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Functional identity of receptors for proteolysis-inducing factor on human and murine skeletal muscle.

Mirza KA, Tisdale MJ - Br. J. Cancer (2014)

Bottom Line: Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody.Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody.These results suggest that the murine and human PIF receptors are identical.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutritional Biomedicine, School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK.

ABSTRACT

Background: Cachexia in both mice and humans is associated with tumour production of a sulphated glycoprotein called proteolysis-inducing factor (PIF). In mice PIF binds with high affinity to a surface receptor in skeletal muscle, but little is known about the human receptor. This study compares the human PIF receptor with the murine.

Methods: Human PIF was isolated from the G361 melanoma and murine PIF from the MAC16 colon adenocarcinoma. The human PIF receptor was isolated from human skeletal muscle myotubes. Protein synthesis and degradation induced by human and murine PIF was studied in human and murine skeletal muscle myotubes.

Results: Both the human and murine PIF receptors showed the same immunoreactivity and Mr 40 000. Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody. Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody.

Conclusions: These results suggest that the murine and human PIF receptors are identical.

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Related in: MedlinePlus

Western blots showing expression of the proteasome 20S α-subunit (A), 19S subunit (B), MuRF1 (C) and MAFbx (D) in human myotubes after treatment with murine PIF (4.2 nM) for 24 h in the absence or presence of the anti-mPIFR antibody (5 μg ml−1) added 2 h prior to the PIF. Actin was used as loading control. The densitometric analysis is based on three separate western blots. Differences from control are indicated as a, P<0.05 or b, P<0.01, while differences in the presence of the anti-mPIFR antibody are shown as d, P<0.05.
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fig6: Western blots showing expression of the proteasome 20S α-subunit (A), 19S subunit (B), MuRF1 (C) and MAFbx (D) in human myotubes after treatment with murine PIF (4.2 nM) for 24 h in the absence or presence of the anti-mPIFR antibody (5 μg ml−1) added 2 h prior to the PIF. Actin was used as loading control. The densitometric analysis is based on three separate western blots. Differences from control are indicated as a, P<0.05 or b, P<0.01, while differences in the presence of the anti-mPIFR antibody are shown as d, P<0.05.

Mentions: Western blotting of components of the ubiquitin–proteasome pathway showed human PIF to increase expression of the 20S and 19S proteasome subunits in murine myotubes (Figure 5A and B), as well as the ubiquitin ligases MuRF1 (Figure 5C) and MAFbx (Figure 5D) and this was attenuated by the anti-murine PIF antibody. Similarly in human myotubes murine PIF increased expression of the 20S and 19S proteasome subunits (Figure 6A and B), MuRF1 (Figure 6C) and MAFbx (Figure 6D), and this was also attenuated by the anti-murine PIF antibody. These results suggest that the murine and human PIFR are essentially identical.


Functional identity of receptors for proteolysis-inducing factor on human and murine skeletal muscle.

Mirza KA, Tisdale MJ - Br. J. Cancer (2014)

Western blots showing expression of the proteasome 20S α-subunit (A), 19S subunit (B), MuRF1 (C) and MAFbx (D) in human myotubes after treatment with murine PIF (4.2 nM) for 24 h in the absence or presence of the anti-mPIFR antibody (5 μg ml−1) added 2 h prior to the PIF. Actin was used as loading control. The densitometric analysis is based on three separate western blots. Differences from control are indicated as a, P<0.05 or b, P<0.01, while differences in the presence of the anti-mPIFR antibody are shown as d, P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150279&req=5

fig6: Western blots showing expression of the proteasome 20S α-subunit (A), 19S subunit (B), MuRF1 (C) and MAFbx (D) in human myotubes after treatment with murine PIF (4.2 nM) for 24 h in the absence or presence of the anti-mPIFR antibody (5 μg ml−1) added 2 h prior to the PIF. Actin was used as loading control. The densitometric analysis is based on three separate western blots. Differences from control are indicated as a, P<0.05 or b, P<0.01, while differences in the presence of the anti-mPIFR antibody are shown as d, P<0.05.
Mentions: Western blotting of components of the ubiquitin–proteasome pathway showed human PIF to increase expression of the 20S and 19S proteasome subunits in murine myotubes (Figure 5A and B), as well as the ubiquitin ligases MuRF1 (Figure 5C) and MAFbx (Figure 5D) and this was attenuated by the anti-murine PIF antibody. Similarly in human myotubes murine PIF increased expression of the 20S and 19S proteasome subunits (Figure 6A and B), MuRF1 (Figure 6C) and MAFbx (Figure 6D), and this was also attenuated by the anti-murine PIF antibody. These results suggest that the murine and human PIFR are essentially identical.

Bottom Line: Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody.Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody.These results suggest that the murine and human PIF receptors are identical.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutritional Biomedicine, School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK.

ABSTRACT

Background: Cachexia in both mice and humans is associated with tumour production of a sulphated glycoprotein called proteolysis-inducing factor (PIF). In mice PIF binds with high affinity to a surface receptor in skeletal muscle, but little is known about the human receptor. This study compares the human PIF receptor with the murine.

Methods: Human PIF was isolated from the G361 melanoma and murine PIF from the MAC16 colon adenocarcinoma. The human PIF receptor was isolated from human skeletal muscle myotubes. Protein synthesis and degradation induced by human and murine PIF was studied in human and murine skeletal muscle myotubes.

Results: Both the human and murine PIF receptors showed the same immunoreactivity and Mr 40 000. Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody. Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody.

Conclusions: These results suggest that the murine and human PIF receptors are identical.

Show MeSH
Related in: MedlinePlus