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Reverse engineering gene network identifies new dysferlin-interacting proteins.

Cacciottolo M, Belcastro V, Laval S, Bushby K, di Bernardo D, Nigro V - J. Biol. Chem. (2010)

Bottom Line: The reverse-engineering algorithm behind the analysis relates genes to each other based on changes in their expression patterns.DYSF and AHNAK were used to query the system and extract lists of potential interacting proteins.Among the 32 predictions the two genes share, we validated the physical interaction between DYSF protein with moesin (MSN) and polymerase I and transcript release factor (PTRF) in mouse heart lysate, thus identifying two novel Dysferlin-interacting proteins.

View Article: PubMed Central - PubMed

Affiliation: TIGEM-Telethon Institute of Genetics and Medicine, 80131 Naples, Italy.

ABSTRACT
Dysferlin (DYSF) is a type II transmembrane protein implicated in surface membrane repair of muscle. Mutations in dysferlin lead to Limb Girdle Muscular Dystrophy 2B (LGMD2B), Miyoshi Myopathy (MM), and Distal Myopathy with Anterior Tibialis onset (DMAT). The DYSF protein complex is not well understood, and only a few protein-binding partners have been identified thus far. To increase the set of interacting protein partners for DYSF we recovered a list of predicted interacting protein through a systems biology approach. The predictions are part of a "reverse-engineered" genome-wide human gene regulatory network obtained from experimental data by computational analysis. The reverse-engineering algorithm behind the analysis relates genes to each other based on changes in their expression patterns. DYSF and AHNAK were used to query the system and extract lists of potential interacting proteins. Among the 32 predictions the two genes share, we validated the physical interaction between DYSF protein with moesin (MSN) and polymerase I and transcript release factor (PTRF) in mouse heart lysate, thus identifying two novel Dysferlin-interacting proteins. Our strategy could be useful to clarify Dysferlin function in intracellular vesicles and its implication in muscle membrane resealing.

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Related in: MedlinePlus

Dysferlin associates with MSN and PTRF in vivo. a, heart muscle homogenates from wt and diseased mice were immunoprecipitated with a monoclonal antibody to Dysferlin (dysf). Immunoprecipitated complexes were separated on SDS-PAGE gels and immunoblotted. Dysferlin precipitates were blotted forDYSF, MSN, PTRF, GSN, and βDG. Immunoblots were also probed with secondary antibodies alone to exclude nonspecific bands. A muscle lysate from a healthy subject was used as internal positive control. b and c, BL10 heart muscle homogenates were immunoprecipitated with a polyclonal antibody to PTRF (b) and with a monoclonal antibody to MSN (c). Immunoprecipitated complexes were separated on SDS-PAGE gels and immunoblotted. PTRF precipitates were blotted for PTRF, DYSF and βDG (b). MSN precipitates were blotted for MSN, DYSF, and βDG (c). A muscle lysate from a healthy subject was used as internal control. TL: total lysate, IP: immunoprecipitation, C: control. Black lines were introduced when more separate gels were used. The results are representative of at least three independent experiments.
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Figure 4: Dysferlin associates with MSN and PTRF in vivo. a, heart muscle homogenates from wt and diseased mice were immunoprecipitated with a monoclonal antibody to Dysferlin (dysf). Immunoprecipitated complexes were separated on SDS-PAGE gels and immunoblotted. Dysferlin precipitates were blotted forDYSF, MSN, PTRF, GSN, and βDG. Immunoblots were also probed with secondary antibodies alone to exclude nonspecific bands. A muscle lysate from a healthy subject was used as internal positive control. b and c, BL10 heart muscle homogenates were immunoprecipitated with a polyclonal antibody to PTRF (b) and with a monoclonal antibody to MSN (c). Immunoprecipitated complexes were separated on SDS-PAGE gels and immunoblotted. PTRF precipitates were blotted for PTRF, DYSF and βDG (b). MSN precipitates were blotted for MSN, DYSF, and βDG (c). A muscle lysate from a healthy subject was used as internal control. TL: total lysate, IP: immunoprecipitation, C: control. Black lines were introduced when more separate gels were used. The results are representative of at least three independent experiments.

Mentions: Because of high expression of candidates in heart muscle (Fig. 1a), immunoprecipitation assay was performed on mouse heart lysate. Heart lysates from wt and Camp mouse were incubated with anti-Dysferlin antibody and after washing the immunoprecipitated protein sample was tested for the presence of selected proteins (Fig. 4a). A lysate from a healthy subject was introduced as an additional positive control, to clarify the nature of positive bands. As shown in the Fig. 4a, positive bands were obtained for MSN and PTRF, but not for GSN in IP samples. Negative controls were introduced: we tested the same samples with (i) the secondary antibody alone to exclude the unspecific reaction, (ii) an unrelated antibody (for β-dystroglycan, βDG) and (iii) the IP on tissue from Dysferlin-deficient mice.


