode to interact with several partners for promiscuous enzymes like GAPDH. We further show that the non-native yeast PGK is stickier than human PGK in our human cytoplasm model, supporting the idea of evolutionary pressure to reduce sticking. Our cytoplasm modeling paves the way toward capturing the atomistic dynamics of an entire enzymatic pathway in-cell.
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Affiliation(s)
- Premila P. Samuel Russell
- Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL61801
- Department of Chemistry, Saint Louis University, Saint Louis, MO63103
| | - Meredith M. Rickard
- Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL61801
| | - Taras V. Pogorelov
- Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL61801
- School of Chemical Sciences, University of Illinois Urbana-Champaign, Urbana, IL61801
- Center for Biophysics and Computational Biology, University of Illinois Urbana-Champaign, Urbana, IL61801
- Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL61801
- National Center for Supercomputing Applications, University of Illinois Urbana-Champaign, Urbana, IL61801
| | - Martin Gruebele
- Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL61801
- Center for Biophysics and Computational Biology, University of Illinois Urbana-Champaign, Urbana, IL61801
- Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL61801
- Department of Physics, University of Illinois Urbana-Champaign, Urbana, IL61801
- Carle-Illinois College of Medicine, University of Illinois Urbana-Champaign, Urbana, IL61801
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4
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Yang W, Wang Y, Liu G, Wang Y, Wu C. TPM4 condensates glycolytic enzymes and facilitates actin reorganization under hyperosmotic stress. Cell Discov 2024; 10:120. [PMID: 39622827 PMCID: PMC11612400 DOI: 10.1038/s41421-024-00744-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2024] [Accepted: 10/20/2024] [Indexed: 12/06/2024] Open
Abstract
Actin homeostasis is fundamental for cell structure and consumes a large portion of cellular ATP. It has been documented in the literature that certain glycolytic enzymes can interact with actin, indicating an intricate interplay between the cytoskeleton and cellular metabolism. Here we report that hyperosmotic stress triggers actin severing and subsequent phase separation of the actin-binding protein tropomyosin 4 (TPM4). TPM4 condensates recruit glycolytic enzymes such as HK2, PFKM, and PKM2, while wetting actin filaments. Notably, the condensates of TPM4 and glycolytic enzymes are enriched of NADH and ATP, suggestive of their functional importance in cell metabolism. At cellular level, actin filament assembly is enhanced upon hyperosmotic stress and TPM4 condensation, while depletion of TPM4 impairs osmolarity-induced actin reorganization. At tissue level, colocalized condensates of TPM4 and glycolytic enzymes are observed in renal tissues subjected to hyperosmotic stress. Together, our findings suggest that stress-induced actin perturbation may act on TPM4 to organize glycolytic hubs that tether energy production to cytoskeletal reorganization.
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Affiliation(s)
- Wenzhong Yang
- Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
- International Cancer Institute, Peking University, Beijing, China
- Institute of Advanced Clinical Medicine, State Key Laboratory of Molecular Oncology, Beijing, China
| | - Yuan Wang
- Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
- International Cancer Institute, Peking University, Beijing, China
- Institute of Advanced Clinical Medicine, State Key Laboratory of Molecular Oncology, Beijing, China
| | - Geyao Liu
- Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
- International Cancer Institute, Peking University, Beijing, China
- Institute of Advanced Clinical Medicine, State Key Laboratory of Molecular Oncology, Beijing, China
| | - Yan Wang
- Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
- International Cancer Institute, Peking University, Beijing, China
- Institute of Advanced Clinical Medicine, State Key Laboratory of Molecular Oncology, Beijing, China
| | - Congying Wu
- Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.
- International Cancer Institute, Peking University, Beijing, China.
- Institute of Advanced Clinical Medicine, State Key Laboratory of Molecular Oncology, Beijing, China.
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5
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Li X, Wen X, Tang W, Wang C, Chen Y, Yang Y, Zhang Z, Zhao Y. Elucidating the spatiotemporal dynamics of glucose metabolism with genetically encoded fluorescent biosensors. CELL REPORTS METHODS 2024; 4:100904. [PMID: 39536758 PMCID: PMC11705769 DOI: 10.1016/j.crmeth.2024.100904] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Revised: 08/20/2024] [Accepted: 10/21/2024] [Indexed: 11/16/2024]
Abstract
Glucose metabolism has been well understood for many years, but some intriguing questions remain regarding the subcellular distribution, transport, and functions of glycolytic metabolites. To address these issues, a living cell metabolic monitoring technology with high spatiotemporal resolution is needed. Genetically encoded fluorescent sensors can achieve specific, sensitive, and spatiotemporally resolved metabolic monitoring in living cells and in vivo, and dozens of glucose metabolite sensors have been developed recently. Here, we highlight the importance of tracking specific intermediate metabolites of glycolysis and glycolytic flux measurements, monitoring the spatiotemporal dynamics, and quantifying metabolite abundance. We then describe the working principles of fluorescent protein sensors and summarize the existing biosensors and their application in understanding glucose metabolism. Finally, we analyze the remaining challenges in developing high-quality biosensors and the huge potential of biosensor-based metabolic monitoring at multiple spatiotemporal scales.
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Affiliation(s)
- Xie Li
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China; Research Unit of New Techniques for Live-Cell Metabolic Imaging, Chinese Academy of Medical Sciences, Beijing 100730, China.
| | - Xueyi Wen
- Research Unit of New Techniques for Live-Cell Metabolic Imaging, Chinese Academy of Medical Sciences, Beijing 100730, China
| | - Weitao Tang
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China
| | - Chengnuo Wang
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China
| | - Yaqiong Chen
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China
| | - Yi Yang
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China
| | - Zhuo Zhang
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China; Research Unit of New Techniques for Live-Cell Metabolic Imaging, Chinese Academy of Medical Sciences, Beijing 100730, China.
| | - Yuzheng Zhao
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China; Research Unit of New Techniques for Live-Cell Metabolic Imaging, Chinese Academy of Medical Sciences, Beijing 100730, China.
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6
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Kierans SJ, Taylor CT. Glycolysis: A multifaceted metabolic pathway and signaling hub. J Biol Chem 2024; 300:107906. [PMID: 39442619 PMCID: PMC11605472 DOI: 10.1016/j.jbc.2024.107906] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2024] [Revised: 10/07/2024] [Accepted: 10/14/2024] [Indexed: 10/25/2024] Open
Abstract
Glycolysis is a highly conserved metabolic pathway responsible for the anaerobic production of adenosine triphosphate (ATP) from the breakdown of glucose molecules. While serving as a primary metabolic pathway in prokaryotes, glycolysis is also utilized by respiring eukaryotic cells, providing pyruvate to fuel oxidative metabolism. Furthermore, glycolysis is the primary source of ATP production in multiple cellular states (e.g., hypoxia) and is particularly important in maintaining bioenergetic homeostasis in the most abundant cell type in the human body, the erythrocyte. Beyond its role in ATP production, glycolysis also functions as a signaling hub, producing several metabolic intermediates which serve roles in both signaling and metabolic processes. These signals emanating from the glycolytic pathway can profoundly impact cell function, phenotype, and fate and have previously been overlooked. In this review, we will discuss the role of the glycolytic pathway as a source of signaling molecules in eukaryotic cells, emphasizing the newfound recognition of glycolysis' multifaceted nature and its importance in maintaining cellular homeostasis, beyond its traditional role in ATP synthesis.
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Affiliation(s)
- Sarah J Kierans
- Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland; UCD School of Medicine, University College Dublin, Dublin, Ireland
| | - Cormac T Taylor
- Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland; UCD School of Medicine, University College Dublin, Dublin, Ireland.
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7
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DeMichele E, Buret AG, Taylor CT. Hypoxia-inducible factor-driven glycolytic adaptations in host-microbe interactions. Pflugers Arch 2024; 476:1353-1368. [PMID: 38570355 PMCID: PMC11310250 DOI: 10.1007/s00424-024-02953-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2024] [Revised: 02/07/2024] [Accepted: 03/22/2024] [Indexed: 04/05/2024]
Abstract
Mammalian cells utilize glucose as a primary carbon source to produce energy for most cellular functions. However, the bioenergetic homeostasis of cells can be perturbed by environmental alterations, such as changes in oxygen levels which can be associated with bacterial infection. Reduction in oxygen availability leads to a state of hypoxia, inducing numerous cellular responses that aim to combat this stress. Importantly, hypoxia strongly augments cellular glycolysis in most cell types to compensate for the loss of aerobic respiration. Understanding how this host cell metabolic adaptation to hypoxia impacts the course of bacterial infection will identify new anti-microbial targets. This review will highlight developments in our understanding of glycolytic substrate channeling and spatiotemporal enzymatic organization in response to hypoxia, shedding light on the integral role of the hypoxia-inducible factor (HIF) during host-pathogen interactions. Furthermore, the ability of intracellular and extracellular bacteria (pathogens and commensals alike) to modulate host cellular glucose metabolism will be discussed.
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Affiliation(s)
- Emily DeMichele
- School of Medicine and Systems Biology Ireland, The Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
- Department of Biological Sciences, University of Calgary, Calgary, AB, Canada
| | - Andre G Buret
- Department of Biological Sciences, University of Calgary, Calgary, AB, Canada
| | - Cormac T Taylor
- School of Medicine and Systems Biology Ireland, The Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
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8
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Wang H, Vant JW, Zhang A, Sanchez RG, Wu Y, Micou ML, Luczak V, Whiddon Z, Carlson NM, Yu SB, Jabbo M, Yoon S, Abushawish AA, Ghassemian M, Masubuchi T, Gan Q, Watanabe S, Griffis ER, Hammarlund M, Singharoy A, Pekkurnaz G. Organization of a functional glycolytic metabolon on mitochondria for metabolic efficiency. Nat Metab 2024; 6:1712-1735. [PMID: 39261628 DOI: 10.1038/s42255-024-01121-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/15/2023] [Accepted: 08/02/2024] [Indexed: 09/13/2024]
Abstract
Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, and is crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyses reversible O-GlcNAcylation, a post-translational modification influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of the regulatory domain of HK1, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances the mitochondrial association with HK1, orchestrating glycolytic and mitochondrial ATP production. Mutation in HK1's O-GlcNAcylation site reduces ATP generation in multiple cell types, specifically affecting metabolic efficiency in neurons. This study reveals a previously unappreciated pathway that links neuronal metabolism and mitochondrial function through OGT and the formation of the glycolytic metabolon, providing potential strategies for tackling metabolic and neurological disorders.
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Affiliation(s)
- Haoming Wang
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
| | - John W Vant
- Biodesign Institute, The School of Molecular Sciences, Arizona State University, Tempe, AZ, USA
| | - Andrew Zhang
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
| | - Richard G Sanchez
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
| | - Youjun Wu
- Department of Genetics and Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA
| | - Mary L Micou
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
- Thomas Jefferson University, Philadelphia, PA, USA
| | - Vincent Luczak
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
- Neurocrine Biosciences, San Diego, CA, USA
| | - Zachary Whiddon
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
| | - Natasha M Carlson
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
| | - Seungyoon B Yu
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
- Denali Therapeutics Inc., South San Francisco, CA, USA
| | - Mirna Jabbo
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
| | - Seokjun Yoon
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
- University of Southern California, Los Angeles, CA, USA
| | - Ahmed A Abushawish
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
| | - Majid Ghassemian
- Biomolecular and Proteomics Mass Spectrometry Facility, University of California San Diego, La Jolla, CA, USA
| | - Takeya Masubuchi
- Cell and Developmental Biology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA
| | - Quan Gan
- Department of Cell Biology, Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, Baltimore, MD, USA
| | - Shigeki Watanabe
- Department of Cell Biology, Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, Baltimore, MD, USA
| | - Eric R Griffis
- Nikon Imaging Center, University of California San Diego, La Jolla, CA, USA
- Altos Labs, San Diego, CA, USA
| | - Marc Hammarlund
- Department of Genetics and Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA
| | - Abhishek Singharoy
- Biodesign Institute, The School of Molecular Sciences, Arizona State University, Tempe, AZ, USA
| | - Gulcin Pekkurnaz
- Neurobiology Department, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA.
