Deletion mutants GST-AnxA6 N1 lacks N terminal tail and repeat domain 1 (residues 1-89); GST-AnxA6 N2 lacks N terminal tail, repeat domains N1 and N2 (residues 1-163); GST-AnxA6 N3 lacks N terminal tail, repeat domains N1, N2 and N3 (residues 1-250); GST-AnxA6 C1 lacks C terminal tail and repeat domain C1 (residues 600-673); GST-AnxA6 C3 lacks C terminal tail and repeat domains C1, C2 and C3 (residues 435-673)

Deletion mutants GST-AnxA6 N1 lacks N terminal tail and repeat domain 1 (residues 1-89); GST-AnxA6 N2 lacks N terminal tail, repeat domains N1 and N2 (residues 1-163); GST-AnxA6 N3 lacks N terminal tail, repeat domains N1, N2 and N3 (residues 1-250); GST-AnxA6 C1 lacks C terminal tail and repeat domain C1 (residues 600-673); GST-AnxA6 C3 lacks C terminal tail and repeat domains C1, C2 and C3 (residues 435-673). 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 null 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 ()-actinin antibody showed the presence of -actinin in the immunoblot which was absent when GST-N terminus erased annexin A6 was utilized for pull down. Overexpression of green fluorescent protein (GFP) tagged full size annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus erased annexin A6 was mostly localized to the nucleus. Overexpression of Istradefylline (KW-6002) GFP-C terminus erased annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Two times immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric -actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component Istradefylline (KW-6002) of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not impact the z-band architecture, as exposed by -actinin immunostaining in shRNA treated cells. Conclusions In overall, the present study demonstrated for the first time that annexin A6 literally interacts with sarcomeric -actinin and alters contractility of cardiomyocytes suggesting that it might play important part Istradefylline (KW-6002) in excitation and contraction process. Background The annexins constitute a family of highly conserved proteins that are characterized by their Ca2+-dependent binding to phospholipids [1]. Annexins are indicated in a wide variety of cells and implicated in various extra- and intracellular processes including mitogenic transmission transduction, differentiation and membrane trafficking events [2]. However, the exact biological role of each annexin remains unfamiliar. In myocardial cells, annexins A2, A5 and A6 are particularly abundant [3-7]. AnxA6 is the most abundant annexin in myocardium [8,9]. It is involved in exocytosis, membrane trafficking and Ca2+ signaling [10]. Conflicting reports demonstrated that it is increased in the onset of heart failure in guinea pig [7] and slightly increased or remain unchanged in faltering human being hearts [11]. Transgenic mice overexpressing AnxA6 developed dilated cardiomyopathy [12], impaired cardiac contractility and showed enhanced intracellular Ca2+ turnover [13]. In contrast, AnxA6 null mice displayed increased rate of Ca2+ removal in myocytes and enhanced contractility [14]. Annexins are exemplified by a bipartite corporation of a unique N terminal website and C terminal core website that varies in length and amino acid composition. The N terminal region is thought to confer practical diversity to the annexin protein. The C terminal domain is definitely formed by either a four or eightfold (in case of AnxA6) repeats of approximately 70 amino acid, each repeat transporting a Ca2+ binding site [15]. The mechanism by which AnxA6 alters contractile functions Rabbit Polyclonal to GCNT7 at the cellular level is not obvious. We hypothesized that AnxA6 might literally interact with the sarcomeric proteins in cardiomyocytes to alter the contractile functions of heart. Consequently, to gain insight into the practical part of AnxA6, we have analysed its interacting partners by mass spectrometry and examined the practical significance of AnxA6 knockdown in cardiomyocytes. Results Binding partners of AnxA6 in heart 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 carried out (Number ?(Figure1).1). The solubilised WHH was applied to GST-AnxA6 bound glutathione-sepharose 4B.