Reverse engineering gene network identifies new dysferlin-interacting proteins.

Cacciottolo M, Belcastro V, Laval S, Bushby K, di Bernardo D, Nigro V - J. Biol. Chem. (2010)

Dysferlin associates with MSN and PTRF in vivo. a, heart muscle homogenates from wt and diseased mice were immunoprecipitated with a monoclonal antibody to Dysferlin (dysf). Immunoprecipitated complexes were separated on SDS-PAGE gels and immunoblotted. Dysferlin precipitates were blotted forDYSF, MSN, PTRF, GSN, and βDG. Immunoblots were also probed with secondary antibodies alone to exclude nonspecific bands. A muscle lysate from a healthy subject was used as internal positive control. b and c, BL10 heart muscle homogenates were immunoprecipitated with a polyclonal antibody to PTRF (b) and with a monoclonal antibody to MSN (c). Immunoprecipitated complexes were separated on SDS-PAGE gels and immunoblotted. PTRF precipitates were blotted for PTRF, DYSF and βDG (b). MSN precipitates were blotted for MSN, DYSF, and βDG (c). A muscle lysate from a healthy subject was used as internal control. TL: total lysate, IP: immunoprecipitation, C: control. Black lines were introduced when more separate gels were used. The results are representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037653&req=5

Figure 4: Dysferlin associates with MSN and PTRF in vivo. a, heart muscle homogenates from wt and diseased mice were immunoprecipitated with a monoclonal antibody to Dysferlin (dysf). Immunoprecipitated complexes were separated on SDS-PAGE gels and immunoblotted. Dysferlin precipitates were blotted forDYSF, MSN, PTRF, GSN, and βDG. Immunoblots were also probed with secondary antibodies alone to exclude nonspecific bands. A muscle lysate from a healthy subject was used as internal positive control. b and c, BL10 heart muscle homogenates were immunoprecipitated with a polyclonal antibody to PTRF (b) and with a monoclonal antibody to MSN (c). Immunoprecipitated complexes were separated on SDS-PAGE gels and immunoblotted. PTRF precipitates were blotted for PTRF, DYSF and βDG (b). MSN precipitates were blotted for MSN, DYSF, and βDG (c). A muscle lysate from a healthy subject was used as internal control. TL: total lysate, IP: immunoprecipitation, C: control. Black lines were introduced when more separate gels were used. The results are representative of at least three independent experiments.
Mentions: Because of high expression of candidates in heart muscle (Fig. 1a), immunoprecipitation assay was performed on mouse heart lysate. Heart lysates from wt and Camp mouse were incubated with anti-Dysferlin antibody and after washing the immunoprecipitated protein sample was tested for the presence of selected proteins (Fig. 4a). A lysate from a healthy subject was introduced as an additional positive control, to clarify the nature of positive bands. As shown in the Fig. 4a, positive bands were obtained for MSN and PTRF, but not for GSN in IP samples. Negative controls were introduced: we tested the same samples with (i) the secondary antibody alone to exclude the unspecific reaction, (ii) an unrelated antibody (for β-dystroglycan, βDG) and (iii) the IP on tissue from Dysferlin-deficient mice.

Bottom Line: The reverse-engineering algorithm behind the analysis relates genes to each other based on changes in their expression patterns.DYSF and AHNAK were used to query the system and extract lists of potential interacting proteins.Among the 32 predictions the two genes share, we validated the physical interaction between DYSF protein with moesin (MSN) and polymerase I and transcript release factor (PTRF) in mouse heart lysate, thus identifying two novel Dysferlin-interacting proteins.

View Article: PubMed Central - PubMed

Affiliation: TIGEM-Telethon Institute of Genetics and Medicine, 80131 Naples, Italy.

ABSTRACT
Dysferlin (DYSF) is a type II transmembrane protein implicated in surface membrane repair of muscle. Mutations in dysferlin lead to Limb Girdle Muscular Dystrophy 2B (LGMD2B), Miyoshi Myopathy (MM), and Distal Myopathy with Anterior Tibialis onset (DMAT). The DYSF protein complex is not well understood, and only a few protein-binding partners have been identified thus far. To increase the set of interacting protein partners for DYSF we recovered a list of predicted interacting protein through a systems biology approach. The predictions are part of a "reverse-engineered" genome-wide human gene regulatory network obtained from experimental data by computational analysis. The reverse-engineering algorithm behind the analysis relates genes to each other based on changes in their expression patterns. DYSF and AHNAK were used to query the system and extract lists of potential interacting proteins. Among the 32 predictions the two genes share, we validated the physical interaction between DYSF protein with moesin (MSN) and polymerase I and transcript release factor (PTRF) in mouse heart lysate, thus identifying two novel Dysferlin-interacting proteins. Our strategy could be useful to clarify Dysferlin function in intracellular vesicles and its implication in muscle membrane resealing.

Show MeSH
Related in: MedlinePlus