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9
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Lynch EM, Hansen H, Salay L, Cooper M, Timr S, Kollman JM, Webb BA. Structural basis for allosteric regulation of human phosphofructokinase-1. Nat Commun 2024; 15:7323. [PMID: 39183237 PMCID: PMC11345425 DOI: 10.1038/s41467-024-51808-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2024] [Accepted: 08/19/2024] [Indexed: 08/27/2024] Open
Abstract
Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively. Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.
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Affiliation(s)
- Eric M Lynch
- Department of Biochemistry, University of Washington, Seattle, WA, USA
| | - Heather Hansen
- Department of Biochemistry and Molecular Medicine, West Virginia University, Morgantown, WV, USA
| | - Lauren Salay
- Department of Biochemistry, University of Washington, Seattle, WA, USA
| | - Madison Cooper
- Department of Biochemistry and Molecular Medicine, West Virginia University, Morgantown, WV, USA
| | - Stepan Timr
- Department of Computational Chemistry, J. Heyrovsky Institute of Physical Chemistry, Czech Academy of Sciences, Prague, Czech Republic
| | - Justin M Kollman
- Department of Biochemistry, University of Washington, Seattle, WA, USA.
| | - Bradley A Webb
- Department of Biochemistry and Molecular Medicine, West Virginia University, Morgantown, WV, USA.
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10
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Kang HW, Nguyen L, An S, Kyoung M. Mechanistic insights into condensate formation of human liver-type phosphofructokinase by stochastic modeling approaches. Sci Rep 2024; 14:19011. [PMID: 39152221 PMCID: PMC11329711 DOI: 10.1038/s41598-024-69534-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2023] [Accepted: 08/06/2024] [Indexed: 08/19/2024] Open
Abstract
Human liver-type phosphofructokinase 1 (PFKL) has been shown to regulate glucose flux as a scaffolder arranging glycolytic and gluconeogenic enzymes into a multienzyme metabolic condensate, the glucosome. However, it has remained elusive of how phase separation of PFKL is governed and initiates glucosome formation in living cells, thus hampering to understand a mechanism of glucosome formation and its functional contribution to human cells. In this work, we developed a stochastic model in silico using the principle of Langevin dynamics to investigate how biological properties of PFKL contribute to the condensate formation. The significance of an intermolecular interaction between PFKLs, an effective concentration of PFKL at a region of interest, and its own self-assembled filaments in formation of PFKL condensates and control of their sizes were demonstrated by molecular dynamics simulation using the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS). Such biological properties that define intracellular dynamics of PFKL appear to be essential for phase separation of PFKL, which may represent an initiation step for the formation of glucosome condensates. Collectively, our computational study provides mechanistic insights of glucosome formation, particularly an initial stage through the formation of PFKL condensates in living human cells.
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Affiliation(s)
- Hye-Won Kang
- Department of Mathematics and Statistics, University of Maryland Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, MD, 21250, USA.
| | - Luan Nguyen
- Department of Mathematics and Statistics, University of Maryland Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, MD, 21250, USA
| | - Songon An
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, MD, 21250, USA
- Program in Oncology, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD, 21201, USA
| | - Minjoung Kyoung
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, MD, 21250, USA.
- Program in Oncology, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD, 21201, USA.
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11
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Shapiro IM, Risbud MV, Landis WJ. Toward understanding the cellular control of vertebrate mineralization: The potential role of mitochondria. Bone 2024; 185:117112. [PMID: 38697384 PMCID: PMC11251007 DOI: 10.1016/j.bone.2024.117112] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/20/2024] [Revised: 04/17/2024] [Accepted: 04/29/2024] [Indexed: 05/05/2024]
Abstract
This review examines the possible role of mitochondria in maintaining calcium and phosphate ion homeostasis and participating in the mineralization of bone, cartilage and other vertebrate hard tissues. The paper builds on the known structural features of mitochondria and the documented observations in these tissues that the organelles contain calcium phosphate granules. Such deposits in mitochondria putatively form to buffer excessively high cytosolic calcium ion concentrations and prevent metabolic deficits and even cell death. While mitochondria protect cytosolic enzyme systems through this buffering capacity, the accumulation of calcium ions by mitochondria promotes the activity of enzymes of the tricarboxylic acid (TCA/Krebs) cycle, increases oxidative phosphorylation and ATP synthesis, and leads to changes in intramitochondrial pH. These pH alterations influence ion solubility and possibly the transitions and composition in the mineral phase structure of the granules. Based on these considerations, mitochondria are proposed to support the mineralization process by providing a mobile store of calcium and phosphate ions, in smaller cluster or larger granule form, while maintaining critical cellular activities. The rise in the mitochondrial calcium level also increases the generation of citrate and other TCA cycle intermediates that contribute to cell function and the development of extracellular mineral. This paper suggests that another key role of the mitochondrion, along with the effects just noted, is to supply phosphate ions, derived from the breakdown of ATP, to endolysosomes and autophagic vesicles originating in the endoplasmic reticulum and Golgi and at the plasma membrane. These many separate but interdependent mitochondrial functions emphasize the critical importance of this organelle in the cellular control of vertebrate mineralization.
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Affiliation(s)
- Irving M Shapiro
- Department of Orthopaedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, United States of America.
| | - Makarand V Risbud
- Department of Orthopaedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, United States of America
| | - William J Landis
- Department of Preventive and Restorative Dental Sciences, School of Dentistry, University of California at San Francisco, San Francisco, CA, United States of America
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12
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Li C, Hao B, Yang H, Wang K, Fan L, Xiao W. Protein aggregation and biomolecular condensation in hypoxic environments (Review). Int J Mol Med 2024; 53:33. [PMID: 38362920 PMCID: PMC10903932 DOI: 10.3892/ijmm.2024.5357] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2023] [Accepted: 01/15/2024] [Indexed: 02/17/2024] Open
Abstract
Due to molecular forces, biomacromolecules assemble into liquid condensates or solid aggregates, and their corresponding formation and dissolution processes are controlled. Protein homeostasis is disrupted by increasing age or environmental stress, leading to irreversible protein aggregation. Hypoxic pressure is an important factor in this process, and uncontrolled protein aggregation has been widely observed in hypoxia‑related conditions such as neurodegenerative disease, cardiovascular disease, hypoxic brain injury and cancer. Biomolecular condensates are also high‑order complexes assembled from macromolecules. Although they exist in different phase from protein aggregates, they are in dynamic balance under certain conditions, and their activation or assembly are considered as important regulatory processes in cell survival with hypoxic pressure. Therefore, a better understanding of the relationship between hypoxic stress, protein aggregation and biomolecular condensation will bring marked benefits in the clinical treatment of various diseases. The aim of the present review was to summarize the underlying mechanisms of aggregate assembly and dissolution induced by hypoxic conditions, and address recent breakthroughs in understanding the role of aggregates in hypoxic‑related diseases, given the hypotheses that hypoxia induces macromolecular assemblage changes from a liquid to a solid phase, and that adenosine triphosphate depletion and ATP‑driven inactivation of multiple protein chaperones play important roles among the process. Moreover, it is anticipated that an improved understanding of the adaptation in hypoxic environments could extend the overall survival of patients and provide new strategies for hypoxic‑related diseases.
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Affiliation(s)
- Chaoqun Li
- School of Exercise and Health, Shanghai University of Sport, Shanghai 200438, P.R. China
- Institute of Energy Metabolism and Health, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China
| | - Bingjie Hao
- Institute of Energy Metabolism and Health, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China
| | - Haiguang Yang
- Institute of Energy Metabolism and Health, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China
| | - Kai Wang
- Institute of Energy Metabolism and Health, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China
| | - Lihong Fan
- School of Exercise and Health, Shanghai University of Sport, Shanghai 200438, P.R. China
- Institute of Energy Metabolism and Health, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China
| | - Weihua Xiao
- School of Exercise and Health, Shanghai University of Sport, Shanghai 200438, P.R. China
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13
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Lynch EM, Hansen H, Salay L, Cooper M, Timr S, Kollman JM, Webb BA. Structural basis for allosteric regulation of human phosphofructokinase-1. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.03.15.585110. [PMID: 38559074 PMCID: PMC10980016 DOI: 10.1101/2024.03.15.585110] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/04/2024]
Abstract
Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.
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Affiliation(s)
- Eric M Lynch
- Department of Biochemistry, University of Washington
| | - Heather Hansen
- Department of Biochemistry and Molecular Medicine, West Virginia University
| | - Lauren Salay
- Department of Biochemistry, University of Washington
| | - Madison Cooper
- Department of Biochemistry and Molecular Medicine, West Virginia University
| | - Stepan Timr
- Department of Computational Chemistry, J. Heyrovsky Institute of Physical Chemistry, Czech Academy of Sciences
| | | | - Bradley A Webb
- Department of Biochemistry and Molecular Medicine, West Virginia University
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14
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Ikari N, Honjo K, Sagami Y, Nakamura Y, Arakawa H. Mieap forms membrane-less organelles involved in cardiolipin metabolism. iScience 2024; 27:108916. [PMID: 38322995 PMCID: PMC10845071 DOI: 10.1016/j.isci.2024.108916] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2023] [Revised: 11/16/2023] [Accepted: 01/11/2024] [Indexed: 02/08/2024] Open
Abstract
Biomolecular condensates (BCs) are formed by proteins with intrinsically disordered regions (IDRs) via liquid-liquid phase separation. Mieap/Spata18, a p53-inducible protein, participates in suppression of colorectal tumors by promoting mitochondrial quality control. However, the regulatory mechanism involved remains unclear. Here, we report that Mieap is an IDR-containing protein that drives formation of BCs involved in cardiolipin metabolism. Mieap BCs specifically phase separate the mitochondrial phospholipid, cardiolipin. Mieap directly binds to cardiolipin in vitro. Lipidomic analysis of cardiolipin suggests that Mieap promotes enzymatic reactions in cardiolipin biosynthesis and remodeling. Accordingly, four cardiolipin biosynthetic enzymes, TAMM41, PGS1, PTPMT1, and CRLS1 and two remodeling enzymes, PLA2G6 and TAZ, are phase-separated by Mieap BCs. Mieap-deficient cells exhibit altered crista structure, leading to decreased respiration activity and ATP production in mitochondria. These results suggest that Mieap may form membrane-less organelles to compartmentalize and facilitate cardiolipin metabolism, thus potentially contributing to mitochondrial quality control.
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Affiliation(s)
- Naoki Ikari
- Division of Cancer Biology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
| | - Katsuko Honjo
- Division of Cancer Biology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
| | - Yoko Sagami
- Division of Cancer Biology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
| | - Yasuyuki Nakamura
- Division of Cancer Biology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
| | - Hirofumi Arakawa
- Division of Cancer Biology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
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15
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Campos M, Albrecht LV. Hitting the Sweet Spot: How Glucose Metabolism Is Orchestrated in Space and Time by Phosphofructokinase-1. Cancers (Basel) 2023; 16:16. [PMID: 38201444 PMCID: PMC10778546 DOI: 10.3390/cancers16010016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2023] [Revised: 12/12/2023] [Accepted: 12/15/2023] [Indexed: 01/12/2024] Open
Abstract
Glycolysis is the central metabolic pathway across all kingdoms of life. Intensive research efforts have been devoted to understanding the tightly orchestrated processes of converting glucose into energy in health and disease. Our review highlights the advances in knowledge of how metabolic and gene networks are integrated through the precise spatiotemporal compartmentalization of rate-limiting enzymes. We provide an overview of technically innovative approaches that have been applied to study phosphofructokinase-1 (PFK1), which represents the fate-determining step of oxidative glucose metabolism. Specifically, we discuss fast-acting chemical biology and optogenetic tools that have delineated new links between metabolite fluxes and transcriptional reprogramming, which operate together to enact tissue-specific processes. Finally, we discuss how recent paradigm-shifting insights into the fundamental basis of glycolytic regulatory control have shed light on the mechanisms of tumorigenesis and could provide insight into new therapeutic vulnerabilities in cancer.
