Limits...
Interaction of annexin A6 with alpha actinin in cardiomyocytes.

Mishra S, Chander V, Banerjee P, Oh JG, Lifirsu E, Park WJ, Kim do H, Bandyopadhyay A - BMC Cell Biol. (2011)

Bottom Line: In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal.Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.In overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Kolkata, India.

ABSTRACT

Background: Annexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state.

Results: To identify the putative binding partners of annexin A6 in the heart, ventricular extracts were subjected to glutathione S-transferase (GST)- annexin A6 pull down assay and the GST- annexin A6 bound proteins were identified by mass spectrometry. The pull down fractions of ventricular extracts with GST-full length annexin A6 as well as GST-C terminus deleted annexin A6 when immunoblotted with anti sarcomeric alpha (α)-actinin antibody showed the presence of α-actinin in the immunoblot which was absent when GST-N terminus deleted annexin A6 was used for pull down. Overexpression of green fluorescent protein (GFP) tagged full length annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus deleted annexin A6 was mostly localized to the nucleus. Overexpression of GFP-C terminus deleted annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Double immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric α-actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.

Conclusions: In overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.

Show MeSH

Related in: MedlinePlus

Identification of putative AnxA6 binding partner(s) in rat heart. A. Purified GST-AnxA6 (A6) affinity beads or GST affinity beads (V) were incubated with NP-40 solubilised WHH (1.5 mg) and the bound proteins (interactome) were separated in 12% SDS-PAGE, stained with Coomassie (lane 2) and analysed via LC MS/MS. B. In vitro binding assay: Solubilised whole heart homogenates (WHH) were incubated with GST affinity beads (vector) or AnxA6-GST affinity beads and the bound proteins collected in elute were separated by 10% SDS-PAGE followed by immunoblotting analysis with anti sarcomeric α-actinin antibody. C. In vitro binding assay with lipid depleted WHH: Solubilised WHH or delipidated WHH incubated either with GST-AnxA6 affinity beads or GST (vector) affinity beads and the bound proteins in elute were separated by 10% SDS-PAGE followed by immunoblotting analysis with anti sarcomeric α-actinin antibody. D. Immunoprecipitation: The whole heart homogenate (300 μg) was subjected to immunoprecipitation with anti sarcomeric α-actinin antibody followed by western blotting with anti-AnxA6 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3037912&req=5

Figure 1: Identification of putative AnxA6 binding partner(s) in rat heart. A. Purified GST-AnxA6 (A6) affinity beads or GST affinity beads (V) were incubated with NP-40 solubilised WHH (1.5 mg) and the bound proteins (interactome) were separated in 12% SDS-PAGE, stained with Coomassie (lane 2) and analysed via LC MS/MS. B. In vitro binding assay: Solubilised whole heart homogenates (WHH) were incubated with GST affinity beads (vector) or AnxA6-GST affinity beads and the bound proteins collected in elute were separated by 10% SDS-PAGE followed by immunoblotting analysis with anti sarcomeric α-actinin antibody. C. In vitro binding assay with lipid depleted WHH: Solubilised WHH or delipidated WHH incubated either with GST-AnxA6 affinity beads or GST (vector) affinity beads and the bound proteins in elute were separated by 10% SDS-PAGE followed by immunoblotting analysis with anti sarcomeric α-actinin antibody. D. Immunoprecipitation: The whole heart homogenate (300 μg) was subjected to immunoprecipitation with anti sarcomeric α-actinin antibody followed by western blotting with anti-AnxA6 antibody.

Mentions: To identify the potential interacting proteins of AnxA6 in heart, in vitro binding of whole heart homogenate (WHH) proteins with GST-AnxA6 fusion protein was conducted (Figure 1). The solubilised WHH was applied to GST-AnxA6 bound glutathione-sepharose 4B beads. The proteins not retained by GST-AnxA6 were mostly removed during washing and those bound to GST-AnxA6 were eluted (Figure 1A, lane 2) and subjected to mass spectrometric analysis. The mass spectrometry analysis showed that α-actinin was one of the major proteins bound to GST-AnxA6 (Additional file 1). Therefore, it is apparent that α-actinin might be a potential interacting partner of AnxA6 in the heart. However, the other proteins obtained by mass spectrometry were also likely to interact with AnxA6 since those too were retained by GST-AnxA6 (Additional file 1).


