Identification of the Immunoproteasome as a Novel Regulator of Skeletal Muscle Differentiation

Abstract

While many of the molecular details of myogenesis have been investigated extensively, the function of immunoproteasomes (iproteasomes) in myogenic differentiation remains unknown. We show here that the mRNA of i-proteasome subunits, the protein levels of constitutive and inducible proteasome subunits, and the proteolytic activities of the 20S and 26S proteasomes were significantly upregulated during differentiation of skeletal muscle C2C12 cells. Knockdown of the i-proteasome catalytic subunit PSMB9 by short hairpin RNA (shRNA) decreased the expression of both PSMB9 and PSMB8 without affecting other catalytic subunits of the proteasome. PSMB9 knockdown and the use of i-proteasome-specific inhibitors both decreased 26S proteasome activities and prevented C2C12 differentiation. Inhibition of the i-proteasome also impaired human skeletal myoblast differentiation. Suppression of the i-proteasome increased protein oxidation, and these oxidized proteins were found to be more susceptible to degradation by exogenous i-proteasomes. Downregulation of the i-proteasome also increased proapoptotic proteins, including Bax, as well as cleaved caspase 3, cleaved caspase 9, and cleaved poly(ADP-ribose) polymerase (PARP), suggesting that impaired differentiation is likely to occur because of significantly increased apoptosis. These results demonstrate for the first time that i-proteasomes, independent of constitutive proteasomes, are critical for skeletal muscle differentiation of mouse C2C12 cells.

Impaired enzymatic defensive activity, mitochondrial dysfunction and proteasome activation are involved in RTT cell oxidative damage

“…Fluorescence were read for 60 min at 10 min intervals at λecc = 360; λemm = 465, gain 60. Results were expressed as RFU/min/mg protein using angular coefficient of interpolate line [21].…”

The Immunoproteasome in Human Lens Epithelial Cells During Oxidative Stress

“…49,53,54 We found that the immunoproteasome subunit LMP7 (b5i) is localized mainly to the cytosol as expected and that the subunit PSMB5 (b5) of the constitutive proteasome is localized mainly to the nucleus. As the constitutive proteasome also degrades proteins in the cytosol, it must be transported from the nucleus to the cytosol on demand.…”

Delineation of Molecular Pathways Involved in Cardiomyopathies Caused by Troponin T Mutations

“…The increased levels of oxidized proteins suggest that many enzymes are likely to be oxidized and have lower enzymatic activity. Increased levels of oxidized proteins in I79N hearts could result from the decreased proteasome activity because the proteasome is involved in degrading oxidized proteins (36). It is also possible that the increased levels of oxidized proteins in these stressed hearts are due to increased energy production.…”

“…The reaction was initiated by adding the specific substrate at a final concentration of 100 M. Final concentrations of bort and specific fluorogenic substrates for each proteolytic proteasome subunit, respectively were: ␤1 -100 M bort, Z-Leu-Leu-Glu-MCA (Peptide Institute, 3179-v), ␤5 -10 M bort, Suc-Leu-Leu-Val-Tyr-AMC (Bachem, Bubendorf, Switzerland, I-1395). These assays have been previously described (35,36).…”

“…The 26S ␤1 (caspase-like) activity of the proteasome was also measured and found to be similar between the three groups of transgenic hearts. Because we have previously shown that the immunoproteasome is important for the removal of oxidized proteins, the immunoproteasome ␤1i and ␤5i activities were determined (35,36) (Fig. 4).…”