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Affiliation(s)
- Melissa Campos
- Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, CA 92697, USA;
| | - Lauren V. Albrecht
- Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, CA 92697, USA;
- Department of Pharmaceutical Sciences, School of Pharmacy & Pharmaceutical Sciences, University of California, Irvine, CA 92697, USA
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16
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Sivadas A, McDonald EF, Shuster SO, Davis CM, Plate L. Site-specific crosslinking reveals Phosphofructokinase-L inhibition drives self-assembly and attenuation of protein interactions. Adv Biol Regul 2023; 90:100987. [PMID: 37806136 PMCID: PMC11108229 DOI: 10.1016/j.jbior.2023.100987] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2023] [Accepted: 09/25/2023] [Indexed: 10/10/2023]
Abstract
Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and inhibition in vitro and in vivo, respectively. Compartmentalized PFKL is hypothesized to modulate metabolic flux consistent with its central role as the rate limiting step in glycolysis. PFKL tetramers self-assemble at two interfaces in the monomer (interface 1 and 2), yet how these interfaces contribute to PFKL compartmentalization and drive protein interactions remains unclear. Here, we used site-specific incorporation of noncanonical photocrosslinking amino acids to identify PFKL interactors at interface 1, 2, and the active site. Tandem mass tag-based quantitative interactomics reveals interface 2 as a hotspot for PFKL interactions, particularly with cytoskeletal, glycolytic, and carbohydrate derivative metabolic proteins. Furthermore, PFKL compartmentalization into puncta was observed in human cells using citrate inhibition. Puncta formation attenuated crosslinked protein-protein interactions with the cytoskeleton at interface 2. This result suggests that PFKL compartmentalization sequesters interface 2, but not interface 1, and may modulate associated protein assemblies with the cytoskeleton.
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Affiliation(s)
- Athira Sivadas
- Department of Chemistry, Vanderbilt University, Nashville, TN, USA
| | - Eli Fritz McDonald
- Department of Chemistry, Vanderbilt University, Nashville, TN, USA; Center for Structural Biology, Vanderbilt University, Nashville, TN, USA
| | | | - Caitlin M Davis
- Department of Chemistry, Yale University, New Haven, CT, USA
| | - Lars Plate
- Department of Chemistry, Vanderbilt University, Nashville, TN, USA; Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA; Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA.
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17
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Jeon M, Schmitt DL, Kyoung M, An S. Size-Specific Modulation of a Multienzyme Glucosome Assembly during the Cell Cycle. ACS BIO & MED CHEM AU 2023; 3:461-470. [PMID: 37876499 PMCID: PMC10591302 DOI: 10.1021/acsbiomedchemau.3c00037] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/09/2023] [Revised: 07/26/2023] [Accepted: 07/26/2023] [Indexed: 10/26/2023]
Abstract
Enzymes in glucose metabolism have been subjected to numerous studies, revealing the importance of their biological roles during the cell cycle. However, due to the lack of viable experimental strategies for measuring enzymatic activities particularly in living human cells, it has been challenging to address whether their enzymatic activities and thus anticipated glucose flux are directly associated with cell cycle progression. It has remained largely elusive how human cells regulate glucose metabolism at a subcellular level to meet the metabolic demands during the cell cycle. Meanwhile, we have characterized that rate-determining enzymes in glucose metabolism are spatially organized into three different sizes of multienzyme metabolic assemblies, termed glucosomes, to regulate the glucose flux between energy metabolism and building block biosynthesis. In this work, we first determined using cell synchronization and flow cytometric techniques that enhanced green fluorescent protein-tagged phosphofructokinase is adequate as an intracellular biomarker to evaluate the state of glucose metabolism during the cell cycle. We then applied fluorescence single-cell imaging strategies and discovered that the percentage of Hs578T cells showing small-sized glucosomes is drastically changed during the cell cycle, whereas the percentage of cells with medium-sized glucosomes is significantly elevated only in the G1 phase, but the percentage of cells showing large-sized glucosomes is barely or minimally altered along the cell cycle. Should we consider our previous localization-function studies that showed assembly size-dependent metabolic roles of glucosomes, this work strongly suggests that glucosome sizes are modulated during the cell cycle to regulate glucose flux between glycolysis and building block biosynthesis. Therefore, we propose the size-specific modulation of glucosomes as a behind-the-scenes mechanism that may explain functional association of glucose metabolism with the cell cycle and, thereby, their metabolic significance in human cell biology.
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Affiliation(s)
- Miji Jeon
- Department
of Chemistry and Biochemistry, University
of Maryland Baltimore County (UMBC); 1000 Hilltop Circle, Baltimore, Maryland 21250, United States
| | - Danielle L. Schmitt
- Department
of Chemistry and Biochemistry, University
of Maryland Baltimore County (UMBC); 1000 Hilltop Circle, Baltimore, Maryland 21250, United States
| | - Minjoung Kyoung
- Department
of Chemistry and Biochemistry, University
of Maryland Baltimore County (UMBC); 1000 Hilltop Circle, Baltimore, Maryland 21250, United States
- Program
in Oncology, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, Maryland 21201, United States
| | - Songon An
- Department
of Chemistry and Biochemistry, University
of Maryland Baltimore County (UMBC); 1000 Hilltop Circle, Baltimore, Maryland 21250, United States
- Program
in Oncology, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, Maryland 21201, United States
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18
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Sivadas A, McDonald EF, Shuster SO, Davis CM, Plate L. Site-Specific Crosslinking Reveals Phosphofructokinase-L Inhibition Drives Self-Assembly and Attenuation of Protein Interactions. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.09.19.558525. [PMID: 37781627 PMCID: PMC10541129 DOI: 10.1101/2023.09.19.558525] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/03/2023]
Abstract
Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and inhibition in vitro and in vivo respectively. Compartmentalized PFKL is hypothesized to modulate metabolic flux consistent with its central role as the rate limiting step in glycolysis. PFKL tetramers self-assemble at two interfaces in the monomer (interface 1 and 2), yet how these interfaces contribute to PFKL compartmentalization and drive protein interactions remains unclear. Here, we used site-specific incorporation of noncanonical photocrosslinking amino acids to identify PFKL interactors at interface 1, 2, and the active site. Tandem mass tag-based quantitative interactomics reveals interface 2 as a hotspot for PFKL interactions, particularly with cytoskeletal, glycolytic, and carbohydrate derivative metabolic proteins. Furthermore, PFKL compartmentalization into puncta was observed in human cells using citrate inhibition. Puncta formation attenuated crosslinked protein-protein interactions with the cytoskeleton at interface 2. This result suggests that PFKL compartmentalization sequesters interface 2, but not interface 1, and may modulate associated protein assemblies with the cytoskeleton.
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Affiliation(s)
- Athira Sivadas
- Department of Chemistry, Vanderbilt University, Nashville, TN, USA
| | - Eli Fritz McDonald
- Department of Chemistry, Vanderbilt University, Nashville, TN, USA
- Center for Structural Biology, Vanderbilt University, Nashville, TN, USA
| | | | | | - Lars Plate
- Department of Chemistry, Vanderbilt University, Nashville, TN, USA
- Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA
- Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA
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19
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Wang H, Vant J, Wu Y, Sanchez R, Micou ML, Zhang A, Luczak V, Yu SB, Jabbo M, Yoon S, Abushawish AA, Ghassemian M, Griffis E, Hammarlund M, Singharoy A, Pekkurnaz G. Functional Organization of Glycolytic Metabolon on Mitochondria. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.26.554955. [PMID: 37662343 PMCID: PMC10473731 DOI: 10.1101/2023.08.26.554955] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/05/2023]
Abstract
Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme Hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis, and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes reversible O-GlcNAcylation, a post-translational modification, influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of HK1's regulatory domain, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances HK1's mitochondrial association, orchestrating glycolytic and mitochondrial ATP production. Mutations in HK1's O-GlcNAcylation site reduce ATP generation, affecting synaptic functions in neurons. The study uncovers a novel pathway that bridges neuronal metabolism and mitochondrial function via OGT and the formation of the glycolytic metabolon, offering new prospects for tackling metabolic and neurological disorders.
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20
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Flood D, Lee ES, Taylor CT. Intracellular energy production and distribution in hypoxia. J Biol Chem 2023; 299:105103. [PMID: 37507013 PMCID: PMC10480318 DOI: 10.1016/j.jbc.2023.105103] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2023] [Revised: 07/17/2023] [Accepted: 07/18/2023] [Indexed: 07/30/2023] Open
Abstract
The hydrolysis of ATP is the primary source of metabolic energy for eukaryotic cells. Under physiological conditions, cells generally produce more than sufficient levels of ATP to fuel the active biological processes necessary to maintain homeostasis. However, mechanisms underpinning the distribution of ATP to subcellular microenvironments with high local demand remain poorly understood. Intracellular distribution of ATP in normal physiological conditions has been proposed to rely on passive diffusion across concentration gradients generated by ATP producing systems such as the mitochondria and the glycolytic pathway. However, subcellular microenvironments can develop with ATP deficiency due to increases in local ATP consumption. Alternatively, ATP production can be reduced during bioenergetic stress during hypoxia. Mammalian cells therefore need to have the capacity to alter their metabolism and energy distribution strategies to compensate for local ATP deficits while also controlling ATP production. It is highly likely that satisfying the bioenergetic requirements of the cell involves the regulated distribution of ATP producing systems to areas of high ATP demand within the cell. Recently, the distribution (both spatially and temporally) of ATP-producing systems has become an area of intense investigation. Here, we review what is known (and unknown) about intracellular energy production and distribution and explore potential mechanisms through which this targeted distribution can be altered in hypoxia, with the aim of stimulating investigation in this important, yet poorly understood field of research.
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Affiliation(s)
- Darragh Flood
- Conway Institute of Biomolecular and Biomedical Research and School of Medicine, University College Dublin, Dublin, Ireland
| | - Eun Sang Lee
- Conway Institute of Biomolecular and Biomedical Research and School of Medicine, University College Dublin, Dublin, Ireland
| | - Cormac T Taylor
- Conway Institute of Biomolecular and Biomedical Research and School of Medicine, University College Dublin, Dublin, Ireland.
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21
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Solis-Miranda J, Chodasiewicz M, Skirycz A, Fernie AR, Moschou PN, Bozhkov PV, Gutierrez-Beltran E. Stress-related biomolecular condensates in plants. THE PLANT CELL 2023; 35:3187-3204. [PMID: 37162152 PMCID: PMC10473214 DOI: 10.1093/plcell/koad127] [Citation(s) in RCA: 32] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/08/2022] [Revised: 04/07/2023] [Accepted: 04/27/2023] [Indexed: 05/11/2023]
Abstract
Biomolecular condensates are membraneless organelle-like structures that can concentrate molecules and often form through liquid-liquid phase separation. Biomolecular condensate assembly is tightly regulated by developmental and environmental cues. Although research on biomolecular condensates has intensified in the past 10 years, our current understanding of the molecular mechanisms and components underlying their formation remains in its infancy, especially in plants. However, recent studies have shown that the formation of biomolecular condensates may be central to plant acclimation to stress conditions. Here, we describe the mechanism, regulation, and properties of stress-related condensates in plants, focusing on stress granules and processing bodies, 2 of the most well-characterized biomolecular condensates. In this regard, we showcase the proteomes of stress granules and processing bodies in an attempt to suggest methods for elucidating the composition and function of biomolecular condensates. Finally, we discuss how biomolecular condensates modulate stress responses and how they might be used as targets for biotechnological efforts to improve stress tolerance.
