Limits...
Proteasome dysfunction induces muscle growth defects and protein aggregation.

Kitajima Y, Tashiro Y, Suzuki N, Warita H, Kato M, Tateyama M, Ando R, Izumi R, Yamazaki M, Abe M, Sakimura K, Ito H, Urushitani M, Nagatomi R, Takahashi R, Aoki M - J. Cell. Sci. (2014)

Bottom Line: The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes of protein and organelle clearance.The autophagy pathway was upregulated, but the process of autophagosome formation was impaired.Our results suggest that appropriate proteasomal activity is important for muscle growth and for maintaining myofiber integrity in collaboration with autophagy pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan Department of Medicine and Science in Sports and Exercise, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

Show MeSH

Related in: MedlinePlus

The effect of Rpt3 knockdown and Rpt3 deletion on proteasomal activity in vitro and in vivo. The chymotrypsin-like (A) and trypsin-like (B) activities of control (con-siRNA)- and Rpt3-siRNA-transfected C2C12 cells were assessed. Proteasomal activity was significantly suppressed in Rpt3-siRNA-transfected cells. Proteasomal activity at 24, 48 and 72 h after transfection is shown. IU, international units. (C) Rpt3 protein expression was suppressed in Rpt3-siRNA-transfected C2C12 cells. (D) Proteasomal activity (trypsin-like and chymotrypsin-like activity) in the tibialis anterior muscles of 2-week-old Rpt3−/− and Rpt3+/+ mice (n = 5). (E) Proteasomal activity (trypsin-like and chymotrypsin-like activity) in the tibialis anterior muscles of 6-week-old Rpt3−/− and Rpt3+/+ mice (n = 6). Trypsin-like and chymotrypsin-like activity were increased in Rpt3−/− mice compared with Rpt3+/+ mice. The highly specific proteasomal inhibitor AdaAhx3L3VS (30 µM) significantly suppressed chymotrypsin-like activity by >70%, and trypsin-like activity by >30% from tibialis anterior muscle homogenate of both 6-week-old Rpt3−/− and Rpt3+/+ mice. (F) Immunoblotting of Rpt3 protein using homogenates of tibialis anterior muscles from 2- and 6-week-old mice. (G) HE staining of tibialis anterior muscles from 2- and 6-week-old mice. Scale bar: 50 µm. (H) Changes in the components of the proteasomal complex of the tibialis anterior muscles. Rpt2 is significantly increased in Rpt3−/− mice compared with its expression in Rpt3+/+ mice. White bars, Rpt3+/+; gray bars, Rpt3−/−. AU, arbitrary units. All quantitative data show the mean+s.e.m.; *P<0.05 (Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4265737&req=5

f04: The effect of Rpt3 knockdown and Rpt3 deletion on proteasomal activity in vitro and in vivo. The chymotrypsin-like (A) and trypsin-like (B) activities of control (con-siRNA)- and Rpt3-siRNA-transfected C2C12 cells were assessed. Proteasomal activity was significantly suppressed in Rpt3-siRNA-transfected cells. Proteasomal activity at 24, 48 and 72 h after transfection is shown. IU, international units. (C) Rpt3 protein expression was suppressed in Rpt3-siRNA-transfected C2C12 cells. (D) Proteasomal activity (trypsin-like and chymotrypsin-like activity) in the tibialis anterior muscles of 2-week-old Rpt3−/− and Rpt3+/+ mice (n = 5). (E) Proteasomal activity (trypsin-like and chymotrypsin-like activity) in the tibialis anterior muscles of 6-week-old Rpt3−/− and Rpt3+/+ mice (n = 6). Trypsin-like and chymotrypsin-like activity were increased in Rpt3−/− mice compared with Rpt3+/+ mice. The highly specific proteasomal inhibitor AdaAhx3L3VS (30 µM) significantly suppressed chymotrypsin-like activity by >70%, and trypsin-like activity by >30% from tibialis anterior muscle homogenate of both 6-week-old Rpt3−/− and Rpt3+/+ mice. (F) Immunoblotting of Rpt3 protein using homogenates of tibialis anterior muscles from 2- and 6-week-old mice. (G) HE staining of tibialis anterior muscles from 2- and 6-week-old mice. Scale bar: 50 µm. (H) Changes in the components of the proteasomal complex of the tibialis anterior muscles. Rpt2 is significantly increased in Rpt3−/− mice compared with its expression in Rpt3+/+ mice. White bars, Rpt3+/+; gray bars, Rpt3−/−. AU, arbitrary units. All quantitative data show the mean+s.e.m.; *P<0.05 (Student's t-test).

Mentions: To investigate the effect of Rpt3 deletion on proteasomal activity in skeletal muscle tissue, a knockdown of Rpt3 by small interfering (si)RNA in C2C12 cultured myoblasts was performed. Proteasomal activity was markedly reduced in C2C12 cells after siRNA-mediated Rpt3 knockdown at 24, 48 and 72 h after transfection (Fig. 4A,B). In the immunoblotting analysis, the Rpt3 band was undetectable after siRNA treatment, indicating that the Rpt3 antibody correctly detected the Rpt3 protein (Fig. 4C).


