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Cited by 7Mitochondrial Function and PathologyMetabolism and Genetic DisordersMetabolism, Diabetes, and Cancer

CLPX regulates mitochondrial fatty acid β-oxidation in liver cells

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Suzuki, Ko, Kubota, Yoshiko, Kaneko, Kiriko, Kamata, Costantine Chasama, Furuyama, Kazumichi·2023·Journal of Biological Chemistry
DOI 10.1016/j.jbc.2023.105210PMC PMC10556790
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Abstract

Mitochondrial fatty acid oxidation (β-oxidation) is an essential metabolic process for energy production in eukaryotic cells, but the regulatory mechanisms of this pathway are largely unknown. In the present study, we found that several enzymes involved in β-oxidation are associated with CLPX, the AAA+ unfoldase that is a component of the mitochondrial matrix protease ClpXP. The suppression of CLPX expression increased β-oxidation activity in the HepG2 cell line and in primary human hepatocytes without glucagon treatment. However, the protein levels of enzymes involved in β-oxidation did not significantly increase in CLPX-deleted HepG2 cells (CLPX-KO cells). Coimmunoprecipitation experiments revealed that the protein level in the immunoprecipitates of each antibody changed after the treatment of WT cells with glucagon, and a part of these changes was also observed in the comparison of WT and CLPX-KO cells without glucagon treatment. Although the exogenous expression of WT or ATP-hydrolysis mutant CLPX suppressed β-oxidation activity in CLPX-KO cells, glucagon treatment induced β-oxidation activity only in CLPX-KO cells expressing WT CLPX. These results suggest that the dissociation of CLPX from its target proteins is essential for the induction of β-oxidation in HepG2 cells. Moreover, specific phosphorylation of AMP-activated protein kinase and a decrease in the expression of acetyl-CoA carboxylase 2 were observed in CLPX-KO cells, suggesting that CLPX might participate in the regulation of the cytosolic signaling pathway for β-oxidation. The mechanism for AMP-activated protein kinase phosphorylation remains elusive; however, our results uncovered the hitherto unknown role of CLPX in mitochondrial β-oxidation in human liver cells.

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  • HepG2 shows functional weakening of mitochondrial β-oxidation of fatty acids.

    “Along with this, the HepG2 cell line shows functional weakening of mitochondrial β-oxidation of fatty acids.”

    From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [101]

    The Results section, “Knockout of CLPX induces β-oxidation in HepG2 cells” (page unavailable), reports that untreated HepG2 cells with CLPX had lower mitochondrial β-oxidation activity than CLPX-knockout HepG2 cells. This supports reduced activity under that specific comparison, but not a general claim that the HepG2 cell line has functionally weakened β-oxidation. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • Basal β-oxidation activity in HepG2 is under strict negative control (e.g., by CLPX) and can be significantly induced (2-3 times) by knocking out the corresponding gene or by glucagon stimulation.

    “Moreover, basal β-oxidation activity in HepG2 is under strict negative control, such as due to the protease CLPX (Caseinolytic Mitochondrial Matrix Peptidase Chaperone Subunit X), and it can be significantly induced (2–3 times) by knocking out the corresponding gene or by stimulation with glucagon [101].”

    From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [101]