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Affiliation(s)
- Jorge Solis-Miranda
- Institutode Bioquimica Vegetal y Fotosintesis, Consejo Superior de Investigaciones Cientificas (CSIC)-Universidad de Sevilla, 41092 Sevilla, Spain
| | - Monika Chodasiewicz
- Biological and Environmental Science and Engineering Division, Center for Desert Agriculture, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia
| | | | - Alisdair R Fernie
- Max-Planck-Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany
| | - Panagiotis N Moschou
- Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, 75007 Uppsala, Sweden
- Department of Biology, University of Crete, Heraklion 71409, Greece
- Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion 70013, Greece
| | - Peter V Bozhkov
- Department of Molecular Sciences, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, 75007 Uppsala, Sweden
| | - Emilio Gutierrez-Beltran
- Institutode Bioquimica Vegetal y Fotosintesis, Consejo Superior de Investigaciones Cientificas (CSIC)-Universidad de Sevilla, 41092 Sevilla, Spain
- Departamento de Bioquimica Vegetal y Biologia Molecular, Facultad de Biologia, Universidad de Sevilla, 41012 Sevilla, Spain
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22
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Kierans SJ, Fagundes RR, Malkov MI, Sparkes R, Dillon ET, Smolenski A, Faber KN, Taylor CT. Hypoxia induces a glycolytic complex in intestinal epithelial cells independent of HIF-1-driven glycolytic gene expression. Proc Natl Acad Sci U S A 2023; 120:e2208117120. [PMID: 37603756 PMCID: PMC10469334 DOI: 10.1073/pnas.2208117120] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Accepted: 07/11/2023] [Indexed: 08/23/2023] Open
Abstract
The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic adenosine triphosphate (ATP) production, an event with consequences for cellular bioenergetics and cell fate. This response is regulated at the transcriptional level by the hypoxia-inducible factor-1(HIF-1)-dependent transcriptional upregulation of glycolytic enzymes (GEs) and glucose transporters. However, this transcriptional upregulation alone is unlikely to account fully for the levels of glycolytic ATP produced during hypoxia. Here, we investigated additional mechanisms regulating glycolysis in hypoxia. We observed that intestinal epithelial cells treated with inhibitors of transcription or translation and human platelets (which lack nuclei and the capacity for canonical transcriptional activity) maintained the capacity for hypoxia-induced glycolysis, a finding which suggests the involvement of a nontranscriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. In our investigations into potential nontranscriptional mechanisms for glycolytic induction, we identified a hypoxia-sensitive formation of complexes comprising GEs and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of such glycolytic complexes occurs independent of HIF-1-driven transcription. Finally, we provide evidence for the presence of HIF-1α in cytosolic fractions of hypoxic cells which physically interacts with the glucose transporter GLUT1 and the GEs in a hypoxia-sensitive manner. In conclusion, we provide insights into the nontranscriptional regulation of hypoxia-induced glycolysis in intestinal epithelial cells.
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Affiliation(s)
- Sarah J. Kierans
- University College Dublin School of Medicine, University College Dublin, DublinD4, Ireland
- Conway Institute of Biomolecular and Biomedical Research, University College Dublin, DublinD4, Ireland
| | - Raphael R. Fagundes
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, GroningenD4, The Netherlands
| | - Mykyta I. Malkov
- University College Dublin School of Medicine, University College Dublin, DublinD4, Ireland
- Conway Institute of Biomolecular and Biomedical Research, University College Dublin, DublinD4, Ireland
| | - Ríona Sparkes
- University College Dublin School of Medicine, University College Dublin, DublinD4, Ireland
- Conway Institute of Biomolecular and Biomedical Research, University College Dublin, DublinD4, Ireland
| | - Eugène T. Dillon
- Conway Institute of Biomolecular and Biomedical Research, University College Dublin, DublinD4, Ireland
| | - Albert Smolenski
- University College Dublin School of Medicine, University College Dublin, DublinD4, Ireland
- Conway Institute of Biomolecular and Biomedical Research, University College Dublin, DublinD4, Ireland
| | - Klaas Nico Faber
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, GroningenD4, The Netherlands
| | - Cormac T. Taylor
- University College Dublin School of Medicine, University College Dublin, DublinD4, Ireland
- Conway Institute of Biomolecular and Biomedical Research, University College Dublin, DublinD4, Ireland
- Systems Biology Ireland, University College Dublin, DublinD4, Ireland
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23
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Schmitt DL, Dranchak P, Parajuli P, Blivis D, Voss T, Kohnhorst CL, Kyoung M, Inglese J, An S. High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells. PLoS One 2023; 18:e0289707. [PMID: 37540718 PMCID: PMC10403072 DOI: 10.1371/journal.pone.0289707] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2023] [Accepted: 07/25/2023] [Indexed: 08/06/2023] Open
Abstract
We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism and thus contribute to human cell biology. We developed a high-content quantitative high-throughput screening (qHTS) assay to identify regulatory mechanisms that control PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs further confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling network that regulates the formation of PFK1-mediated glucosome assembly in human cells.
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Affiliation(s)
- Danielle L. Schmitt
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, United States of America
| | - Patricia Dranchak
- National Institutes of Health, National Center for Advancing Translational Sciences, Rockville, Maryland, United States of America
| | - Prakash Parajuli
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, United States of America
| | - Dvir Blivis
- National Institutes of Health, National Center for Advancing Translational Sciences, Rockville, Maryland, United States of America
| | - Ty Voss
- National Institutes of Health, National Center for Advancing Translational Sciences, Rockville, Maryland, United States of America
| | - Casey L. Kohnhorst
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, United States of America
| | - Minjoung Kyoung
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, United States of America
- Program in Oncology, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, Maryland, United States of America
| | - James Inglese
- National Institutes of Health, National Center for Advancing Translational Sciences, Rockville, Maryland, United States of America
- National Institutes of Health, National Human Genome Research Institute, Bethesda, Maryland, United States of America
| | - Songon An
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, United States of America
- Program in Oncology, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, Maryland, United States of America
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Binder MJ, Pedley AM. The roles of molecular chaperones in regulating cell metabolism. FEBS Lett 2023; 597:1681-1701. [PMID: 37287189 PMCID: PMC10984649 DOI: 10.1002/1873-3468.14682] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2023] [Revised: 05/22/2023] [Accepted: 05/29/2023] [Indexed: 06/09/2023]
Abstract
Fluctuations in nutrient and biomass availability, often as a result of disease, impart metabolic challenges that must be overcome in order to sustain cell survival and promote proliferation. Cells adapt to these environmental changes and stresses by adjusting their metabolic networks through a series of regulatory mechanisms. Our understanding of these rewiring events has largely been focused on those genetic transformations that alter protein expression and the biochemical mechanisms that change protein behavior, such as post-translational modifications and metabolite-based allosteric modulators. Mounting evidence suggests that a class of proteome surveillance proteins called molecular chaperones also can influence metabolic processes. Here, we summarize several ways the Hsp90 and Hsp70 chaperone families act on human metabolic enzymes and their supramolecular assemblies to change enzymatic activities and metabolite flux. We further highlight how these chaperones can assist in the translocation and degradation of metabolic enzymes. Collectively, these studies provide a new view for how metabolic processes are regulated to meet cellular demand and inspire new avenues for therapeutic intervention.
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Jin S, Campbell EJ, Ip CK, Layfield S, Bathgate RAD, Herzog H, Lawrence AJ. Molecular Profiling of VGluT1 AND VGluT2 Ventral Subiculum to Nucleus Accumbens Shell Projections. Neurochem Res 2023:10.1007/s11064-023-03921-z. [PMID: 37017888 DOI: 10.1007/s11064-023-03921-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2022] [Revised: 03/14/2023] [Accepted: 03/24/2023] [Indexed: 04/06/2023]
Abstract
The nucleus accumbens shell is a critical node in reward circuitry, encoding environments associated with reward. Long-range inputs from the ventral hippocampus (ventral subiculum) to the nucleus accumbens shell have been identified, yet their precise molecular phenotype remains to be determined. Here we used retrograde tracing to identify the ventral subiculum as the brain region with the densest glutamatergic (VGluT1-Slc17a7) input to the shell. We then used circuit-directed translating ribosome affinity purification to examine the molecular characteristics of distinct glutamatergic (VGluT1, VGluT2-Slc17a6) ventral subiculum to nucleus accumbens shell projections. We immunoprecipitated translating ribosomes from this population of projection neurons and analysed molecular connectomic information using RNA sequencing. We found differential gene enrichment across both glutamatergic projection neuron subtypes. In VGluT1 projections, we found enrichment of Pfkl, a gene involved in glucose metabolism. In VGluT2 projections, we found a depletion of Sparcl1 and Dlg1, genes known to play a role in depression- and addiction-related behaviours. These findings highlight potential glutamatergic neuronal-projection-specific differences in ventral subiculum to nucleus accumbens shell projections. Together these data advance our understanding of the phenotype of a defined brain circuit.
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Affiliation(s)
- Shubo Jin
- The Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, The University of Melbourne, Parkville, Melbourne, VIC, 3052, Australia
- Florey Department of Neuroscience and Mental Health, The University of Melbourne, Parkville, Melbourne, VIC, 3010, Australia
| | - Erin J Campbell
- The Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, The University of Melbourne, Parkville, Melbourne, VIC, 3052, Australia.
- Florey Department of Neuroscience and Mental Health, The University of Melbourne, Parkville, Melbourne, VIC, 3010, Australia.
| | - Chi Kin Ip
- Neuroscience Division, Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW, 2010, Australia
- Faculty of Medicine, University of New South Wales, Sydney, NSW, 2052, Australia
| | - Sharon Layfield
- The Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, The University of Melbourne, Parkville, Melbourne, VIC, 3052, Australia
- Florey Department of Neuroscience and Mental Health, The University of Melbourne, Parkville, Melbourne, VIC, 3010, Australia
| | - Ross A D Bathgate
- The Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, The University of Melbourne, Parkville, Melbourne, VIC, 3052, Australia
- Florey Department of Neuroscience and Mental Health, The University of Melbourne, Parkville, Melbourne, VIC, 3010, Australia
- Department of Biochemistry and Pharmacology, The University of Melbourne, Parkville, Melbourne, VIC, 3010, Australia
| | - Herbert Herzog
- Neuroscience Division, Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW, 2010, Australia
- Faculty of Medicine, University of New South Wales, Sydney, NSW, 2052, Australia
| | - Andrew J Lawrence
- The Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, The University of Melbourne, Parkville, Melbourne, VIC, 3052, Australia.
- Florey Department of Neuroscience and Mental Health, The University of Melbourne, Parkville, Melbourne, VIC, 3010, Australia.
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26
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Zhang Y, Li W, Bian Y, Li Y, Cong L. Multifaceted roles of aerobic glycolysis and oxidative phosphorylation in hepatocellular carcinoma. PeerJ 2023; 11:e14797. [PMID: 36748090 PMCID: PMC9899054 DOI: 10.7717/peerj.14797] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Accepted: 01/04/2023] [Indexed: 02/04/2023] Open
Abstract
Liver cancer is a common malignancy with high morbidity and mortality rates. Changes in liver metabolism are key factors in the development of primary hepatic carcinoma, and mitochondrial dysfunction is closely related to the occurrence and development of tumours. Accordingly, the study of the metabolic mechanism of mitochondria in primary hepatic carcinomas has gained increasing attention. A growing body of research suggests that defects in mitochondrial respiration are not generally responsible for aerobic glycolysis, nor are they typically selected during tumour evolution. Conversely, the dysfunction of mitochondrial oxidative phosphorylation (OXPHOS) may promote the proliferation, metastasis, and invasion of primary hepatic carcinoma. This review presents the current paradigm of the roles of aerobic glycolysis and OXPHOS in the occurrence and development of hepatocellular carcinoma (HCC). Mitochondrial OXPHOS and cytoplasmic glycolysis cooperate to maintain the energy balance in HCC cells. Our study provides evidence for the targeting of mitochondrial metabolism as a potential therapy for HCC.
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Affiliation(s)
- Ying Zhang
- Department of Oncology, Shandong Provincial Hospital affiliated to Shandong First Medical University, Jinan, China
| | - Wenhuan Li
- Department of Oncology, Shandong Provincial Hospital affiliated to Shandong First Medical University, Jinan, China
| | - Yuan Bian
- Department of Emergency Medicine, Qilu Hospital of Shandong University, Jinan, China
| | - Yan Li
- Department of Oncology, Shandong Provincial Hospital affiliated to Shandong First Medical University, Jinan, China
| | - Lei Cong
- Department of Oncology, Shandong Provincial Hospital affiliated to Shandong First Medical University, Jinan, China,Department of Oncology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China
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27
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Utsumi R, Murata Y, Ito-Harashima S, Akai M, Miura N, Kuroda K, Ueda M, Kataoka M. Foci-forming regions of pyruvate kinase and enolase at the molecular surface incorporate proteins into yeast cytoplasmic metabolic enzymes transiently assembling (META) bodies. PLoS One 2023; 18:e0283002. [PMID: 37053166 PMCID: PMC10101385 DOI: 10.1371/journal.pone.0283002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2022] [Accepted: 02/28/2023] [Indexed: 04/14/2023] Open
Abstract
Spatial reorganization of metabolic enzymes to form the "metabolic enzymes transiently assembling (META) body" is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27-42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed "META body-forming sequences (METAfos)." METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation.
