Following Ariadne's thread: a new perspective on RBR ubiquitin ligases

Ubiquitin signaling pathways rely on E3 ligases for effecting the final transfer of ubiquitin from E2 ubiquitin conjugating enzymes to a protein target. Here we re-evaluate the hybrid RING/HECT mechanism used by the E3 family RING-between-RINGs (RBRs) to transfer ubiquitin to substrates. We place RBRs into the context of current knowledge of HECT and RING E3s. Although not as abundant as the other types of E3s (there are only slightly more than a dozen RBR E3s in the human genome), RBRs are conserved in all eukaryotes and play important roles in biology. Re-evaluation of RBR ligases as RING/HECT E3s provokes new questions and challenges the field

Differential expression of insulin genes 1 and 2 in MIN6 cells and pseudoislets

There is some evidence that the two rodent insulin genes are differentially regulated in mice, although there is no satisfactory consensus on the relative levels and patterns of expression for the two genes. Using the mouse insulinoma cell line MIN6, we have demonstrated by quantitative RT-PCR, differential patterns of expression for the two genes. In mouse islets and early passage MIN6 cells, expression of ins I and ins 2 were found to be approximately equal, but levels of ins I mRNA diminished rapidly with continued passage. Furthermore, the ins I gene was found to be up-regulated in response to glucose stimulation and as a result of increased cell-cell contact, but no effect on the ins 2 gene was observed. Since the MIN6 cell line is frequently used as a P-cell model for gene expression studies, consideration should be given to both insulin genes. (C) 2002 Elsevier Science (USA). All rights reserved

The role of cytosolic phospholipase A(2) in insulin secretion

Cytosolic phospholipase A(2)(cPLA(2)) comprises a widely expressed family of enzymes, some members of which have the properties required of signal transduction elements in electrically excitable cells. Thus, alpha-, and beta-isoforms of cPLA(2) are activated by the increases in intracellular Ca2+ concentration ([Ca2+](i)) achieved in depolarized cells.. Activation is associated with a redistribution of the enzyme within the cell; activation of cPLA(2) generates arachidonic acid (AA), a biologically active unsaturated fatty acid that can be further metabolized to generate a plethora of biologically active molecules. Studies using relatively nonselective pharmacological inhibitors have implicated cPLA(2) in insulin secretory responses to stimuli that elevate P-cell [Ca2+](i); therefore, we have investigated the role of cPLA(2) in beta-cell function by generating beta-cell lines that under- or overexpress the alpha-isoform of cPLA(2). The functional phenotype of the modified cells was assessed by observation of cellular ultrastructure, by measuring insulin gene expression and insulin protein content, and by measuring the effects of insulin secretagogues on cPLA(2) distribution, on changes in [Ca2+](i), and on the rate and pattern of insulin secretion. Our results suggest that. cPLA(2) is not required for the initiation of insulin secretion from beta-cells, but that it plays an important role in the maintenance of beta-cell insulin stores. Our data also demonstrate that excessive production of, or exposure to, AA is deleterious to normal beta-cell secretory function through metabolic dysfunction

BDE-47 and 6-OH-BDE-47 modulate calcium homeostasis in primary fetal human neural progenitor cells via ryanodine receptor-independent mechanisms

Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants found in rising concentrations in human tissue. Epidemiological and animal studies have raised concern for their potential to induce developmental neurotoxicity (DNT). Considering the essential role of calcium homeostasis in neurodevelopment, PBDE-induced disturbance of intracellular calcium concentration ([Ca(2+)]i) may underlie PBDE-induced DNT. To test this hypothesis, we investigated acute effects of BDE-47 and 6-OH-BDE-47 on [Ca(2+)]i in human neural progenitor cells (hNPCs) and unraveled involved signaling pathways. Short-time differentiated hNPCs were exposed to BDE-47, 6-OH-BDE-47, and multiple inhibitors/stimulators of presumably involved signaling pathways to determine possible effects on [Ca(2+)]i by single-cell microscopy with the fluorescent dye Fura-2. Initial characterization of calcium signaling pathways confirmed the early developmental stage of hNPCs. In these cells, BDE-47 (2 μM) and 6-OH-BDE-47 (0.2 μM) induce [Ca(2+)]i transients. This increase in [Ca(2+)]i is due to extracellular Ca(2+) influx and intracellular release of Ca(2+), mainly from the endoplasmic reticulum (ER). While extracellular Ca(2+) seems to enter the cytoplasm upon 6-OH-BDE-47 by interfering with the cell membrane and independent of Ca(2+) ion channels, ER-derived Ca(2+) is released following activation of protein lipase C and inositol 1,4,5-trisphosphate receptor, but independently of ryanodine receptors. These findings illustrate that immature developing hNPCs respond to low concentrations of 6-OH-BDE-47 by an increase in [Ca(2+)]i and provide new mechanistic explanations for such BDE-induced calcium disruption. Thus, these data support the possibility of a critical window of PBDE exposure, i.e., early human brain development, which has to be acknowledged in risk assessment

Role of adenine nucleotides in insulin secretion from MIN6 pseudoislets

Insulin secretion from MIN6 cells configured as cell aggregates by culture on a gelatin substrate (pseudoislets) is enhanced compared to that of MIN6 cells grown as monolayers on tissue culture plastic, indicating the importance of beta-cell-to-beta-cell proximity for insulin release. In this study we have shown that glucose induced a biphasic release of insulin from pseudoislets, whereas the amplitude and duration of the responses of equivalent monolayer cells were much reduced. Purinergic agonists have been implicated in intercellular communication between beta-cells, so we investigated whether adenine nucleotides co-released with insulin are responsible for the enhanced secretory responses of pseudoislets. We have demonstrated that MIN6 cells express purinergic A(1) and P2Y receptors, and that adenine nucleotides increased [Ca2+](i) with an efficacy of agonists being ATP > ADP > AMP. However, neither suramin nor the more selective A, antagonist 1,3-dipropyl-8-cyclopentylxanthine reduced glucose-induced insulin secretion from pseudoislets, and stimulation of monolayer cells with a range of adenine nucleotides did not enhance glucose-induced secretion. These results suggest that enhanced secretion from MIN6 pseudoislets is not due to increased paracrine/autocrine action of adenine nucleotides. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved

Role of adenine nucleotides in insulin secretion from MIN6 pseudoislets

Insulin secretion from MIN6 cells configured as cell aggregates by culture on a gelatin substrate (pseudoislets) is enhanced compared to that of MIN6 cells grown as monolayers on tissue culture plastic, indicating the importance of ?-cell-to-?-cell proximity for insulin release. In this study we have shown that glucose induced a biphasic release of insulin from pseudoislets, whereas the amplitude and duration of the responses of equivalent monolayer cells were much reduced. Purinergic aqonists have been implicated in intercellular communication between ?-cells, so we investigated whether adenine nucleotides co-released with insulin are responsible for the enhanced secretory responses of pseudoislets. We have demonstrated that MIN6 cells express purinergic A1 and P2Y receptors, and that adenine nucleotides increased [Ca2+] with an efficacy of agonists being ATP>ADP>AMP. However, neither suramin nor the more selective A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine reduced glucose-induced insulin secretion from pseudoislets, and stimulation of monolayer cells with a range of adenine nucleotides did not enhance glucose-induced secretion. These results suggest that enhanced secretion from MIN6 pseudoislets is not due to increased paracrine/autocrine action of adenine nucleotides. © 2002 Elsevier Science Ireland Ltd. All rights reserved.</p