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References 59 · Cited by 7

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  1. [1]Martinez-Reyes I. Chandel N.S. Mitochondrial TCA cycle metabolites control physiology and disease Nat. Commun. 11 2020 102 31900386 PMC6941980
  2. [2]Cheong A. Archambault D. Degani R. Iverson E. Tremblay K.D. Mager J. Nuclear-encoded mitochondrial ribosomal proteins are required to initiate gastrulation Development 147 2020 PMC7272334 32376682
  3. [3]Fillmore N. Mori J. Lopaschuk G.D. Mitochondrial fatty acid oxidation alterations in heart failure, ischaemic heart disease and diabetic cardiomyopathy Br. J. Pharmacol. 171 2014 2080 2090 24147975 PMC3976623
  4. [4]Gaude E. Frezza C. Defects in mitochondrial metabolism and cancer Cancer Metab. 2 2014 10 25057353 PMC4108232
  5. [5]Lin M.T. Beal M.F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases Nature 443 2006 787 795 17051205
  6. [6]Quiros P.M. Langer T. Lopez-Otin C. New roles for mitochondrial proteases in health, ageing and disease Nat. Rev. 16 2015 345 359 25970558
  7. [7]Baker B.M. Nargund A.M. Sun T. Haynes C.M. Protective coupling of mitochondrial function and protein synthesis via the eIF2alpha kinase GCN-2 PLoS Genet. 8 2012 e1002760 PMC3375257 22719267
  8. [8]Voos W. Pollecker K. The mitochondrial lon protease: novel functions off the beaten track? Biomolecules 10 2020 253 32046155 PMC7072132
  9. [9]Bezawork-Geleta A. Brodie E.J. Dougan D.A. Truscott K.N. LON is the master protease that protects against protein aggregation in human mitochondria through direct degradation of misfolded proteins Sci. Rep. 5 2015 17397 PMC4667172 26627475
  10. [11]Pryde K.R. Taanman J.W. Schapira A.H. A LON-ClpP proteolytic Axis degrades complex I to extinguish ROS production in depolarized mitochondria Cell Rep. 17 2016 2522 2531 27926857 PMC5177631
  11. [12]Lee Y.G. Kim H.W. Nam Y. Shin K.J. Lee Y.J. Park D.H. LONP1 and ClpP cooperatively regulate mitochondrial proteostasis for cancer cell survival Oncogenesis 10 2021 18 33637676 PMC7910295
  12. [13]Kubota Y. Nomura K. Katoh Y. Yamashita R. Kaneko K. Furuyama K. Novel mechanisms for heme-dependent degradation of ALAS1 protein as a component of negative feedback regulation of heme biosynthesis J. Biol. Chem. 291 2016 20516 20529 27496948 PMC5034046
  13. [14]Tian Q. Li T. Hou W. Zheng J. Schrum L.W. Bonkovsky H.L. Lon peptidase 1 (LONP1)-dependent breakdown of mitochondrial 5-aminolevulinic acid synthase protein by heme in human liver cells J. Biol. Chem. 286 2011 26424 26430 21659532 PMC3143606
  14. [15]Baker T.A. Sauer R.T. ClpXP, an ATP-powered unfolding and protein-degradation machine Biochim. Biophys. Acta 1823 2012 15 28 21736903 PMC3209554
  15. [16]Lowth B.R. Kirstein-Miles J. Saiyed T. Brotz-Oesterhelt H. Morimoto R.I. Truscott K.N. Substrate recognition and processing by a Walker B mutant of the human mitochondrial AAA+ protein CLPX J. Struct. Biol. 179 2012 193 201 22710082
  16. [17]Flynn J.M. Neher S.B. Kim Y.I. Sauer R.T. Baker T.A. Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals Mol. Cell 11 2003 671 683 12667450
  17. [18]Neher S.B. Villen J. Oakes E.C. Bakalarski C.E. Sauer R.T. Gygi S.P. Proteomic profiling of ClpXP substrates after DNA damage reveals extensive instability within SOS regulon Mol. Cell 22 2006 193 204 16630889
  18. [19]Al-Furoukh N. Ianni A. Nolte H. Holper S. Kruger M. Wanrooij S. ClpX stimulates the mitochondrial unfolded protein response (UPRmt) in mammalian cells Biochim. Biophys. Acta 1853 2015 2580 2591 26142927
  19. [20]Fischer F. Langer J.D. Osiewacz H.D. Identification of potential mitochondrial CLPXP protease interactors and substrates suggests its central role in energy metabolism Sci. Rep. 5 2015 18375 PMC4683621 26679294
  20. [21]Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis Cell 161 2015 858 867 25957689 PMC4467794
  21. [22]Szczepanowska K. Maiti P. Kukat A. Hofsetz E. Nolte H. Senft K. CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels EMBO J. 35 2016 2566 2583 27797820 PMC5283601