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Affiliation(s)
- Ryotaro Utsumi
- Department of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan
| | - Yuki Murata
- Department of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan
| | - Sayoko Ito-Harashima
- Department of Applied Biological Chemistry, Graduate School of Agriculture, Osaka Metropolitan University, Sakai, Japan
| | - Misaki Akai
- School of Applied Life Sciences, College of Life, Environment, and Advanced Sciences, Osaka Prefecture University, Sakai, Japan
| | - Natsuko Miura
- Department of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan
- Department of Applied Biological Chemistry, Graduate School of Agriculture, Osaka Metropolitan University, Sakai, Japan
- School of Applied Life Sciences, College of Life, Environment, and Advanced Sciences, Osaka Prefecture University, Sakai, Japan
- Research Institute for LAC-SYS (RILACS), Osaka Metropolitan University, Sakai, Japan
| | - Kouichi Kuroda
- Department of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Mitsuyoshi Ueda
- Department of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Michihiko Kataoka
- Department of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan
- Department of Applied Biological Chemistry, Graduate School of Agriculture, Osaka Metropolitan University, Sakai, Japan
- School of Applied Life Sciences, College of Life, Environment, and Advanced Sciences, Osaka Prefecture University, Sakai, Japan
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Human cytomegalovirus induces neuronal enolase to support virally mediated metabolic remodeling. Proc Natl Acad Sci U S A 2022; 119:e2205789119. [PMID: 36459650 PMCID: PMC9894225 DOI: 10.1073/pnas.2205789119] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/05/2022] Open
Abstract
Viruses depend on cellular metabolic resources to supply the energy and biomolecular building blocks necessary for their replication. Human cytomegalovirus (HCMV), a leading cause of birth defects and morbidity in immunosuppressed individuals, induces numerous metabolic activities that are important for productive infection. However, many of the mechanisms through which these metabolic activities are induced and how they contribute to infection are unclear. We find that HCMV infection of fibroblasts induces a neuronal gene signature as well as the expression of several metabolic enzyme isoforms that are typically expressed in other tissue types. Of these, the most substantially induced glycolytic gene was the neuron-specific isoform of enolase 2 (ENO2). Induction of ENO2 expression is important for HCMV-mediated glycolytic activation as well as for the virally induced remodeling of pyrimidine-sugar metabolism, which provides the glycosyl subunits necessary for protein glycosylation. Inhibition of ENO2 expression or activity reduced uridine diphosphate (UDP)-sugar pools, attenuated the accumulation of viral glycoproteins, and induced the accumulation of noninfectious viral particles. In addition, our data indicate that the induction of ENO2 expression depends on the HCMV UL38 protein. Collectively, our data indicate that HCMV infection induces a tissue atypical neuronal glycolytic enzyme to activate glycolysis and UDP-sugar metabolism, increase the accumulation of glycosyl building blocks, and enable the expression of an essential viral glycoprotein and the production of infectious virions.
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Lin JMG, Kourtis S, Ghose R, Pardo Lorente N, Kubicek S, Sdelci S. Metabolic modulation of transcription: The role of one-carbon metabolism. Cell Chem Biol 2022; 29:S2451-9456(22)00415-9. [PMID: 36513079 DOI: 10.1016/j.chembiol.2022.11.009] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2022] [Revised: 10/05/2022] [Accepted: 11/18/2022] [Indexed: 12/15/2022]
Abstract
While it is well known that expression levels of metabolic enzymes regulate the metabolic state of the cell, there is mounting evidence that the converse is also true, that metabolite levels themselves can modulate gene expression via epigenetic modifications and transcriptional regulation. Here we focus on the one-carbon metabolic pathway, which provides the essential building blocks of many classes of biomolecules, including purine nucleotides, thymidylate, serine, and methionine. We review the epigenetic roles of one-carbon metabolic enzymes and their associated metabolites and introduce an interactive computational resource that places enzyme essentiality in the context of metabolic pathway topology. Therefore, we briefly discuss examples of metabolic condensates and higher-order complexes of metabolic enzymes downstream of one-carbon metabolism. We speculate that they may be required to the formation of transcriptional condensates and gene expression control. Finally, we discuss new ways to exploit metabolic pathway compartmentalization to selectively target these enzymes in cancer.
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Affiliation(s)
- Jung-Ming G Lin
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Vienna 1090, Austria
| | - Savvas Kourtis
- Centre for Genomic Regulation (CRG), the Barcelona Institute of Science and Technology, Barcelona, Catalonia 08003, Spain
| | - Ritobrata Ghose
- Centre for Genomic Regulation (CRG), the Barcelona Institute of Science and Technology, Barcelona, Catalonia 08003, Spain
| | - Natalia Pardo Lorente
- Centre for Genomic Regulation (CRG), the Barcelona Institute of Science and Technology, Barcelona, Catalonia 08003, Spain
| | - Stefan Kubicek
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Vienna 1090, Austria
| | - Sara Sdelci
- Centre for Genomic Regulation (CRG), the Barcelona Institute of Science and Technology, Barcelona, Catalonia 08003, Spain.
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30
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Orofiamma LA, Vural D, Antonescu CN. Control of cell metabolism by the epidermal growth factor receptor. BIOCHIMICA ET BIOPHYSICA ACTA. MOLECULAR CELL RESEARCH 2022; 1869:119359. [PMID: 36089077 DOI: 10.1016/j.bbamcr.2022.119359] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/01/2022] [Revised: 08/24/2022] [Accepted: 09/01/2022] [Indexed: 06/15/2023]
Abstract
The epidermal growth factor receptor (EGFR) triggers the activation of many intracellular signals that control cell proliferation, growth, survival, migration, and differentiation. Given its wide expression, EGFR has many functions in development and tissue homeostasis. Some of the cellular outcomes of EGFR signaling involve alterations of specific aspects of cellular metabolism, and alterations of cell metabolism are emerging as driving influences in many physiological and pathophysiological contexts. Here we review the mechanisms by which EGFR regulates cell metabolism, including by modulation of gene expression and protein function leading to control of glucose uptake, glycolysis, biosynthetic pathways branching from glucose metabolism, amino acid metabolism, lipogenesis, and mitochondrial function. We further examine how this regulation of cell metabolism by EGFR may contribute to cell proliferation and differentiation and how EGFR-driven control of metabolism can impact certain diseases and therapy outcomes.
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Affiliation(s)
- Laura A Orofiamma
- Department of Chemistry and Biology, Toronto Metropolitan University, Toronto, Ontario M5B 2K3, Canada; Graduate Program in Molecular Science, Toronto Metropolitan University, Toronto, Ontario M5B 2K3, Canada
| | - Dafne Vural
- Department of Chemistry and Biology, Toronto Metropolitan University, Toronto, Ontario M5B 2K3, Canada; Graduate Program in Molecular Science, Toronto Metropolitan University, Toronto, Ontario M5B 2K3, Canada
| | - Costin N Antonescu
- Department of Chemistry and Biology, Toronto Metropolitan University, Toronto, Ontario M5B 2K3, Canada; Graduate Program in Molecular Science, Toronto Metropolitan University, Toronto, Ontario M5B 2K3, Canada.
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31
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Sun L, Liu XP, Yan X, Wu S, Tang X, Chen C, Li G, Hu H, Wang D, Li S. Identification of molecular subtypes based on liquid-liquid phase separation and cross-talk with immunological phenotype in bladder cancer. Front Immunol 2022; 13:1059568. [PMID: 36518754 PMCID: PMC9742536 DOI: 10.3389/fimmu.2022.1059568] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2022] [Accepted: 11/14/2022] [Indexed: 11/30/2022] Open
Abstract
Background Mounting evidence has demonstrated that an imbalance in liquid-liquid phase separation (LLPS) can induce alteration in the spatiotemporal coordination of biomolecular condensates, which plays a role in carcinogenesis and cachexia. However, the role of LLPS in the occurrence and progression of bladder cancer (BLCA) remains to be elucidated. Identifying the role of LLPS in carcinogenesis may aid in cancer therapeutics. Methods A total of 1,351 BLCA samples from six cohorts were retrieved from publicly available databases like The Cancer Genome Atlas, Gene Expression Omnibus, and ArrayExpress. The samples were divided into three distinct clusters, and their multi-dimensional heterogeneities were explored. The LLPS patterns of all patients were determined based on the LLPS-related risk score (LLPSRS), and its multifaceted landscape was depicted and experimentally validated at the multi-omics level. Finally, a cytotoxicity-related and LLPSRS-based classifier was established to predict the patient's response to immune checkpoint blockade (ICB) treatment. Results Three LLPS-related subtypes were identified and validated. The differences in prognosis, tumor microenvironment (TME) features, cancer hallmarks, and certain signatures of the three LLPS-related subtypes were validated. LLPSRS was calculated, which could be used as a prognostic biomarker. A close correlation was observed between clinicopathological features, genomic variations, biological mechanisms, immune infiltration in TME, chemosensitivity, and LLPSRS. Furthermore, our classifier could effectively predict immunotherapy response in patients with BLCA. Conclusions Our study identified a novel categorization of BLCA patients based on LLPS. The LLPSRS could predict the prognosis of patients and aid in designing personalized medicine. Further, our binary classifier could effectively predict patients' sensitivity to immunotherapy.
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Affiliation(s)
- Le Sun
- Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Xiao-Ping Liu
- Department of Biological Repositories, Cancer Precision Diagnosis and Treatment and Translational Medicine Hubei Engineering Research Center, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Xin Yan
- Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Shaojie Wu
- Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Xiaoyu Tang
- Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Chen Chen
- Department of Biological Repositories, Cancer Precision Diagnosis and Treatment and Translational Medicine Hubei Engineering Research Center, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Gang Li
- Department of Biological Repositories, Cancer Precision Diagnosis and Treatment and Translational Medicine Hubei Engineering Research Center, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Hankun Hu
- Department of Pharmacy, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Du Wang
- The Institute of Technological Sciences, Wuhan University, Wuhan, China
| | - Sheng Li
- Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China,Department of Biological Repositories, Cancer Precision Diagnosis and Treatment and Translational Medicine Hubei Engineering Research Center, Zhongnan Hospital of Wuhan University, Wuhan, China,*Correspondence: Sheng Li,
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Shuib S, Nazir MYM, Ibrahim I, Song Y, Ratledge C, Hamid AA. Co-existence of type I fatty acid synthase and polyketide synthase metabolons in Aurantiochytrium SW1 and their implications for lipid biosynthesis. Biochim Biophys Acta Mol Cell Biol Lipids 2022; 1867:159224. [PMID: 36007759 DOI: 10.1016/j.bbalip.2022.159224] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2022] [Revised: 08/16/2022] [Accepted: 08/16/2022] [Indexed: 11/26/2022]
Abstract
The key enzymes of lipid biosynthesis in oleaginous filamentous fungi exist as metabolons. However, the existence of a similar organization in other groups of oleaginous microorganisms is still unknown. In this study, we confirmed the occurrence of two separate and distinct lipogenic metabolons in a thraustochytrid, Aurantiochytrium SW1. These involve the Type I Fatty Acid Synthase (FAS) pathway, consisting of six enzymes: fatty acid synthase, malic enzyme (ME), ATP: citrate lyase (ACL), acetyl-CoA carboxylase (ACC), malate dehydrogenase (MD) and pyruvate carboxylase (PC), and the Polyketide Synthase-like (PKS) pathway, consisting of PKS subunits a, b, c, glucose-6-phosphate dehydrogenase (G6PDH) 6-phosphogluconate dehydrogenase (6PGDH), ACL and ACC. This suggests that the NADPH requirement for the FAS pathway is primarily generated and channelled by ME whereas G6PDH and 6PGDH fulfil this role for the PKS pathway. Diminished biosynthesis of palmitic acid (16:0), docosahexaenoic acid (22:6 n-3, DHA) and docosapentaenoic acid (22:5 n-6, DPA) correlated with the dissociation of their respective metabolons thereby suggesting that regulation of the pathways is achieved through the formation and dissociation of the metabolons.
