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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

Proteasome ‘chymotrypsin-like' enzyme activity in C2C12 myotubes after treatment with either murine (A) or human (B) PIF (4.2 nM) and in human myotubes in the presence of murine (C) and human (D) PIF (4.2 nM) for 24 h in the absence, or presence of the anti-mPIFR antibody (rAb; 5 μg ml−1) added 2 h prior to the PIF. The experiment was repeated three times. Differences from control are indicated as b, P<0.01 or c, P<0.001, while differences in the presence of the anti-mPIFR antibody are shown as d, P<0.05, e, P<0.01 or f, P<0.001.
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fig4: Proteasome ‘chymotrypsin-like' enzyme activity in C2C12 myotubes after treatment with either murine (A) or human (B) PIF (4.2 nM) and in human myotubes in the presence of murine (C) and human (D) PIF (4.2 nM) for 24 h in the absence, or presence of the anti-mPIFR antibody (rAb; 5 μg ml−1) added 2 h prior to the PIF. The experiment was repeated three times. Differences from control are indicated as b, P<0.01 or c, P<0.001, while differences in the presence of the anti-mPIFR antibody are shown as d, P<0.05, e, P<0.01 or f, P<0.001.

Mentions: The predominant catabolic pathway for protein degradation in skeletal muscle is the ubiquitin–proteasome pathway (Lecker et al, 1999). The functional activity of the proteasome can be measured as the ‘chymotrypsin-like' enzyme activity, which is located on the β-5 subunit of the 20S proteasome. As with total protein degradation the proteasome ‘chymotrypsin-like' enzyme activity was increased by both murine and human PIF in murine myotubes, and attenuated by the polyclonal antisera to the mPIFR (Figure 4A and B). Both murine and human PIF also increased the ‘chymotrypsin-like' enzyme activity of human muscle to about the same extent (Figure 4C and D), and in both cases this was attenuated by the rabbit anti-mPIFR antibody.


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

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

Proteasome ‘chymotrypsin-like' enzyme activity in C2C12 myotubes after treatment with either murine (A) or human (B) PIF (4.2 nM) and in human myotubes in the presence of murine (C) and human (D) PIF (4.2 nM) for 24 h in the absence, or presence of the anti-mPIFR antibody (rAb; 5 μg ml−1) added 2 h prior to the PIF. The experiment was repeated three times. Differences from control are indicated as b, P<0.01 or c, P<0.001, while differences in the presence of the anti-mPIFR antibody are shown as d, P<0.05, e, P<0.01 or f, P<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Proteasome ‘chymotrypsin-like' enzyme activity in C2C12 myotubes after treatment with either murine (A) or human (B) PIF (4.2 nM) and in human myotubes in the presence of murine (C) and human (D) PIF (4.2 nM) for 24 h in the absence, or presence of the anti-mPIFR antibody (rAb; 5 μg ml−1) added 2 h prior to the PIF. The experiment was repeated three times. Differences from control are indicated as b, P<0.01 or c, P<0.001, while differences in the presence of the anti-mPIFR antibody are shown as d, P<0.05, e, P<0.01 or f, P<0.001.
Mentions: The predominant catabolic pathway for protein degradation in skeletal muscle is the ubiquitin–proteasome pathway (Lecker et al, 1999). The functional activity of the proteasome can be measured as the ‘chymotrypsin-like' enzyme activity, which is located on the β-5 subunit of the 20S proteasome. As with total protein degradation the proteasome ‘chymotrypsin-like' enzyme activity was increased by both murine and human PIF in murine myotubes, and attenuated by the polyclonal antisera to the mPIFR (Figure 4A and B). Both murine and human PIF also increased the ‘chymotrypsin-like' enzyme activity of human muscle to about the same extent (Figure 4C and D), and in both cases this was attenuated by the rabbit anti-mPIFR antibody.

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