Limits...
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.

Show MeSH

Related in: MedlinePlus

Dysferlin co-localizes with MSN and PTRF. COS7 cells were grown on glass coverslips in 6-well plates and transfected with the specific construct. After 36 h, cells were fixed in 4% paraformaldehyde/PBS for 10 min at room temperature. a and b, COS7 cells were transfected with Myc-Dysferlin alone and followed by a monoclonal anti-Myc antibody. c–f, COS7 cells were transfected with MSN construct (c and d) or PTRF (e and f) alone followed by a polyclonal anti-Ha antibody. g–l, COS7 cell were transfected with EGFP-DYSF construct together with HA-MSN (g, h, k) or HA-PTRF (i, j, l). An antibody against the HA epitope was used to detect MSN and PTRF constructs, while Dysferlin expression was followed by EGFP fluorescence. The results are representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3037653&req=5

Figure 2: Dysferlin co-localizes with MSN and PTRF. COS7 cells were grown on glass coverslips in 6-well plates and transfected with the specific construct. After 36 h, cells were fixed in 4% paraformaldehyde/PBS for 10 min at room temperature. a and b, COS7 cells were transfected with Myc-Dysferlin alone and followed by a monoclonal anti-Myc antibody. c–f, COS7 cells were transfected with MSN construct (c and d) or PTRF (e and f) alone followed by a polyclonal anti-Ha antibody. g–l, COS7 cell were transfected with EGFP-DYSF construct together with HA-MSN (g, h, k) or HA-PTRF (i, j, l). An antibody against the HA epitope was used to detect MSN and PTRF constructs, while Dysferlin expression was followed by EGFP fluorescence. The results are representative of at least three independent experiments.

Mentions: To gain information about the subcellular localization and validate any potential interaction, human MSN and PTRF coding sequence were cloned into the eukaryotic expression vector pcDNA3-HA and transfected into COS7 cells alone and in association with Myc-EGFP-DFL construct. First, we tested the efficacy of transfection using the single construct. PTRF and MSN were followed using the polyclonal antibody against HA epitope, while DYSF through the monoclonal anti-Myc antibody. Both proteins showed both cytoplasmatic and submembrane expression (Fig. 2, a–f). Then, we co-transfected both construct and followed DYSF by the GFP while MSN and PTRF using the monoclonal antibody for the HA epitope. IF staining (Fig. 2, g–k) showed a perfect merge of Dysferlin with both MSN and PTRF. The signal was observed both in the cytoplasm and along the plasma membrane (Fig. 2, a–k). We previously described the use of skin biopsies to analyze muscle proteins and obtain information about their subcellular localization in dystrophic as well as control samples (26). So, to get more evidence of a co-localization of Dysferlin and both PTRF and MSN, we performed an immunofluorescence assay on both a skin biopsy taken from a normal control (Fig. 3a) and muscle sections from a wild type mouse (Fig. 3b). As seen in Fig. 3, a and b, the proteins show a common pattern of expression of sarcolemmal staining in common with many muscle proteins. The IF assay, on both cells and tissues samples, is consistent with a possible interaction of DYSF with MSN and PTRF. To determine whether the absence of dysferlin affected PTRF localization, we performed immunofluorescence on muscle samples from an LGMD2B patient (Fig. 3c) and Camp mouse (Fig. 3d). As shown in Fig. 3, c and d, the absence of dysferlin did not alter the staining pattern of PTRF, which resembles the control in both mouse and patient tissues. As a negative control, the secondary antibodies alone were used (data not shown).


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 co-localizes with MSN and PTRF. COS7 cells were grown on glass coverslips in 6-well plates and transfected with the specific construct. After 36 h, cells were fixed in 4% paraformaldehyde/PBS for 10 min at room temperature. a and b, COS7 cells were transfected with Myc-Dysferlin alone and followed by a monoclonal anti-Myc antibody. c–f, COS7 cells were transfected with MSN construct (c and d) or PTRF (e and f) alone followed by a polyclonal anti-Ha antibody. g–l, COS7 cell were transfected with EGFP-DYSF construct together with HA-MSN (g, h, k) or HA-PTRF (i, j, l). An antibody against the HA epitope was used to detect MSN and PTRF constructs, while Dysferlin expression was followed by EGFP fluorescence. 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 2: Dysferlin co-localizes with MSN and PTRF. COS7 cells were grown on glass coverslips in 6-well plates and transfected with the specific construct. After 36 h, cells were fixed in 4% paraformaldehyde/PBS for 10 min at room temperature. a and b, COS7 cells were transfected with Myc-Dysferlin alone and followed by a monoclonal anti-Myc antibody. c–f, COS7 cells were transfected with MSN construct (c and d) or PTRF (e and f) alone followed by a polyclonal anti-Ha antibody. g–l, COS7 cell were transfected with EGFP-DYSF construct together with HA-MSN (g, h, k) or HA-PTRF (i, j, l). An antibody against the HA epitope was used to detect MSN and PTRF constructs, while Dysferlin expression was followed by EGFP fluorescence. The results are representative of at least three independent experiments.
Mentions: To gain information about the subcellular localization and validate any potential interaction, human MSN and PTRF coding sequence were cloned into the eukaryotic expression vector pcDNA3-HA and transfected into COS7 cells alone and in association with Myc-EGFP-DFL construct. First, we tested the efficacy of transfection using the single construct. PTRF and MSN were followed using the polyclonal antibody against HA epitope, while DYSF through the monoclonal anti-Myc antibody. Both proteins showed both cytoplasmatic and submembrane expression (Fig. 2, a–f). Then, we co-transfected both construct and followed DYSF by the GFP while MSN and PTRF using the monoclonal antibody for the HA epitope. IF staining (Fig. 2, g–k) showed a perfect merge of Dysferlin with both MSN and PTRF. The signal was observed both in the cytoplasm and along the plasma membrane (Fig. 2, a–k). We previously described the use of skin biopsies to analyze muscle proteins and obtain information about their subcellular localization in dystrophic as well as control samples (26). So, to get more evidence of a co-localization of Dysferlin and both PTRF and MSN, we performed an immunofluorescence assay on both a skin biopsy taken from a normal control (Fig. 3a) and muscle sections from a wild type mouse (Fig. 3b). As seen in Fig. 3, a and b, the proteins show a common pattern of expression of sarcolemmal staining in common with many muscle proteins. The IF assay, on both cells and tissues samples, is consistent with a possible interaction of DYSF with MSN and PTRF. To determine whether the absence of dysferlin affected PTRF localization, we performed immunofluorescence on muscle samples from an LGMD2B patient (Fig. 3c) and Camp mouse (Fig. 3d). As shown in Fig. 3, c and d, the absence of dysferlin did not alter the staining pattern of PTRF, which resembles the control in both mouse and patient tissues. As a negative control, the secondary antibodies alone were used (data not shown).

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