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Affiliation(s)
- Shuwahida Shuib
- Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia; Autoimmune Unit, Allergy and Immunology Research Centre, Institute for Medical Research (IMR), National Institute of Health (NIH) Malaysia, No. 1, Jalan Setia Murni U13/52, Bandar Setia Alam, 40170 Shah Alam, Selangor, Malaysia
| | - Mohamed Yusuf Mohamed Nazir
- Department of Food Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia; Innovation Centre for Confectionery Technology (MANIS), Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
| | - Izyanti Ibrahim
- Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
| | - Yuanda Song
- Colin Ratledge Center for Microbial Lipids, School of Agriculture Engineering and Food Sciences, Shandong University of Technology, 266 Xincun Rd., Zibo, Shandong, PR China
| | - Colin Ratledge
- Department of Biological Sciences, University of Hull, Kingston upon Hull HU6 7RX, United Kingdom
| | - Aidil Abdul Hamid
- Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
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Zhang S, Sun X, Mou M, Amahong K, Sun H, Zhang W, Shi S, Li Z, Gao J, Zhu F. REGLIV: Molecular regulation data of diverse living systems facilitating current multiomics research. Comput Biol Med 2022; 148:105825. [PMID: 35872412 DOI: 10.1016/j.compbiomed.2022.105825] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2022] [Revised: 06/29/2022] [Accepted: 07/03/2022] [Indexed: 12/24/2022]
Abstract
Multiomics is a powerful technique in molecular biology that facilitates the identification of new associations among different molecules (genes, proteins & metabolites). It has attracted tremendous research interest from the scientists worldwide and has led to an explosive number of published studies. Most of these studies are based on the regulation data provided in available databases. Therefore, it is essential to have molecular regulation data that are strictly validated in the living systems of various cell lines and in vivo models. However, no database has been developed yet to provide comprehensive molecular regulation information validated by living systems. Herein, a new database, Molecular Regulation Data of Living System Facilitating Multiomics Study (REGLIV) is introduced to describe various types of molecular regulation tested by the living systems. (1) A total of 2996 regulations describe the changes in 1109 metabolites triggered by alterations in 284 genes or proteins, and (2) 1179 regulations describe the variations in 926 proteins induced by 125 endogenous metabolites. Overall, REGLIV is unique in (a) providing the molecular regulation of a clearly defined regulatory direction other than simple correlation, (b) focusing on molecular regulations that are validated in a living system not simply in an in vitro test, and (c) describing the disease/tissue/species specific property underlying each regulation. Therefore, REGLIV has important implications for the future practice of not only multiomics, but also other fields relevant to molecular regulation. REGLIV is freely accessible at: https://idrblab.org/regliv/.
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Affiliation(s)
- Song Zhang
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China
| | - Xiuna Sun
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China
| | - Minjie Mou
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China
| | - Kuerbannisha Amahong
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China
| | - Huaicheng Sun
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China
| | - Wei Zhang
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China
| | - Shuiyang Shi
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China
| | - Zhaorong Li
- Innovation Institute for Artificial Intelligence in Medicine of Zhejiang University, Alibaba-Zhejiang University Joint Research Center of Future Digital Healthcare, Hangzhou, 330110, China
| | - Jianqing Gao
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China; Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, China
| | - Feng Zhu
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China; Innovation Institute for Artificial Intelligence in Medicine of Zhejiang University, Alibaba-Zhejiang University Joint Research Center of Future Digital Healthcare, Hangzhou, 330110, China.
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Ciesla J, Moreno I, Munger J. TNFα-induced metabolic reprogramming drives an intrinsic anti-viral state. PLoS Pathog 2022; 18:e1010722. [PMID: 35834576 PMCID: PMC9321404 DOI: 10.1371/journal.ppat.1010722] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2022] [Revised: 07/26/2022] [Accepted: 07/01/2022] [Indexed: 11/22/2022] Open
Abstract
Cytokines induce an anti-viral state, yet many of the functional determinants responsible for limiting viral infection are poorly understood. Here, we find that TNFα induces significant metabolic remodeling that is critical for its anti-viral activity. Our data demonstrate that TNFα activates glycolysis through the induction of hexokinase 2 (HK2), the isoform predominantly expressed in muscle. Further, we show that glycolysis is broadly important for TNFα-mediated anti-viral defense, as its inhibition attenuates TNFα’s ability to limit the replication of evolutionarily divergent viruses. TNFα was also found to modulate the metabolism of UDP-sugars, which are essential precursor substrates for glycosylation. Our data indicate that TNFα increases the concentration of UDP-glucose, as well as the glucose-derived labeling of UDP-glucose and UDP-N-acetyl-glucosamine in a glycolytically-dependent manner. Glycolysis was also necessary for the TNFα-mediated accumulation of several glycosylated anti-viral proteins. Consistent with the importance of glucose-driven glycosylation, glycosyl-transferase inhibition attenuated TNFα’s ability to promote the anti-viral cell state. Collectively, our data indicate that cytokine-mediated metabolic remodeling is an essential component of the anti-viral response. Viral infection often activates a host cell’s intrinsic immune response resulting in the cellular secretion of cytokines, important host-defense molecules. These cytokines act on neighboring cells to make them less permissive to viral infection. Many of the mechanisms through which cytokines promote a less permissive cell state remain unclear. Our data indicate that treatment with the anti-viral cytokine TNFα induces substantial changes to cellular metabolic activity, including activating glucose metabolism. We find that these TNFα-induced metabolic changes are critical for TNFα to limit the replication of diverse viruses including Human Cytomegalovirus and two Coronaviruses, OC43 and SARS-CoV-2. Inhibition of glucose metabolism during TNFα treatment prevented the expression of a variety of known cellular anti-viral proteins. Collectively, our data indicate that cytokine-induced metabolic remodeling is an important component of TNFα’s ability to promote a less permissive cell state and raises further questions about the mechanisms through which specific cytokine-induced metabolic activities contribute to various aspects of anti-viral defense.
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Affiliation(s)
- Jessica Ciesla
- Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America
| | - Isreal Moreno
- Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America
| | - Joshua Munger
- Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America
- * E-mail:
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Zappa F, Muniozguren NL, Wilson MZ, Costello MS, Ponce-Rojas JC, Acosta-Alvear D. Signaling by the integrated stress response kinase PKR is fine-tuned by dynamic clustering. J Cell Biol 2022; 221:e202111100. [PMID: 35522180 PMCID: PMC9086502 DOI: 10.1083/jcb.202111100] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2021] [Revised: 02/15/2022] [Accepted: 04/05/2022] [Indexed: 12/16/2022] Open
Abstract
The double-stranded RNA sensor kinase PKR is one of four integrated stress response (ISR) sensor kinases that phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2α) in response to stress. The current model of PKR activation considers the formation of back-to-back PKR dimers as a prerequisite for signal propagation. Here we show that PKR signaling involves the assembly of dynamic PKR clusters. PKR clustering is driven by ligand binding to PKR's sensor domain and by front-to-front interfaces between PKR's kinase domains. PKR clusters are discrete, heterogeneous, autonomous coalescences that share some protein components with processing bodies. Strikingly, eIF2α is not recruited to PKR clusters, and PKR cluster disruption enhances eIF2α phosphorylation. Together, these results support a model in which PKR clustering may limit encounters between PKR and eIF2α to buffer downstream signaling and prevent the ISR from misfiring.
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Affiliation(s)
- Francesca Zappa
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA
| | - Nerea L. Muniozguren
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA
| | - Maxwell Z. Wilson
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA
| | - Michael S. Costello
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA
| | - Jose Carlos Ponce-Rojas
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA
| | - Diego Acosta-Alvear
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA
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Hu ZK, Niu JL, Lin JJ, Guo Y, Dong LH. Proteomics of restenosis model in LDLR-deficient hamsters coupled with the proliferative rat vascular smooth muscle cells reveals a new mechanism of vascular remodeling diseases. J Proteomics 2022; 264:104634. [PMID: 35661764 DOI: 10.1016/j.jprot.2022.104634] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2021] [Revised: 05/25/2022] [Accepted: 05/26/2022] [Indexed: 12/21/2022]
Abstract
A major pathological mechanism involved in vascular remodeling diseases is the proliferation and migration of vascular smooth muscle cells. The lipid distribution of golden hamsters is similar to that of humans, which makes them an excellent study model for studying the pathogenesis and molecular characteristics of vascular remodeling diseases. We performed proteomic analysis on Sprague Dawley rat VSMCs (rVSMCs) and restenosis hamsters with low-density lipoprotein receptor (LDLR) deficiency as part of this study. We have also performed the enrichment analysis of differentially modified proteins in regards to Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein domain. 1070 differentially abundant proteins were assessed in rVSMCs before and after platelet-derived growth factor-BB (PDGF-BB) stimulation. Specifically, 1246 proteins displayed significant differences in the restenosis model in LDLR-deficient hamsters. An analysis of crosstalk between LDLR+/- hamsters artery restenosis and proliferating rVSMCs revealed 130 differentially expressed proteins, including 67 up-regulated proteins and 63 downregulated proteins. Enrichment analysis with KEGG showed differential proteins to be mainly concentrated in metabolic pathways. There are numerous differentially abundant proteins but particularly two proteins (phosphofructokinase 1 of liver type and lactate dehydrogenase A) were found to be up-regulated by PDGF-BB stimulation of rVSMCs and in a restenosis model of hamsters with LDLR+/- expression. SIGNIFICANCE: Based on bioinformatics, we have found glycolysis pathway plays an important role in both the LDLR+/- hamsters restenosis model and the proliferation of rVSMCs. Some key glycolysis enzymes may likely be developed either as new biomarkers or drug targets for vascular remodeling diseases.
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Affiliation(s)
- Zhao-Kun Hu
- Department of Biochemistry and Molecular Biology, College of Basic Medicine, Cardiovascular Medical Science Center, Key Laboratory of Medical Biotechnology of Hebei Province, The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang, 050017, China
| | - Jiang-Ling Niu
- Department of Biochemistry and Molecular Biology, College of Basic Medicine, Cardiovascular Medical Science Center, Key Laboratory of Medical Biotechnology of Hebei Province, The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang, 050017, China
| | - Jia-Jie Lin
- Department of Biochemistry and Molecular Biology, College of Basic Medicine, Cardiovascular Medical Science Center, Key Laboratory of Medical Biotechnology of Hebei Province, The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang, 050017, China
| | - Yu Guo
- Department of Biochemistry and Molecular Biology, College of Basic Medicine, Cardiovascular Medical Science Center, Key Laboratory of Medical Biotechnology of Hebei Province, The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang, 050017, China
| | - Li-Hua Dong
- Department of Biochemistry and Molecular Biology, College of Basic Medicine, Cardiovascular Medical Science Center, Key Laboratory of Medical Biotechnology of Hebei Province, The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang, 050017, China.
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Conversion of Hyperpolarized [1- 13C]Pyruvate in Breast Cancer Cells Depends on Their Malignancy, Metabolic Program and Nutrient Microenvironment. Cancers (Basel) 2022; 14:cancers14071845. [PMID: 35406616 PMCID: PMC8997828 DOI: 10.3390/cancers14071845] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2022] [Revised: 03/25/2022] [Accepted: 04/01/2022] [Indexed: 12/19/2022] Open
Abstract
Hyperpolarized magnetic resonance spectroscopy (MRS) is a technology for characterizing tumors in vivo based on their metabolic activities. The conversion rates (kpl) of hyperpolarized [1-13C]pyruvate to [1-13C]lactate depend on monocarboxylate transporters (MCT) and lactate dehydrogenase (LDH); these are also indicators of tumor malignancy. An unresolved issue is how glucose and glutamine availability in the tumor microenvironment affects metabolic characteristics of the cancer and how this relates to kpl-values. Two breast cancer cells of different malignancy (MCF-7, MDA-MB-231) were cultured in media containing defined combinations of low glucose (1 mM; 2.5 mM) and glutamine (0.1 mM; 1 mM) and analyzed for pyruvate uptake, intracellular metabolite levels, LDH and pyruvate kinase activities, and 13C6-glucose-derived metabolomics. The results show variability of kpl with the different glucose/glutamine conditions, congruent with glycolytic activity, but not with LDH activity or the Warburg effect; this suggests metabolic compartmentation. Remarkably, kpl-values were almost two-fold higher in MCF-7 than in the more malignant MDA-MB-231 cells, the latter showing a higher flux of 13C-glucose-derived pyruvate to the TCA-cycle metabolites 13C2-citrate and 13C3-malate, i.e., pyruvate decarboxylation and carboxylation, respectively. Thus, MRS with hyperpolarized [1-13C-pyruvate] is sensitive to both the metabolic program and the nutritional state of cancer cells.
