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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 and PTRF co-sedimented into the same fractions. Skeletal muscles from wild type and diseased (Camp ko) mice were collected and homogenized. Intracellular vesicle compartments were isolated by differential centrifugation. Supernatant medium was layered onto a linear 10–50% sucrose-optiprep density gradient and subjected to ultracentrifugation (Sw41Ti rotor, 27,000 rpm for 4 h at 4 °C). Starting from the top of the gradient, fractions were collected and separated on SDS-PAGE gels and then immunobloted with DYSF, Caveolin3 (CAV3), and PTRF antibodies to evaluate the relative expression. 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 5: Dysferlin and PTRF co-sedimented into the same fractions. Skeletal muscles from wild type and diseased (Camp ko) mice were collected and homogenized. Intracellular vesicle compartments were isolated by differential centrifugation. Supernatant medium was layered onto a linear 10–50% sucrose-optiprep density gradient and subjected to ultracentrifugation (Sw41Ti rotor, 27,000 rpm for 4 h at 4 °C). Starting from the top of the gradient, fractions were collected and separated on SDS-PAGE gels and then immunobloted with DYSF, Caveolin3 (CAV3), and PTRF antibodies to evaluate the relative expression. Black lines were introduced when more separate gels were used. The results are representative of at least three independent experiments.

Mentions: Because of the evidence of an intracellular vesicular localization of both PTRF (28) and DYSF (5, 7, 29) and their relationship to CAV3 (28, 30), we decided to analyze the distribution of both proteins in a linear gradient. Muscles collected from wt mouse lower limbs were homogenized with a 0.25 m sucrose solution to disrupt cellular but not vesicle membrane and centrifuged to obtain a microsomal sample, enriched in intracellular vesicles. This sample was loaded on a density gradient and centrifuged to allow the sample to equilibrate in the density gradient with the consequent separation of vesicles by buoyant density. Thirty fractions were collected from the gradient starting from the top and analyzed for the expression of DYSF, CAV3, as a positive marker, and PTRF through Western blot. As shown in Fig. 5, Dysferlin-positive vesicles concentrated in the middle part of the gradient. Most of fractions with an intense Dysferlin signal also showed a strong signal for caveolin3, a known Dysferlin-interacting protein, identifying the correct vesicle compartment. We observed that the same fractions were also positive for the expression of PTRF protein, supporting the hypothesis of a physical interaction and localization in the same vesicle compartment consistent with a common function in the muscle fiber.


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 and PTRF co-sedimented into the same fractions. Skeletal muscles from wild type and diseased (Camp ko) mice were collected and homogenized. Intracellular vesicle compartments were isolated by differential centrifugation. Supernatant medium was layered onto a linear 10–50% sucrose-optiprep density gradient and subjected to ultracentrifugation (Sw41Ti rotor, 27,000 rpm for 4 h at 4 °C). Starting from the top of the gradient, fractions were collected and separated on SDS-PAGE gels and then immunobloted with DYSF, Caveolin3 (CAV3), and PTRF antibodies to evaluate the relative expression. 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 5: Dysferlin and PTRF co-sedimented into the same fractions. Skeletal muscles from wild type and diseased (Camp ko) mice were collected and homogenized. Intracellular vesicle compartments were isolated by differential centrifugation. Supernatant medium was layered onto a linear 10–50% sucrose-optiprep density gradient and subjected to ultracentrifugation (Sw41Ti rotor, 27,000 rpm for 4 h at 4 °C). Starting from the top of the gradient, fractions were collected and separated on SDS-PAGE gels and then immunobloted with DYSF, Caveolin3 (CAV3), and PTRF antibodies to evaluate the relative expression. Black lines were introduced when more separate gels were used. The results are representative of at least three independent experiments.
Mentions: Because of the evidence of an intracellular vesicular localization of both PTRF (28) and DYSF (5, 7, 29) and their relationship to CAV3 (28, 30), we decided to analyze the distribution of both proteins in a linear gradient. Muscles collected from wt mouse lower limbs were homogenized with a 0.25 m sucrose solution to disrupt cellular but not vesicle membrane and centrifuged to obtain a microsomal sample, enriched in intracellular vesicles. This sample was loaded on a density gradient and centrifuged to allow the sample to equilibrate in the density gradient with the consequent separation of vesicles by buoyant density. Thirty fractions were collected from the gradient starting from the top and analyzed for the expression of DYSF, CAV3, as a positive marker, and PTRF through Western blot. As shown in Fig. 5, Dysferlin-positive vesicles concentrated in the middle part of the gradient. Most of fractions with an intense Dysferlin signal also showed a strong signal for caveolin3, a known Dysferlin-interacting protein, identifying the correct vesicle compartment. We observed that the same fractions were also positive for the expression of PTRF protein, supporting the hypothesis of a physical interaction and localization in the same vesicle compartment consistent with a common function in the muscle fiber.

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