Advertisement

Skeletal Muscle Lysosomal Function via Cathepsin Activity Measurement

  • Kristyn Gumpper
  • Matthew Sermersheim
  • Michael X. Zhu
  • Pei-Hui Lin
Part of the Methods in Molecular Biology book series (MIMB, volume 1854)

Abstract

Muscle wasting or cachexia is commonly associated with aging and many diseases such as cancer, infection, autoimmune disorders, and trauma. Decrease in muscle mass, or muscle atrophy, is often caused by dysfunction of protein proteolytic systems, such as lysosomes, which regulate protein turnover and homeostasis. Lysosomes contain many hydrolases and proteases and, thus, represent the major organelle that control protein turnover. Recently, lysosomes have emerged as a signaling hub to integrate cellular functions of nutrient sensing and metabolism, autophagy, phagocytosis, and endocytosis, which are all related to tissue homeostasis. In this chapter, we describe the protocol used to measure lysosomal proteinase (cathepsins) activity in the skeletal muscle. A better understanding of lysosomal function in muscle homeostasis is critical in developing new therapeutic approaches to prevent muscle wasting.

Keywords

Atrophy Autophagy Enzyme kinetics Fluorimeter Muscle acid lysates (MAL) Protein degradation Skeletal muscle function 

Notes

Acknowledgments

This work was supported by NIH grant R01GM092759 (to M.X.Z) and an Ohio State University intramural Lockwood Research grant (to P.H.L).

References

  1. 1.
    Bonaldo P, Sandri M (2013) Cellular and molecular mechanisms of muscle atrophy. Dis Model Mech 6:25–39CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Sakuma K, Aoi W, Yamaguchi A (2017) Molecular mechanism of sarcopenia and cachexia: recent research advances. Pflugers Arch 469(5-6):573–591CrossRefPubMedGoogle Scholar
  3. 3.
    Narici MV, de Boer MD (2011) Disuse of the musculo-skeletal system in space and on earth. Eur J Appl Physiol 111:403–420CrossRefPubMedGoogle Scholar
  4. 4.
    Chopard A, Hillock S, Jasmin BJ (2009) Molecular events and signalling pathways involved in skeletal muscle disuse-induced atrophy and the impact of countermeasures. J Cell Mol Med 13:3032–3050CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bialek P, Morris C, Parkington J, St Andre M, Owens J, Yaworsky P, Seeherman H, Jelinsky SA (2011) Distinct protein degradation profiles are induced by different disuse models of skeletal muscle atrophy. Physiol Genomics 43:1075–1086CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Xu H, Ren D (2015) Lysosomal physiology. Annu Rev Physiol 77:57–80CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Xiong J, Zhu MX (2016) Regulation of lysosomal ion homeostasis by channels and transporters. Sci China Life Sci 59:777–791CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Stoka V, Turk V, Turk B (2016) Lysosomal cathepsins and their regulation in aging and neurodegeneration. Ageing Res Rev 32:22–37CrossRefPubMedGoogle Scholar
  9. 9.
    Aversa Z, Pin F, Lucia S, Penna F, Verzaro R, Fazi M, Colasante G, Tirone A, Rossi Fanelli F, Ramaccini C, Costelli P, Muscaritoli M (2016) Autophagy is induced in the skeletal muscle of cachectic cancer patients. Sci Rep 6:30340CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Eskelinen EL, Saftig P (2009) Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta 1793:664–673CrossRefPubMedGoogle Scholar
  11. 11.
    Reiser J, Adair B, Reinheckel T (2010) Specialized roles for cysteine cathepsins in health and disease. J Clin Invest 120:3421–3431CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Mohamed MM, Sloane BF (2006) Cysteine cathepsins: multifunctional enzymes in cancer. Nat Rev Cancer 6:764–775CrossRefPubMedGoogle Scholar
  13. 13.
    Choe Y, Leonetti F, Greenbaum DC, Lecaille F, Bogyo M, Bromme D, Ellman JA, Craik CS (2006) Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities. J Biol Chem 281:12824–12832CrossRefPubMedGoogle Scholar
  14. 14.
    Schroter J, Schott KJ, Purtill MA, Neuhoff V (1986) Lysosomal protein degradation in experimental hyperphenylalaninaemia. J Inherit Metab Dis 9:273–282CrossRefPubMedGoogle Scholar
  15. 15.
    Creasy BM, Hartmann CB, White FK, McCoy KL (2007) New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations. Cytometry A 71:114–123CrossRefPubMedGoogle Scholar
  16. 16.
    Rossman MD, Maida BT, Douglas SD (1990) Monocyte-derived macrophage and alveolar macrophage fibronectin production and cathepsin D activity. Cell Immunol 126:268–277CrossRefPubMedGoogle Scholar
  17. 17.
    Wilder CL, Park KY, Keegan PM, Platt MO (2011) Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues. Arch Biochem Biophys 516:52–57CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Jang BG, Choi BY, Kim JH, Kim MJ, Sohn M, Suh SW (2013) Impairment of autophagic flux promotes glucose reperfusion-induced neuro2A cell death after glucose deprivation. PLoS One 8:e76466CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Lin PH, Duann P, Komazaki S, Park KH, Li H, Sun M, Sermersheim M, Gumpper K, Parrington J, Galione A, Evans AM, Zhu MX, Ma J (2015) Lysosomal two-pore channel subtype 2 (TPC2) regulates skeletal muscle autophagic signaling. J Biol Chem 290:3377–3389CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Kristyn Gumpper
    • 1
    • 2
  • Matthew Sermersheim
    • 1
    • 2
  • Michael X. Zhu
    • 3
  • Pei-Hui Lin
    • 1
    • 2
  1. 1.Davis Heart and Lung Research InstituteThe Ohio State UniversityColumbusUSA
  2. 2.Department of SurgeryThe Ohio State University Wexner Medical CenterColumbusUSA
  3. 3.Department of Integrative Biology and Pharmacology, McGovern Medical SchoolThe University of Texas Health Science Center at HoustonHoustonUSA

Personalised recommendations