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Petty HR. Enzyme Trafficking and Co-Clustering Precede and Accurately Predict Human Breast Cancer Recurrences: An Interdisciplinary Review. Am J Physiol Cell Physiol 2022; 322:C991-C1010. [PMID: 35385324 DOI: 10.1152/ajpcell.00042.2022] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Although great effort has been expended to understand cancer's origins, less attention has been given to the primary cause of cancer deaths - cancer recurrences and their sequelae. This interdisciplinary review addresses mechanistic features of aggressive cancer by studying metabolic enzyme patterns within ductal carcinoma in situ (DCIS) of the breast lesions. DCIS lesions from patients who did or did not experience a breast cancer recurrence were compared. Several proteins, including phospho-Ser226-glucose transporter type 1, phosphofructokinase type L and phosphofructokinase/fructose 2,6-bisphosphatase type 4 are found in nucleoli of ductal epithelial cells in samples from patients who will not subsequently recur, but traffic to the cell periphery in samples from patients who will experience a cancer recurrence. Large co-clusters of enzymes near plasmalemmata will enhance product formation because enzyme concentrations in clusters are very high while solvent molecules and solutes diffuse through small channels. These structural changes will accelerate aerobic glycolysis. Agglomerations of pentose phosphate pathway and glutathione synthesis enzymes enhance GSH formation. As aggressive cancer lesions are incomplete at early stages, they may be unrecognizable. We have found that machine learning provides superior analyses of tissue images and may be used to identify biomarker patterns associated with recurrent and non-recurrent patients with high accuracy. This suggests a new prognostic test to predict DCIS patients who are likely to recur and those who are at low risk for recurrence. Mechanistic interpretations provide a deeper understanding of anti-cancer drug action and suggest that aggressive metastatic cancer cells are sensitive to reductive chemotherapy.
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Affiliation(s)
- Howard R Petty
- Dept. of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, MI, United States
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Igelmann S, Lessard F, Ferbeyre G. Liquid-Liquid Phase Separation in Cancer Signaling, Metabolism and Anticancer Therapy. Cancers (Basel) 2022; 14:cancers14071830. [PMID: 35406602 PMCID: PMC8997759 DOI: 10.3390/cancers14071830] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2022] [Revised: 03/30/2022] [Accepted: 03/31/2022] [Indexed: 01/07/2023] Open
Abstract
The cancer state is thought to be maintained by genetic and epigenetic changes that drive a cancer-promoting gene expression program. However, recent results show that cellular states can be also stably maintained by the reorganization of cell structure leading to the formation of biological condensates via the process of liquid-liquid phase separation. Here, we review the data showing cancer-specific biological condensates initiated by mutant oncoproteins, RNA-binding proteins, or lincRNAs that regulate oncogenic gene expression programs and cancer metabolism. Effective anticancer drugs may specifically partition into oncogenic biological condensates (OBC).
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Affiliation(s)
- Sebastian Igelmann
- Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC H3C 3J7, Canada;
- Montreal Cancer Institute, CR-CHUM, Université de Montréal, Montréal, QC H2X 0A9, Canada
| | - Frédéric Lessard
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, QC G1R 2J6, Canada;
| | - Gerardo Ferbeyre
- Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC H3C 3J7, Canada;
- Montreal Cancer Institute, CR-CHUM, Université de Montréal, Montréal, QC H2X 0A9, Canada
- Correspondence: ; Tel.: +1-514-343-7571
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Mehta S, Zhang J. Liquid-liquid phase separation drives cellular function and dysfunction in cancer. Nat Rev Cancer 2022; 22:239-252. [PMID: 35149762 PMCID: PMC10036213 DOI: 10.1038/s41568-022-00444-7] [Citation(s) in RCA: 203] [Impact Index Per Article: 67.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 01/12/2022] [Indexed: 12/11/2022]
Abstract
Cancer is a disease of uncontrollably reproducing cells. It is governed by biochemical pathways that have escaped the regulatory bounds of normal homeostatic balance. This balance is maintained through precise spatiotemporal regulation of these pathways. The formation of biomolecular condensates via liquid-liquid phase separation (LLPS) has recently emerged as a widespread mechanism underlying the spatiotemporal coordination of biological activities in cells. Biomolecular condensates are widely observed to directly regulate key cellular processes involved in cancer cell pathology, and the dysregulation of LLPS is increasingly implicated as a previously hidden driver of oncogenic activity. In this Perspective, we discuss how LLPS shapes the biochemical landscape of cancer cells.
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Affiliation(s)
- Sohum Mehta
- Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA
| | - Jin Zhang
- Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA.
- Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA.
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Garde A, Kenny IW, Kelley LC, Chi Q, Mutlu AS, Wang MC, Sherwood DR. Localized glucose import, glycolytic processing, and mitochondria generate a focused ATP burst to power basement-membrane invasion. Dev Cell 2022; 57:732-749.e7. [PMID: 35316617 PMCID: PMC8969095 DOI: 10.1016/j.devcel.2022.02.019] [Citation(s) in RCA: 35] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2021] [Revised: 01/18/2022] [Accepted: 02/18/2022] [Indexed: 11/27/2022]
Abstract
Invasive cells use transient, energy-consuming protrusions to breach basement membrane (BM) barriers. Using the ATP sensor PercevalHR during anchor cell (AC) invasion in Caenorhabditis elegans, we show that BM invasion is accompanied by an ATP burst from mitochondria at the invasive front. RNAi screening and visualization of a glucose biosensor identified two glucose transporters, FGT-1 and FGT-2, which bathe invasive front mitochondria with glucose and facilitate the ATP burst to form protrusions. FGT-1 localizes at high levels along the invasive membrane, while FGT-2 is adaptive, enriching most strongly during BM breaching and when FGT-1 is absent. Cytosolic glycolytic enzymes that process glucose for mitochondrial ATP production cluster with invasive front mitochondria and promote higher mitochondrial membrane potential and ATP levels. Finally, we show that UNC-6 (netrin), which polarizes invasive protrusions, also orients FGT-1. These studies reveal a robust and integrated energy acquisition, processing, and delivery network that powers BM breaching.
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Affiliation(s)
- Aastha Garde
- Department of Biology, Duke University, Box 90338, Durham, NC 27708, USA; Department of Cell Biology, Duke University Medical Center, Durham, NC 27708, USA
| | - Isabel W Kenny
- Department of Biology, Duke University, Box 90338, Durham, NC 27708, USA
| | - Laura C Kelley
- Department of Biology, Duke University, Box 90338, Durham, NC 27708, USA
| | - Qiuyi Chi
- Department of Biology, Duke University, Box 90338, Durham, NC 27708, USA
| | - Ayse Sena Mutlu
- Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA
| | - Meng C Wang
- Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA; Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA
| | - David R Sherwood
- Department of Biology, Duke University, Box 90338, Durham, NC 27708, USA.
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Michels PAM, Gualdrón-López M. Biogenesis and metabolic homeostasis of trypanosomatid glycosomes: new insights and new questions. J Eukaryot Microbiol 2022; 69:e12897. [PMID: 35175680 DOI: 10.1111/jeu.12897] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Revised: 02/14/2022] [Accepted: 02/14/2022] [Indexed: 11/28/2022]
Abstract
Kinetoplastea and Diplonemea possess peroxisome-related organelles that, uniquely, contain most of the enzymes of the glycolytic pathway and are hence called glycosomes. Enzymes of several other core metabolic pathways have also been located in glycosomes, in addition to some characteristic peroxisomal systems such as pathways of lipid metabolism. A considerable amount of research has been performed on glycosomes of trypanosomes since their discovery four decades ago. Not only the role of the glycosomal enzyme systems in the overall cell metabolism appeared to be unique, but the organelles display also remarkable features regarding their biogenesis and structural properties. These features are similar to those of the well-studied peroxisomes of mammalian and plant cells and yeasts yet exhibit also differences reflecting the large evolutionary distance between these protists and the representatives of other major eukaryotic lineages. Despite all research performed, many questions remain about various properties and the biological roles of glycosomes and peroxisomes. Here we review the current knowledge about glycosomes, often comparing it with information about peroxisomes. Furthermore, we highlight particularly many questions that remain about the biogenesis, and the heterogeneity in structure and content of these enigmatic organelles, and the properties of their boundary membrane.
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Affiliation(s)
- Paul A M Michels
- Centre for Immunity, Infection and Evolution and Centre for Translational and Chemical Biology, The University of Edinburgh, Edinburgh, United Kingdom
| | - Melisa Gualdrón-López
- Instituto Salud Global, Hospital Clinic-Universitat de Barcelona, and Institute for Health Sciences Trias i Pujol, Barcelona, Spain
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Jeon M, Chauhan KM, Szeto GL, Kyoung M, An S. Subcellular regulation of glucose metabolism through multienzyme glucosome assemblies by EGF-ERK1/2 signaling pathways. J Biol Chem 2022; 298:101675. [PMID: 35122791 PMCID: PMC8892083 DOI: 10.1016/j.jbc.2022.101675] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Revised: 01/26/2022] [Accepted: 01/28/2022] [Indexed: 12/12/2022] Open
Abstract
A multienzyme metabolic assembly for human glucose metabolism, namely the glucosome, has been previously demonstrated to partition glucose flux between glycolysis and building block biosynthesis in an assembly size-dependent manner. Among three different sizes of glucosome assemblies, we have shown that large-sized glucosomes are functionally associated with the promotion of serine biosynthesis in the presence of epidermal growth factor (EGF). However, due to multifunctional roles of EGF in signaling pathways, it is unclear which EGF-mediated signaling pathways promote these large glucosome assemblies in cancer cells. In this study, we used Luminex multiplexing assays and high-content single-cell imaging to demonstrate that EGF triggers temporal activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in Hs578T cells. Subsequently, we found that treatments with a pharmacological inhibitor of ERK1/2, SCH772984, or short-hairpin RNAs targeting ERK1/2 promote the dissociation of large-sized assemblies to medium-sized assemblies in Hs578T cells. In addition, our Western blot analyses revealed that EGF treatment does not increase the expression levels of enzymes that are involved in both glucose metabolism and serine biosynthesis. The observed spatial transition of glucosome assemblies between large and medium sizes appears to be mediated by the degree of dynamic partitioning of glucosome enzymes without changing their expression levels. Collectively, our study demonstrates that EGF–ERK1/2 signaling pathways play an important role in the upregulation of large-sized glucosomes in cancer cells, thus functionally governing the promotion of glycolysis-derived serine biosynthesis.
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Affiliation(s)
- Miji Jeon
- Departments of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, 21250
| | - Krishna M Chauhan
- Departments of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, 21250
| | - Gregory L Szeto
- Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, 21250; Program in Oncology, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD 21201
| | - Minjoung Kyoung
- Departments of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, 21250; Program in Oncology, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD 21201
| | - Songon An
- Departments of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, 21250; Program in Oncology, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD 21201.
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Miura N. Condensate Formation by Metabolic Enzymes in Saccharomyces cerevisiae. Microorganisms 2022; 10:232. [PMID: 35208686 PMCID: PMC8876316 DOI: 10.3390/microorganisms10020232] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2021] [Revised: 01/17/2022] [Accepted: 01/18/2022] [Indexed: 12/31/2022] Open
Abstract
Condensate formation by a group of metabolic enzymes in the cell is an efficient way of regulating cell metabolism through the formation of "membrane-less organelles." Because of the use of green fluorescent protein (GFP) for investigating protein localization, various enzymes were found to form condensates or filaments in living Saccharomyces cerevisiae, mammalian cells, and in other organisms, thereby regulating cell metabolism in the certain status of the cells. Among different environmental stresses, hypoxia triggers the spatial reorganization of many proteins, including more than 20 metabolic enzymes, to form numerous condensates, including "Glycolytic body (G-body)" and "Purinosome." These individual condensates are collectively named "Metabolic Enzymes Transiently Assembling (META) body". This review overviews condensate or filament formation by metabolic enzymes in S. cerevisiae, focusing on the META body, and recent reports in elucidating regulatory machinery of META body formation.