  22. [23]Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 20 1999 3551 3567 10612281
  23. [24]Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources Nat. Protoc. 4 2009 44 57 19131956
  24. [25]Huang da W. Sherman B.T. Lempicki R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists Nucleic Acids Res. 37 2009 1 13 19033363 PMC2615629
  25. [26]Paredes F. Sheldon K. Lassegue B. Williams H.C. Faidley E.A. Benavides G.A. Poldip2 is an oxygen-sensitive protein that controls PDH and alphaKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer Proc. Natl. Acad. Sci. U. S. A. 115 2018 1789 1794 29434038 PMC5828627
  26. [27]Nakayama T. Al-Maawali A. El-Quessny M. Rajab A. Khalil S. Stoler J.M. Mutations in PYCR2, encoding pyrroline-5-carboxylate reductase 2, cause microcephaly and hypomyelination Am. J. Hum. Genet. 96 2015 709 719 25865492 PMC4570282
  27. [28]Becker C. Kukat A. Szczepanowska K. Hermans S. Senft K. Brandscheid C.P. CLPP deficiency protects against metabolic syndrome but hinders adaptive thermogenesis EMBO Rep. 19 2018 e45126 PMC5934779 29588285
  28. [29]Bhaskaran S. Pharaoh G. Ranjit R. Murphy A. Matsuzaki S. Nair B.C. Loss of mitochondrial protease ClpP protects mice from diet-induced obesity and insulin resistance EMBO Rep. 19 2018 e45009 PMC5836096 29420235
  29. [30]Uchinomiya S. Matsunaga N. Kamoda K. Kawagoe R. Tsuruta A. Ohdo S. Fluorescence detection of metabolic activity of the fatty acid beta oxidation pathway in living cells Chem. Commun. (Camb) 56 2020 3023 3026 32048639
  30. [31]Zhang W.S. Pan A. Zhang X. Ying A. Ma G. Liu B.L. Inactivation of NF-kappaB2 (p52) restrains hepatic glucagon response via preserving PDE4B induction Nat. Commun. 10 2019 4303 31541100 PMC6754499
  31. [32]Islam R. Kim J.G. Park Y. Cho J.Y. Cap K.C. Kho A.R. Insulin induces phosphorylation of pyruvate dehydrogenase through RhoA activation pathway in HepG2 cells FASEB J. 33 2019 2072 2083 30226812
  32. [33]Sidossis L.S. Wolfe R.R. Glucose and insulin-induced inhibition of fatty acid oxidation: the glucose-fatty acid cycle reversed Am. J. Physiol. 270 1996 E733 738 8928782
  33. [34]Houten S.M. Violante S. Ventura F.V. Wanders R.J. The biochemistry and physiology of mitochondrial fatty acid beta-oxidation and its genetic disorders Annu. Rev. Physiol. 78 2016 23 44 26474213
  34. [35]Liang K. Li N. Wang X. Dai J. Liu P. Wang C. Cryo-EM structure of human mitochondrial trifunctional protein Proc. Natl. Acad. Sci. U. S. A. 115 2018 7039 7044 29915090 PMC6142257
  35. [36]Xia C. Fu Z. Battaile K.P. Kim J.P. Crystal structure of human mitochondrial trifunctional protein, a fatty acid beta-oxidation metabolon Proc. Natl. Acad. Sci. U. S. A. 116 2019 6069 6074 30850536 PMC6442613
  36. [37]Viollet B. Foretz M. Guigas B. Horman S. Dentin R. Bertrand L. Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders J. Physiol. 574 2006 41 53 16644802 PMC1817784
  37. [38]Wang Q. Liu S. Zhai A. Zhang B. Tian G. AMPK-mediated regulation of lipid metabolism by phosphorylation Biol. Pharm. Bull. 41 2018 985 993 29709897
  38. [39]Fang G. Zhang P. Liu J. Zhang X. Zhu X. Li R. Inhibition of GSK-3beta activity suppresses HCC malignant phenotype by inhibiting glycolysis via activating AMPK/mTOR signaling Cancer Lett. 463 2019 11 26 31404613
  39. [40]Oh S.Y. Park S.K. Kim J.W. Ahn Y.H. Park S.W. Kim K.S. Acetyl-CoA carboxylase beta gene is regulated by sterol regulatory element-binding protein-1 in liver J. Biol. Chem. 278 2003 28410 28417 12764144
  40. [41]Abu-Elheiga L. Brinkley W.R. Zhong L. Chirala S.S. Woldegiorgis G. Wakil S.J. The subcellular localization of acetyl-CoA carboxylase 2 Proc. Natl. Acad. Sci. U. S. A. 97 2000 1444 1449 10677481 PMC26453
  41. [42]Shpilka T. Haynes C.M. The mitochondrial UPR: mechanisms, physiological functions and implications in ageing Nat. Rev. 19 2018 109 120 29165426
  42. [43]Munoz-Carvajal F. Sanhueza M. The mitochondrial unfolded protein response: a hinge between healthy and pathological aging Front. Aging Neurosci. 12 2020 581849 PMC7518384 33061907
