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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 blot of murine (lane 1) and human (lane 2) PIF receptors on 12% SDS–PAGE using rabbit polyclonal antiserum to the murine PIF receptor. The PIF receptors were purified as detailed in methods.
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fig1: Western blot of murine (lane 1) and human (lane 2) PIF receptors on 12% SDS–PAGE using rabbit polyclonal antiserum to the murine PIF receptor. The PIF receptors were purified as detailed in methods.

Mentions: The human PIFR (hPIFR) was isolated from human skeletal muscle myotubes, produced by differentiation of skeletal muscle myoblasts in vitro, using a WGA affinity column, as previously described for the mPIFR; Todorov et al, 2007). Western blotting showed that the hPIFR showed immunoreactivity to the rabbit polyclonal antibody raised against the mPIFR (Figure 1), and that both the hPIFR and the mPIFR had the same Mr 40 000 on SDS–PAGE. We have previously reported that in skeletal muscle PIF inhibits protein synthesis, as well as stimulating protein degradation, both in vitro (Eley and Tisdale, 2007) and in vivo (Lorite et al, 1997). In this study human PIF, isolated from the G361 human melanoma (Todorov et al, 1999) and murine PIF isolated from the MAC16 colon adenocarcinoma (Todorov et al, 1997), when used at the optimum concentration (4.2 nM), previously observed (Eley and Tisdale, 2007; Whitehouse and Tisdale, 2003), both significantly inhibited protein synthesis in murine C2C12 myotubes (Figure 2A and B), and in both cases this was attenuated by a rabbit polyclonal antibody to the mPIFR, but not by a non-specific rabbit polyclonal antibody. A previous study (Todorov et al, 2007) established that a concentration of 5 μg ml−1 of antibody gave optimal results. A similar situation was observed in human myotubes (Figure 2C and D) with both murine and human PIF inhibiting protein synthesis, which was attenuated by the anti-mPIFR antibody, but not by a non-specific polyclonal antibody.


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

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

Western blot of murine (lane 1) and human (lane 2) PIF receptors on 12% SDS–PAGE using rabbit polyclonal antiserum to the murine PIF receptor. The PIF receptors were purified as detailed in methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Western blot of murine (lane 1) and human (lane 2) PIF receptors on 12% SDS–PAGE using rabbit polyclonal antiserum to the murine PIF receptor. The PIF receptors were purified as detailed in methods.
Mentions: The human PIFR (hPIFR) was isolated from human skeletal muscle myotubes, produced by differentiation of skeletal muscle myoblasts in vitro, using a WGA affinity column, as previously described for the mPIFR; Todorov et al, 2007). Western blotting showed that the hPIFR showed immunoreactivity to the rabbit polyclonal antibody raised against the mPIFR (Figure 1), and that both the hPIFR and the mPIFR had the same Mr 40 000 on SDS–PAGE. We have previously reported that in skeletal muscle PIF inhibits protein synthesis, as well as stimulating protein degradation, both in vitro (Eley and Tisdale, 2007) and in vivo (Lorite et al, 1997). In this study human PIF, isolated from the G361 human melanoma (Todorov et al, 1999) and murine PIF isolated from the MAC16 colon adenocarcinoma (Todorov et al, 1997), when used at the optimum concentration (4.2 nM), previously observed (Eley and Tisdale, 2007; Whitehouse and Tisdale, 2003), both significantly inhibited protein synthesis in murine C2C12 myotubes (Figure 2A and B), and in both cases this was attenuated by a rabbit polyclonal antibody to the mPIFR, but not by a non-specific rabbit polyclonal antibody. A previous study (Todorov et al, 2007) established that a concentration of 5 μg ml−1 of antibody gave optimal results. A similar situation was observed in human myotubes (Figure 2C and D) with both murine and human PIF inhibiting protein synthesis, which was attenuated by the anti-mPIFR antibody, but not by a non-specific polyclonal 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