Proteasome dysfunction induces muscle growth defects and protein aggregation.

Kitajima Y, Tashiro Y, Suzuki N, Warita H, Kato M, Tateyama M, Ando R, Izumi R, Yamazaki M, Abe M, Sakimura K, Ito H, Urushitani M, Nagatomi R, Takahashi R, Aoki M - J. Cell. Sci. (2014)

The effect of Rpt3 knockdown and Rpt3 deletion on proteasomal activity in vitro and in vivo. The chymotrypsin-like (A) and trypsin-like (B) activities of control (con-siRNA)- and Rpt3-siRNA-transfected C2C12 cells were assessed. Proteasomal activity was significantly suppressed in Rpt3-siRNA-transfected cells. Proteasomal activity at 24, 48 and 72 h after transfection is shown. IU, international units. (C) Rpt3 protein expression was suppressed in Rpt3-siRNA-transfected C2C12 cells. (D) Proteasomal activity (trypsin-like and chymotrypsin-like activity) in the tibialis anterior muscles of 2-week-old Rpt3−/− and Rpt3+/+ mice (n = 5). (E) Proteasomal activity (trypsin-like and chymotrypsin-like activity) in the tibialis anterior muscles of 6-week-old Rpt3−/− and Rpt3+/+ mice (n = 6). Trypsin-like and chymotrypsin-like activity were increased in Rpt3−/− mice compared with Rpt3+/+ mice. The highly specific proteasomal inhibitor AdaAhx3L3VS (30 µM) significantly suppressed chymotrypsin-like activity by >70%, and trypsin-like activity by >30% from tibialis anterior muscle homogenate of both 6-week-old Rpt3−/− and Rpt3+/+ mice. (F) Immunoblotting of Rpt3 protein using homogenates of tibialis anterior muscles from 2- and 6-week-old mice. (G) HE staining of tibialis anterior muscles from 2- and 6-week-old mice. Scale bar: 50 µm. (H) Changes in the components of the proteasomal complex of the tibialis anterior muscles. Rpt2 is significantly increased in Rpt3−/− mice compared with its expression in Rpt3+/+ mice. White bars, Rpt3+/+; gray bars, Rpt3−/−. AU, arbitrary units. All quantitative data show the mean+s.e.m.; *P<0.05 (Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265737&req=5

f04: The effect of Rpt3 knockdown and Rpt3 deletion on proteasomal activity in vitro and in vivo. The chymotrypsin-like (A) and trypsin-like (B) activities of control (con-siRNA)- and Rpt3-siRNA-transfected C2C12 cells were assessed. Proteasomal activity was significantly suppressed in Rpt3-siRNA-transfected cells. Proteasomal activity at 24, 48 and 72 h after transfection is shown. IU, international units. (C) Rpt3 protein expression was suppressed in Rpt3-siRNA-transfected C2C12 cells. (D) Proteasomal activity (trypsin-like and chymotrypsin-like activity) in the tibialis anterior muscles of 2-week-old Rpt3−/− and Rpt3+/+ mice (n = 5). (E) Proteasomal activity (trypsin-like and chymotrypsin-like activity) in the tibialis anterior muscles of 6-week-old Rpt3−/− and Rpt3+/+ mice (n = 6). Trypsin-like and chymotrypsin-like activity were increased in Rpt3−/− mice compared with Rpt3+/+ mice. The highly specific proteasomal inhibitor AdaAhx3L3VS (30 µM) significantly suppressed chymotrypsin-like activity by >70%, and trypsin-like activity by >30% from tibialis anterior muscle homogenate of both 6-week-old Rpt3−/− and Rpt3+/+ mice. (F) Immunoblotting of Rpt3 protein using homogenates of tibialis anterior muscles from 2- and 6-week-old mice. (G) HE staining of tibialis anterior muscles from 2- and 6-week-old mice. Scale bar: 50 µm. (H) Changes in the components of the proteasomal complex of the tibialis anterior muscles. Rpt2 is significantly increased in Rpt3−/− mice compared with its expression in Rpt3+/+ mice. White bars, Rpt3+/+; gray bars, Rpt3−/−. AU, arbitrary units. All quantitative data show the mean+s.e.m.; *P<0.05 (Student's t-test).
Mentions: To investigate the effect of Rpt3 deletion on proteasomal activity in skeletal muscle tissue, a knockdown of Rpt3 by small interfering (si)RNA in C2C12 cultured myoblasts was performed. Proteasomal activity was markedly reduced in C2C12 cells after siRNA-mediated Rpt3 knockdown at 24, 48 and 72 h after transfection (Fig. 4A,B). In the immunoblotting analysis, the Rpt3 band was undetectable after siRNA treatment, indicating that the Rpt3 antibody correctly detected the Rpt3 protein (Fig. 4C).

Bottom Line: The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes of protein and organelle clearance.The autophagy pathway was upregulated, but the process of autophagosome formation was impaired.Our results suggest that appropriate proteasomal activity is important for muscle growth and for maintaining myofiber integrity in collaboration with autophagy pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan Department of Medicine and Science in Sports and Exercise, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

Show MeSH
Related in: MedlinePlus