  43. [44]Carling D. Mayer F.V. Sanders M.J. Gamblin S.J. AMP-activated protein kinase: nature's energy sensor Nat. Chem. Biol. 7 2011 512 518 21769098
  44. [46]Zhou C. Lyu L.H. Miao H.K. Bahr T. Zhang Q.Y. Liang T. Redox regulation by SOD2 modulates colorectal cancer tumorigenesis through AMPK-mediated energy metabolism Mol. Carcinog. 59 2020 545 556 32149414
  45. [47]Hinchy E.C. Gruszczyk A.V. Willows R. Navaratnam N. Hall A.R. Bates G. Mitochondria-derived ROS activate AMP-activated protein kinase (AMPK) indirectly J. Biol. Chem. 293 2018 17208 17217 30232152 PMC6222118
  46. [48]Mogk A. Huber D. Bukau B. Integrating protein homeostasis strategies in prokaryotes Cold Spring Harb. Perspect. Biol. 3 2011 a004366 PMC3062218 21441580
  47. [49]Levchenko I. Luo L. Baker T.A. Disassembly of the Mu transposase tetramer by the ClpX chaperone Genes Dev. 9 1995 2399 2408 7557391
  48. [50]Burton B.M. Baker T.A. Remodeling protein complexes: insights from the AAA+ unfoldase ClpX and Mu transposase Protein Sci. 14 2005 1945 1954 16046622 PMC2279306
  49. [51]Sheka A.C. Adeyi O. Thompson J. Hameed B. Crawford P.A. Ikramuddin S. Nonalcoholic steatohepatitis: a review JAMA 323 2020 1175 1183 32207804
  50. [52]Bence K.K. Birnbaum M.J. Metabolic drivers of non-alcoholic fatty liver disease Mol. Metab. 50 2021 101143 PMC8324696 33346069
  51. [53]Koo S.H. Nonalcoholic fatty liver disease: molecular mechanisms for the hepatic steatosis Clin. Mol. Hepatol. 19 2013 210 215 24133660 PMC3796672
  52. [54]Moore M.P. Cunningham R.P. Meers G.M. Johnson S.A. Wheeler A.A. Ganga R.R. Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD Hepatology 76 2022 1452 1465 35000203 PMC9270503
  53. [55]Kimura T. Hosokawa T. Taoka M. Tsutsumi K. Ando K. Ishiguro K. Quantitative and combinatory determination of in situ phosphorylation of tau and its FTDP-17 mutants Sci. Rep. 6 2016 33479 PMC5027580 27641626
  54. [56]Kaneko K. Kubota Y. Nomura K. Hayashimoto H. Chida T. Yoshino N. Establishment of a cell model of X-linked sideroblastic anemia using genome editing Exp. Hematol. 65 2018 57 68.e52 29908199
  55. [57]Kakiuchi K. Yamauchi Y. Taoka M. Iwago M. Fujita T. Ito T. Proteomic analysis of in vivo 14-3-3 interactions in the yeast Saccharomyces cerevisiae Biochemistry 46 2007 7781 7792 17559233
  56. [58]Horwich A.L. Fenton W.A. Williams K.R. Kalousek F. Kraus J.P. Doolittle R.F. Structure and expression of a complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase Science 224 1984 1068 1074 6372096
  57. [59]Kadirvel S. Furuyama K. Harigae H. Kaneko K. Tamai Y. Ishida Y. The carboxyl-terminal region of erythroid-specific 5-aminolevulinate synthase acts as an intrinsic modifier for its catalytic activity and protein stability Exp. Hematol. 40 2012 477 486 e471 22269113

Authors
Suzuki, K., Kubota, Y., Kaneko, K., Kamata, C. C., & Furuyama, K.
Affiliations
Iwate Medical University (JP)
Corresponding author
Furuyama, Kazumichi
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2023-10
Volume
299
Issue
10
Pages
105210
ISSN
0021-9258 (ISSN-L)
DOI
10.1016/j.jbc.2023.105210
PMC
PMC10556790
OpenAlex
W4386400754
Cited by
7
References
59
Citations by year
2026: 5 · 2025: 2
FWCI
0.62
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Ministry of Education, Culture, Sports, Science and Technology
  • Iwate Medical University
  • Japan Society for the Promotion of Science (JP17K08640, JP21K08375, JP21K06874)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Metabolism and Genetic Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
Keywords
ClpX, AAA+ unfoldase, mitochondrial beta-oxidation, ClpXP protease, hepatocytes, glucagon, AMPK, acetyl-CoA carboxylase 2, protein phosphorylation, energy metabolism regulation
MeSH terms
Fatty Acids, Glucagon, Humans, Mitochondria, Mitochondria, Liver, Oxidation-Reduction, Phosphorylation, Hepatocytes, Endopeptidase Clp, AMP-Activated Protein Kinases, Hep G2 Cells

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