FKBP65-dependent peptidyl-prolyl isomerase activity potentiates the lysyl hydroxylase 2-driven collagen cross-link switch

Bruck Syndrome is a connective tissue disease associated with inactivating mutations in lysyl hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10-null and –wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde–derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities

Interaction of annexin A6 with alpha actinin in cardiomyocytes

<p>Abstract</p> <p>Background</p> <p>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 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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Lysyl Hydroxylase 2 Is Secreted by Tumor Cells and Can Modify Collagen in the Extracellular Space

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147–1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology

Epithelial-to-mesenchymal transition drives a pro-metastatic Golgi compaction process through scaffolding protein PAQR11

Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3-dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the prometastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking. Ectopic expression of the EMT-activating transcription factor ZEB1 stimulated Golgi compaction and relieved microRNA-mediated repression of the Golgi scaffolding protein PAQR11. Depletion of PAQR11 dispersed Golgi organelles and impaired anterograde vesicle transport to the plasma membrane as well as retrograde vesicle tethering to the Golgi. The N-terminal scaffolding domain of PAQR11 was associated with key regulators of Golgi compaction and vesicle transport in pull-down assays and was required to reconstitute Golgi compaction in PAQR11-deficient tumor cells. Finally, high PAQR11 levels were correlated with EMT and shorter survival in human cancers, and PAQR11 was found to be essential for tumor cell migration and metastasis in EMT-driven lung adenocarcinoma models. We conclude that EMT initiates a PAQR11-mediated Golgi compaction process that drives metastasis

Understanding The Dynamics Of Annexin A6 In Cardiomyocytes

Deregulations in signaling pathways that control cell-size are prominent in maladaptive cardiomyocyte hypertrophy, a major risk factor in heart failure.Present work explores the intracellular dynamics of annexin A6 (Anxa6), a protein differentially expressed in cardiomyocyte hypertrophy. Induction of prohypertrophic signaling by acute and chronic exposure of H9c2(2-1) cardiomyocytes to chemical agonists Phenylephrine (PE), Angiotensin II (Ang II) or Isoproterenol (Iso) resulted in profound increment in cell-size and hypertrophy associated phenotypes, as revealed by microscopic and biochemical examinations. Chronic treatments also compromised mitochondrial dynamics and activated mitochondrial pathways of apoptosis. Anxa6 was upregulated in a phasic fashion in course of acute treatments and in a more linear fashion in course of chronic treatments. Biophysical and microscopic experiments revealed an oscillatory cytosolic dynamics of the protein in hypertrophied cells that resulted in a differential localization pattern. Anxa6 was progressively distributed from perinuclear compartment to the plasma membrane under acute treatment. Chronic treatments concentrated the protein more to the perinuclear space. In either scenario, the appearance was granular punctate. Ectopic expression of Anxa6 at controlled levels significantly protected the cardiomyocytes from hypertrophy-associated increase in cell size and conserved mitochondrial dynamics. However, such expression also rendered the cells vulnerable to apoptosis. Anxa6 knockdown augmented hypertrophic responses and severely abrogated mitochondrial dynamics in cardiomyocytes. At molecular levels,Anxa6 was found to interact with the anti-hypertrophic candidate ANP, proapoptotic nuclear enzyme Poly-(ADP-ribose) polymerase 1 (Parp1) and the survival kinase Akt. Capability to interact with such diversely functional candidates enabled Anxa6 to participate in multiple scaffolding events simultaneously, thereby exhibiting the oscillatory pattern of dynamics. Mutagenesis studies confirmed that Ca2+ was essential for Anxa6 interactions, which occasionally required cholesterol. The Anxa6-ANP interaction was crucial for ANP mediated counter hypertrophic responses whereas Anxa6-Parp1 and Anxa6-Akt interactions have implications in apoptosis and mitochondrial dynamics. Structurally, interactions of Anxa6 were mediated mostly by its Nterminus whereas C-terminus was more catalytic, presumably serving to bind Ca2+ required for the interactions. Together, present study depicts a novel mechanistic spectrum of Anxa6 dynamics that helps to regulate equilibrium state of the homeostatic machineries that dictate life and death decisions in cardiomyocytes

Songs of Dissent and Consciousness: Pronouncements of the Bauls of Rural Bengal

Bauls, the wandering minstrels of rural Bengal (of both Bangladesh and India), are a socio-religiously marginalized cultural group. While the ritualistic practices and spiritual discourses of the Bauls have received scholarly attention, scholarship on Bauls’ songs about material and communicative adversities and their emancipatory visions is lacking. Bauls’ performances and discourses are precursors to envisioning alternative emancipatory possibilities that question dominant intolerances, oppressions, and exploitations. This article documents and reflects on the works of two contemporary Bauls—Shah Abdul Karim and Manimohan Das. Through their songs and performances, they (i) question the power structure and legitimize the sufferings and struggles of the downtrodden, and (ii) seek to raise societal consciousness in imagining a free and just society