Binding partners of Annexin A2 in MDA-MB-231 cells
Ontology highlight
ABSTRACT: A mass-spec of proteins found bound to Annexin A2 following a immunoprecipitation of Annexin A2 from cell lysate of MDAMB231 cells plated 1) on plastic or 2) on collagen-1
Project description:We have previously established two sibling glioma subclones, J3T-1 and J3T-2, showing distinct invasive and angiogenic phenotypes. J3T-1, expressing high annexin A2, demonstrates robust angiogenesis and tumor invasion around neovasculature. J3T-2, expressing low annexin A2, demonstrates diffuse invasion along white matter tracts. Knockdown of annexin A2 in J3T-1 (J3T-1shA) resulted in diffuse invasion pattern, and overexpression of annexin A2 in J3T-2 (J3T-2A) showed prominent angiogenesis. We used microarrays to identify genes which promote the phenotypic transition regulated by annexin A2.
Project description:Whole genome expression profiling in the presence and absence of annexin A2 [shRNA] identified fundamentally altered transcriptional programming that changes the radioresponsive transcriptome. Bioinformatics predicted that silencing AnxA2 may enhance cell death responses to stress in association with reduced activation of pro-survival signals such as nuclear factor kappa B. This prediction was validated by demonstrating a significant increase in sensitivity toward tumor necrosis factor alpha induced cell death in annexin A2 silenced cells, relative to vector controls, associated with reduced nuclear translocation of RelA (p65) following tumor necrosis factor alpha treatment. Murine progenitor cells, JB6 cell line, were stably transformed with Annexin A2 shRNA or vector only control and then treated with ionizing radiation at two doses, 10cGy and 100cGy. Samples were collected at 2hr, 6hr and 24hr for RNA isolation. Two untreated time points were collected also: 2hr and 24hr.
Project description:Whole genome expression profiling in the presence and absence of annexin A2 [shRNA] identified fundamentally altered transcriptional programming that changes the radioresponsive transcriptome. Bioinformatics predicted that silencing AnxA2 may enhance cell death responses to stress in association with reduced activation of pro-survival signals such as nuclear factor kappa B. This prediction was validated by demonstrating a significant increase in sensitivity toward tumor necrosis factor alpha induced cell death in annexin A2 silenced cells, relative to vector controls, associated with reduced nuclear translocation of RelA (p65) following tumor necrosis factor alpha treatment.
Project description:Plasma membrane repair is critical for tissue integrity, especially for elongated contractile muscle cells. Genetically-mediated defects in plasma membrane resealing produce persistent leak, leading to a disordered extracellular matrix. Loss of the membrane repair protein dysferlin slows sarcolemmal resealing and promotes excess leak. Annexin A6 is also implicated in sarcolemmal repair, forming repair caps at the site of membrane disruption. On its own, deletion of the gene for annexin A6, Anxa6, had little effect on muscle health. In contrast, combined loss of dysferlin and annexin A6 (DysfA6) generated muscle fibers with profoundly defective membrane leak. Strikingly, Anxa6 deletion in the context of loss of dystrophin (mdxA6) did not exacerbate muscle defects. The persistent membrane leak in DysfA6 muscle resulted in marked macrophage infiltration with disordered macrophage polarization. Injured muscle fibers were targets of macrophage efferocytosis. Loss of Anxa6 was associated with increased expression of annexins A1 and A2, both of which were heavily deposited into the extracellular matrix. In vitro, macrophages exposed to annexins A1 and A2 expressed increased Csf1, consistent with a model where excess leak results in annexins A1 and A2 in the extracellular matrix, where this protein composition elicits macrophage proliferation and efferocytosis.
Project description:Plasma membrane repair is critical for tissue integrity, especially for elongated contractile muscle cells. Genetically-mediated defects in plasma membrane resealing produce persistent leak, leading to a disordered extracellular matrix. Loss of the membrane repair protein dysferlin slows sarcolemmal resealing and promotes excess leak. Annexin A6 is also implicated in sarcolemmal repair, forming repair caps at the site of membrane disruption. On its own, deletion of the gene for annexin A6, Anxa6, had little effect on muscle health. In contrast, combined loss of dysferlin and annexin A6 (DysfA6) generated muscle fibers with profoundly defective membrane leak. Strikingly, Anxa6 deletion in the context of loss of dystrophin (mdxA6) did not exacerbate muscle defects. The persistent membrane leak in DysfA6 muscle resulted in marked macrophage infiltration with disordered macrophage polarization. Injured muscle fibers were targets of macrophage efferocytosis. Loss of Anxa6 was associated with increased expression of annexins A1 and A2, both of which were heavily deposited into the extracellular matrix. In vitro, macrophages exposed to annexins A1 and A2 expressed increased Csf1, consistent with a model where excess leak results in annexins A1 and A2 in the extracellular matrix, where this protein composition elicits macrophage proliferation and efferocytosis.
Project description:To investigate the genes differentially expressed upon plating on top of matrixes with different stiffness, we compared the expression profiles of MDA-MB-231 breast cancer cells plated on a stiff substrate (plastic) with the same cells plated on a soft substrate (hydrogels 0.7 kPa). Keywords: expression profiling by array
Project description:In this work we use stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 by reciprocal pull down. This indicates that SopD2 and PipB2 likely both interact with annexin A2 through protein-protein interactions to establish the S. Typhimurium intracellular replicativeniche. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.
Project description:Repair of injured muscle involves repair of injured myofibers through the involvement of dysferlin and its interacting partners, including annexin. Studies with mice and patients have established that dysferlin deficit leads to chronic inflammation and adipogenic replacement of the diseased muscle. However, longitudinal analysis of annexin deficit on muscle pathology and function is lacking. Here we show that unlike annexin A1, but similar to dysferlin, lack of annexin A2 (AnxA2) causes poor myofiber repair and progressive weakening with age. However, unlike dysferlin-deficient muscle, AnxA2-deficient muscles do not exhibit chronic inflammation or adipogenic replacement. Deletion of AnxA2 in dysferlin deficient mice reduces inflammation, adipogenic replacement, and loss in muscle function caused by dysferlin deficit. These results show that: a) AnxA2 facilitates myofiber repair, b) chronic inflammation and adipogenic replacement of dysferlinopathic muscle requires AnxA2, and c) inhibiting AnxA2-mediated inflammation is a novel therapeutic avenue for dysferlinopathy.