Duolink Proximity Ligation Assay Research Articles

14-3-3 beta in the healthy and diseased male reproductive system

Dysfunction of cellular processes in the testes can lead to infertility, tumourigenesis or other testicular disorders. 14-3-3 proteins are known to play pivotal roles in cellular communication, signal transduction, intracellular trafficking, cell-cycle control, transcription and cytoskeletal structure and have been implicated in several diseases including tumourigenesis. Here we investigated the expression of the 14-3-3 beta isoform in healthy testicular tissues of humans, rats and mice as well as in tissues of Sertoli-cell-only (SCO) syndrome, intratubular germ cell neoplasia (IGCN) and classical seminoma. Samples of healthy and diseased testes from humans, rats and mice were analysed by immunohistochemistry. For PCR, human testis cell lysates were used. Immunoblot analyses of rats and humans healthy testes were performed. Duolink proximity ligation assay (PLA) and co-immunoprecipitation (Co-IP) were carried out to investigate interactions between 14-3-3 beta and vimentin in human, rat and mouse testes. In healthy testes and SCO syndrome, strong 14-3-3 beta-positive cells could be identified as Sertoli cells. Furthermore, 14-3-3 beta proteins were detected in cells of the peritubular stroma. In samples of IGCN and classical seminoma, the malignant transformed cells stained positive for 14-3-3 beta antigen. Immunoblot analyses revealed the presence of 14-3-3 beta in healthy testicular tissues. 14-3-3 beta mRNA transcripts were detected in cell lysates of healthy human testes. Interaction of 14-3-3 beta with the intermediate filament vimentin was revealed by Duolink PLA and Co-IP. Co-IP experiments identified tubulin as another 14-3-3 beta binding partner. Our data suggest that 14-3-3 beta expression is essential for normal spermatogenesis by interacting with vimentin in Sertoli cells. Additionally, 14-3-3 beta expression in malignant transformed cells in IGCN and classical seminoma may lead to tumourigenesis and cell survival.

Bortezomib Induces G-CSF-Triggered Granulocytic Differentiation of CD34+ Cells From CN Patients by Rescue of STAT5-Dependent Degradation of LEF-1 Protein

AbstractAbstract 388LEF-1 is a decisive transcription factor involved in G-CSFR triggered granulopoiesis and is downregulated in severe congenital neutropenia (CN). However the exact mechanism of LEF-1 downregulation is still unclear. CN patients show response to G-CSF therapy, however therapeutically high doses of G-CSF are required to increase the neutrophil counts. Also CN patients are found to be at increased risk of developing AML (cumulative incidence ∼ 20%). Since LEF-1 is normally expressed in monocytes and lymphocytes whose differentiation is not affected in CN, it must be a granulopoiesis-specific mechanism downstream of G-CSFR signalling leading to LEF-1 down-regulation. STAT5 is one of the factors activated by G-CSF signalling pathway and has also been found to be hyper-activated in AML blasts. Also STAT5 is known to suppress LEF-1 target gene C/EBPα. Therefore we investigated the effects of activated STAT5 on LEF-1 gene expression and functions. We performed chromatin immunoprecipitation (ChIP) assay from CD34+ cell nuclear lysates and found two putative STAT5 binding sites. Reporter gene assays performed using LEF-1 gene promoter, which contains these STAT5 binding sites co-transfected with LEF-1 and STAT5a cDNA showed significant down-regulation of LEF-1 dependent activation in presence of constitutive active STAT5a (caSTAT5a). However, when we introduced deletion mutations in both STAT5 binding regions and performed the reporter gene assays we found that STAT5 dependent down regulation was independent of its direct binding to the LEF-1 gene promoter. Therefore we further checked the effects of activated STAT5 on LEF-1 protein levels. We found that co-transfection of caSTAT5a cDNA with LEF-1 significantly reduces the LEF-1 protein levels. This reduction in LEF-1 protein levels could be normalised by treating the cells with proteosome inhibitors, bortezomib and MG132. Moreover addition of bortezomib or MG132 to the reporter assays could rescue the effect of STAT5 dependent downregulation on LEF-1 gene autoregulation. We indentified a novel endogenous interaction of STAT5 with LEF-1 protein in the co-immunoprecipites from jurkat cell lysates and this interaction was confirmed insitu using Duolink proximity ligation assay. Furthermore we analysed G-CSF dependent granulocytic differentiation of CD34+ from CN patients treated additionally with bortezomib. Granulocytic differentiation was assessed by FACS using myeloid-granulocytic surface markers CD15, CD16 and CD11b. Interestingly, we found that addition of bortezomib to G-CSF resulted in granulocytic differentiation of CD34+ cells of CN patients in vitro, which was abolished in CN cells treated with G-CSF alone. This granulocytic differentiation was accompanied by restoration of LEF-1 protein levels by bortezomib. Taken together we identified a novel mechanism of LEF-1 downregulation in CN patients, which is via elevated degradation of LEF-1 protein mediated by hyper-activated STAT5. This in turn leads to abrogated autoregulation of LEF-1 mRNA expression. Along with, this study opens gates to a new therapeutic approach using bortezomib in combination with G-CSF for CN patients who do not respond or need high doses of G-CSF. Disclosures:No relevant conflicts of interest to declare.

Functional Interaction of piGST and Peroxiredoxin 6 (Prdx6) in Cultured Cells

Prdx6 is a moonlighting protein that has both PLA2 and peroxidase activities. Partial purification of native enzyme from bovine lung revealed the presence of πGST in an active preparation and we have observed that purified recombinant Prdx6 from E coli generally lacks peroxidase activity unless incubated with GSH‐loaded πGST. We provided direct evidence for the formation of a complex between Prdx6 and πGST in vitro (Biochemistry, 2006, Vol. 45, p360), and showed that delivery of recombinant Prdx6 into cells (MCF7) that lack πGST did not increase the phospholipid hydroperoxide peroxidase activity unless πGST was co‐delivered (PNAS, 2004, Vol. 101, p3780). These data indicate an in vitro interaction between Prdx6 and πGST that is required for the peroxidase activity of Prdx6. To evaluate this functional interaction in vivo, we used the Duolink Proximity Ligation Assay to explore the interaction of πGST and Prdx6 in vivo. There is a dramatic increase in interaction between endogenous πGST and Prdx6 in mouse pulmonary microvascular endothelial cells (MPMVEC) upon their treatment with tert‐butyl hydroperoxide. Furthermore, transfection of MCF7 cells with a vector expressing πGST increases the phospholipid hydroperoxide peroxidase activity in the cells, presumably due to activation of Prdx6 by πGST. These in vivo studies provide evidence that πGST functionally interacts with Prdx6 in the intact cell.