Interaction of annexin A6 with alpha actinin in cardiomyocytes.

Mishra S, Chander V, Banerjee P, Oh JG, Lifirsu E, Park WJ, Kim do H, Bandyopadhyay A - BMC Cell Biol. (2011)

Identification of putative AnxA6 binding partner(s) in rat heart. A. Purified GST-AnxA6 (A6) affinity beads or GST affinity beads (V) were incubated with NP-40 solubilised WHH (1.5 mg) and the bound proteins (interactome) were separated in 12% SDS-PAGE, stained with Coomassie (lane 2) and analysed via LC MS/MS. B. In vitro binding assay: Solubilised whole heart homogenates (WHH) were incubated with GST affinity beads (vector) or AnxA6-GST affinity beads and the bound proteins collected in elute were separated by 10% SDS-PAGE followed by immunoblotting analysis with anti sarcomeric α-actinin antibody. C. In vitro binding assay with lipid depleted WHH: Solubilised WHH or delipidated WHH incubated either with GST-AnxA6 affinity beads or GST (vector) affinity beads and the bound proteins in elute were separated by 10% SDS-PAGE followed by immunoblotting analysis with anti sarcomeric α-actinin antibody. D. Immunoprecipitation: The whole heart homogenate (300 μg) was subjected to immunoprecipitation with anti sarcomeric α-actinin antibody followed by western blotting with anti-AnxA6 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037912&req=5

Figure 1: Identification of putative AnxA6 binding partner(s) in rat heart. A. Purified GST-AnxA6 (A6) affinity beads or GST affinity beads (V) were incubated with NP-40 solubilised WHH (1.5 mg) and the bound proteins (interactome) were separated in 12% SDS-PAGE, stained with Coomassie (lane 2) and analysed via LC MS/MS. B. In vitro binding assay: Solubilised whole heart homogenates (WHH) were incubated with GST affinity beads (vector) or AnxA6-GST affinity beads and the bound proteins collected in elute were separated by 10% SDS-PAGE followed by immunoblotting analysis with anti sarcomeric α-actinin antibody. C. In vitro binding assay with lipid depleted WHH: Solubilised WHH or delipidated WHH incubated either with GST-AnxA6 affinity beads or GST (vector) affinity beads and the bound proteins in elute were separated by 10% SDS-PAGE followed by immunoblotting analysis with anti sarcomeric α-actinin antibody. D. Immunoprecipitation: The whole heart homogenate (300 μg) was subjected to immunoprecipitation with anti sarcomeric α-actinin antibody followed by western blotting with anti-AnxA6 antibody.
Mentions: To identify the potential interacting proteins of AnxA6 in heart, in vitro binding of whole heart homogenate (WHH) proteins with GST-AnxA6 fusion protein was conducted (Figure 1). The solubilised WHH was applied to GST-AnxA6 bound glutathione-sepharose 4B beads. The proteins not retained by GST-AnxA6 were mostly removed during washing and those bound to GST-AnxA6 were eluted (Figure 1A, lane 2) and subjected to mass spectrometric analysis. The mass spectrometry analysis showed that α-actinin was one of the major proteins bound to GST-AnxA6 (Additional file 1). Therefore, it is apparent that α-actinin might be a potential interacting partner of AnxA6 in the heart. However, the other proteins obtained by mass spectrometry were also likely to interact with AnxA6 since those too were retained by GST-AnxA6 (Additional file 1).

Bottom Line: In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal.Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.In overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Kolkata, India.

ABSTRACT

Background: Annexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state.

Results: To identify the putative binding partners of annexin A6 in the heart, ventricular extracts were subjected to glutathione S-transferase (GST)- annexin A6 pull down assay and the GST- annexin A6 bound proteins were identified by mass spectrometry. The pull down fractions of ventricular extracts with GST-full length annexin A6 as well as GST-C terminus deleted annexin A6 when immunoblotted with anti sarcomeric alpha (α)-actinin antibody showed the presence of α-actinin in the immunoblot which was absent when GST-N terminus deleted annexin A6 was used for pull down. Overexpression of green fluorescent protein (GFP) tagged full length annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus deleted annexin A6 was mostly localized to the nucleus. Overexpression of GFP-C terminus deleted annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Double immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric α-actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.

Conclusions: In overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.

Show MeSH
Related in: MedlinePlus