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Affiliation(s)
- Natsuko Miura
- Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai 599-8531, Japan
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45
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Li X, Han M, Zhang H, Liu F, Pan Y, Zhu J, Liao Z, Chen X, Zhang B. Structures and biological functions of zinc finger proteins and their roles in hepatocellular carcinoma. Biomark Res 2022; 10:2. [PMID: 35000617 PMCID: PMC8744215 DOI: 10.1186/s40364-021-00345-1] [Citation(s) in RCA: 58] [Impact Index Per Article: 19.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Accepted: 11/23/2021] [Indexed: 12/15/2022] Open
Abstract
Zinc finger proteins are transcription factors with the finger domain, which plays a significant role in gene regulation. As the largest family of transcription factors in the human genome, zinc finger (ZNF) proteins are characterized by their different DNA binding motifs, such as C2H2 and Gag knuckle. Different kinds of zinc finger motifs exhibit a wide variety of biological functions. Zinc finger proteins have been reported in various diseases, especially in several cancers. Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated death worldwide, especially in China. Most of HCC patients have suffered from hepatitis B virus (HBV) and hepatitis C virus (HCV) injection for a long time. Although the surgical operation of HCC has been extremely developed, the prognosis of HCC is still very poor, and the underlying mechanisms in HCC tumorigenesis are still not completely understood. Here, we summarize multiple functions and recent research of zinc finger proteins in HCC tumorigenesis and progression. We also discuss the significance of zinc finger proteins in HCC diagnosis and prognostic evaluation.
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Affiliation(s)
- Xinxin Li
- Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China.,Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Wuhan, 430030, China
| | - Mengzhen Han
- Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China.,Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Wuhan, 430030, China
| | - Hongwei Zhang
- Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China.,Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Wuhan, 430030, China
| | - Furong Liu
- Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China.,Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Wuhan, 430030, China
| | - Yonglong Pan
- Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China.,Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Wuhan, 430030, China
| | - Jinghan Zhu
- Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China.,Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Wuhan, 430030, China
| | - Zhibin Liao
- Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China. .,Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Wuhan, 430030, China.
| | - Xiaoping Chen
- Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China. .,Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Wuhan, 430030, China.
| | - Bixiang Zhang
- Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China. .,Hubei Key Laboratory of Hepato-Pancreato-Biliary Diseases, Wuhan, 430030, China.
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Liu M, Chen X, Xia J. Multienzyme Catalysis in Phase-Separated Protein Condensates. Methods Mol Biol 2022; 2487:345-354. [PMID: 35687245 DOI: 10.1007/978-1-0716-2269-8_20] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
Liquid-liquid phase separation forms condensates that feature a highly concentrated liquid phase, a defined yet dynamic boundary, and dynamic exchange at and across the boundary. Phase transition drives the formation of dynamic multienzyme complexes in cells, and understanding how phase separation regulates multienzyme catalysis may need the help of in vitro investigations. Recently we have constructed synthetic versions of multienzyme biosynthetic systems by assembling enzymes in protein condensates. Here, we describe the methods for checking the enzyme assembly using fluorescent microscopy and centrifugation assay. We further provide steps for analysis of the cascade enzyme catalytic efficiencies inside the condensates, using enzymes from terpene biosynthesis pathway.
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Affiliation(s)
- Miao Liu
- Department of Chemistry, The Chinese University of Hong Kong, Hong Kong, SAR, China
| | - Xi Chen
- Department of Chemistry, The Chinese University of Hong Kong, Hong Kong, SAR, China
| | - Jiang Xia
- Department of Chemistry, The Chinese University of Hong Kong, Hong Kong, SAR, China.
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An S, Parajuli P, Kennedy EL, Kyoung M. Multi-dimensional Fluorescence Live-Cell Imaging for Glucosome Dynamics in Living Human Cells. Methods Mol Biol 2022; 2487:15-26. [PMID: 35687227 PMCID: PMC9191769 DOI: 10.1007/978-1-0716-2269-8_2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Fluorescence live-cell imaging that has contributed to our understanding of cell biology is now at the frontline of studying quantitative biochemistry in a cell. Particularly, technological advancements of fluorescence live-cell imaging and associated strategies in recent years have allowed us to discover various subcellular macromolecular assemblies in living human cells. Here we describe how real-time dynamics of a multienzyme metabolic assembly, the "glucosome," that is responsible for regulating glucose flux at subcellular levels, has been investigated in both 2- and 3-dimensional space of single human cells. We envision that such multi-dimensional fluorescence live-cell imaging will continue to revolutionize our understanding of how intracellular metabolic pathways and their network are functionally orchestrated at single-cell levels.
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Affiliation(s)
- Songon An
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, MD 21250,Program in Oncology, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD 21201,Corresponding authors: &
| | - Prakash Parajuli
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, MD 21250
| | - Erin L. Kennedy
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, MD 21250
| | - Minjoung Kyoung
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, MD 21250,Program in Oncology, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD 21201,Corresponding authors: &
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Gad S, Ayakar S. Protein scaffolds: A tool for multi-enzyme assembly. BIOTECHNOLOGY REPORTS (AMSTERDAM, NETHERLANDS) 2021; 32:e00670. [PMID: 34824995 PMCID: PMC8605239 DOI: 10.1016/j.btre.2021.e00670] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/15/2021] [Revised: 08/13/2021] [Accepted: 09/03/2021] [Indexed: 12/31/2022]
Abstract
The synthesis of complex molecules using multiple enzymes simultaneously in one reaction vessel has rapidly emerged as a new frontier in the field of bioprocess technology. However, operating different enzymes together in a single vessel limits their operational performance which needs to be addressed. With this respect, scaffolding proteins play an immense role in bringing different enzymes together in a specific manner. The scaffolding improves the catalytic performance, enzyme stability and provides an optimal micro-environment for biochemical reactions. This review describes the components of protein scaffolds, different ways of constructing a protein scaffold-based multi-enzyme complex, and their effects on enzyme kinetics. Moreover, different conjugation strategies viz; dockerin-cohesin interaction, SpyTag-SpyCatcher system, peptide linker-based ligation, affibody, and sortase-mediated ligation are discussed in detail. Various analytical and characterization tools that have enabled the development of these scaffolding strategies are also reviewed. Such mega-enzyme complexes promise wider applications in the field of biotechnology and bioengineering.
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Affiliation(s)
- Shubhada Gad
- Department of Biotechnology, Institute of Chemical Technology - IndianOil Odisha Campus Bhubaneswar, Odisha 751013, India
| | - Sonal Ayakar
- Department of Biotechnology, Institute of Chemical Technology - IndianOil Odisha Campus Bhubaneswar, Odisha 751013, India
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49
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Zhang S, Hua Z, Ba G, Xu N, Miao J, Zhao G, Gong W, Liu Z, Thiele CJ, Li Z. Antitumor effects of the small molecule DMAMCL in neuroblastoma via suppressing aerobic glycolysis and targeting PFKL. Cancer Cell Int 2021; 21:619. [PMID: 34819091 PMCID: PMC8613996 DOI: 10.1186/s12935-021-02330-y] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2021] [Accepted: 11/10/2021] [Indexed: 12/20/2022] Open
Abstract
Background Neuroblastoma (NB) is a common solid malignancy in children that is associated with a poor prognosis. Although the novel small molecular compound Dimethylaminomicheliolide (DMAMCL) has been shown to induce cell death in some tumors, little is known about its role in NB. Methods We examined the effect of DMAMCL on four NB cell lines (NPG, AS, KCNR, BE2). Cellular confluence, survival, apoptosis, and glycolysis were detected using Incucyte ZOOM, CCK-8 assays, Annexin V-PE/7-AAD flow cytometry, and Seahorse XFe96, respectively. Synergistic effects between agents were evaluated using CompuSyn and the effect of DMAMCL in vivo was evaluated using a xenograft mouse model. Phosphofructokinase-1, liver type (PFKL) expression was up- and down-regulated using overexpression plasmids or siRNA. Results When administered as a single agent, DMAMCL decreased cell proliferation in a time- and dose-dependent manner, increased the percentage of cells in SubG1 phase, and induced apoptosis in vitro, as well as inhibiting tumor growth and prolonging survival in tumor-bearing mice (NGP, BE2) in vivo. In addition, DMAMCL exerted synergistic effects when combined with etoposide or cisplatin in vitro and displayed increased antitumor effects when combined with etoposide in vivo compared to either agent alone. Mechanistically, DMAMCL suppressed aerobic glycolysis by decreasing glucose consumption, lactate excretion, and ATP production, as well as reducing the expression of PFKL, a key glycolysis enzyme, in vitro and in vivo. Furthermore, PFKL overexpression attenuated DMAMCL-induced cell death, whereas PFKL silencing promoted NB cell death. Conclusions The results of this study suggest that DMAMCL exerts antitumor effects on NB both in vitro and in vivo by suppressing aerobic glycolysis and that PFKL could be a potential target of DMAMCL in NB. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-021-02330-y.
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Affiliation(s)
- Simeng Zhang
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, China.,Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004, China
| | - Zhongyan Hua
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, China.,Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004, China
| | - Gen Ba
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, China.,Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004, China
| | - Ning Xu
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, China.,Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004, China
| | - Jianing Miao
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, China.,Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004, China
| | - Guifeng Zhao
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, China.,Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004, China
| | - Wei Gong
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, China.,Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004, China
| | - Zhihui Liu
- Cellular & Molecular Biology Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health Bethesda, Bethesda, MD, 20892, USA
| | - Carol J Thiele
- Cellular & Molecular Biology Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health Bethesda, Bethesda, MD, 20892, USA
| | - Zhijie Li
- Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, China. .,Medical Research Center, Liaoning Key Laboratory of Research and Application of Animal Models for Environment and Metabolic Diseases, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004, China.
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50
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Mammes A, Pasquier J, Mammes O, Conti M, Douard R, Loric S. Extracellular vesicles: General features and usefulness in diagnosis and therapeutic management of colorectal cancer. World J Gastrointest Oncol 2021; 13:1561-1598. [PMID: 34853637 PMCID: PMC8603448 DOI: 10.4251/wjgo.v13.i11.1561] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/22/2021] [Revised: 06/29/2021] [Accepted: 09/08/2021] [Indexed: 02/06/2023] Open
Abstract
In the world, among all type of cancers, colorectal cancer (CRC) is the third most commonly diagnosed in males and the second in females. In most of cases, (RP1) patients’ prognosis limitation with malignant tumors can be attributed to delayed diagnosis of the disease. Identification of patients with early-stage disease leads to more effective therapeutic interventions. Therefore, new screening methods and further innovative treatment approaches are mandatory as they may lead to an increase in progression-free and overall survival rates. For the last decade, the interest in extracellular vesicles (EVs) research has exponentially increased as EVs generation appears to be a universal feature of every cell that is strongly involved in many mechanisms of cell-cell communication either in physiological or pathological situations. EVs can cargo biomolecules, such as lipids, proteins, nucleic acids and generate transmission signal through the intercellular transfer of their content. By this mechanism, tumor cells can recruit and modify the adjacent and systemic microenvironment to support further invasion and dissemination. This review intends to cover the most recent literature on the role of EVs production in colorectal normal and cancer tissues. Specific attention is paid to the use of EVs for early CRC diagnosis, follow-up, and prognosis as EVs have come into the spotlight of research as a high potential source of ‘liquid biopsies’. The use of EVs as new targets or nanovectors as drug delivery systems for CRC therapy is also summarized.
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Affiliation(s)
- Aurelien Mammes
- INSERM UMR-938, Cancer Biology and Therapeutics Unit, Saint-Antoine Research Center, Saint Antoine University Hospital, Paris 75012, France
| | - Jennifer Pasquier
- INSERM UMR-938, Cancer Biology and Therapeutics Unit, Saint-Antoine Research Center, Saint Antoine University Hospital, Paris 75012, France
| | | | - Marc Conti
- INSERM UMR-938, Cancer Biology and Therapeutics Unit, Saint-Antoine Research Center, Saint Antoine University Hospital, Paris 75012, France
- Metabolism Research Unit, Integracell SAS, Longjumeau 91160, France
| | - Richard Douard
- UCBM, Necker University Hospital, Paris 75015, France
- Gastrointestinal Surgery Department, Clinique Bizet, Paris 75016, France
| | - Sylvain Loric
- INSERM UMR-938, Cancer Biology and Therapeutics Unit, Saint-Antoine Research Center, Saint Antoine University Hospital, Paris 75012, France
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