Identification and characterization of a gain-of-function RAG-1 mutant

RAG-1 and RAG-2 initiate V(D)J recombination by cleaving DNA at recombination signal sequences through sequential nicking and transesterification reactions to yield blunt signal ends and coding ends terminating in a DNA hairpin structure. Ubiquitous DNA repair factors then mediate the rejoining of broken DNA. V(D)J recombination adheres to the 12/23 rule, which limits rearrangement to signal sequences bearing different lengths of DNA (12 or 23 base pairs) between the conserved heptamer and nonamer sequences to which the RAG proteins bind. Both RAG proteins have been subjected to extensive mutagenesis, revealing residues required for one or both cleavage steps or involved in the DNA end-joining process. Gain-of-function RAG mutants remain unidentified. Here, we report a novel RAG-1 mutation, E649A, that supports elevated cleavage activity in vitro by preferentially enhancing hairpin formation. DNA binding activity and the catalysis of other DNA strand transfer reactions, such as transposition, are not substantially affected by the RAG-1 mutation. However, 12/23-regulated synapsis does not strongly stimulate the cleavage activity of a RAG complex containing E649A RAG-1, unlike its wild-type counterpart. Interestingly, wild-type and E649A RAG-1 support similar levels of cleavage and recombination of plasmid substrates containing a 12/23 pair of signal sequences in cell culture; however, E649A RAG-1 supports about threefold more cleavage and recombination than wild-type RAG-1 on 12/12 plasmid substrates. These data suggest that the E649A RAG-1 mutation may interfere with the RAG proteins' ability to sense 12/23-regulated synapsis.     

Metabolic characteristics of pancreatic beta-cells exposed to calcium-transporting ionophores.

The effects of the ionophores A-23187 and X-537 A on glucose metabolism, ATP content and sucrose permeability in pancreatic islets microdissected from obese-hyperglycemic mice were studied. The formation of 14CO2 from 10 mM D-[U-14C] GLUCOSE WAS INHIBITED BY OMISSION OF Ca2+ from the medium. A-23187 (10 muM) induced a further decrease of 14CO2 formation whereas X-537 A (10 muM) had no effect. At 20 mM glucose both A-23187 (48 muM) and X-537 A (43 muM) decreased the 14CO2 formation in the absence of Ca2+ whereas only X-537 A inhibited in the presence of Ca2+. X-537 A (43 muM) also decreased the formation of 3H2O from 20 mM D-[5-3H] glucose. The islet content of ATP was not changed after incubation in media deficient in either Mg2+ or Ca2+. However, omission of both Mg2+ and Ca2+ resulted in about 50% decrease of the ATP content. A-23187 and X-537 A induced dose-dependent decreases of the islet ATP content. X-537 A was much more potent than A-23187. Both ionophores induced stronger depression of the ATP content when Ca2+ was omitted. X-537 A (43 muM) but not A-23187 (48 muM) increased the beta-cell membrane permeability as indicated by an increased sucrose space in relation to the urea space of islets. Such an effect was not obtained with X-537 A at 1 muM or by omission of Ca2+. It is suggested that the marked metabolic effects of the ionophores reflect an impaired mitochondrial metabolism. These metabolic changes should be considered in interpretations of ionophore action on insulin secretion.     

Metabolic activation-driven mitochondrial hyperpolarization predicts insulin secretion in human pancreatic beta-cells

Mitochondrial metabolism plays a central role in insulin secretion in pancreatic beta-cells. Generation of protonmotive force and ATP synthesis from glucose-originated pyruvate are critical steps in the canonical pathway of glucose-stimulated insulin secretion. Mitochondrial metabolism is intertwined with pathways that are thought to amplify insulin secretion with mechanisms distinct from the canonical pathway, and the relative importance of these two pathways is controversial. Here I show that glucose-induced mitochondrial membrane potential (MMP) hyperpolarization is necessary for, and predicts, the rate of insulin secretion in primary cultured human beta-cells. When glucose concentration is elevated, increased metabolism results in a substantial MMP hyperpolarization, as well as in increased rates of ATP synthesis and turnover marked by faster cell respiration. Using modular kinetic analysis I explored what properties of cellular energy metabolism enable a large glucose-induced change in MMP in human beta-cells. I found that an ATP-dependent pathway activates glucose or substrate oxidation, acting as a positive feedback in energy metabolism. This activation mechanism is essential for concomitant fast respiration and high MMP, and for a high magnitude glucose-induced MMP hyperpolarization and therefore for insulin secretion.     

Rab27a in pancreatic beta-cells, a busy protein in membrane trafficking

The small GTPases have the ‘active’ GTP-bound and ‘inactive’ GDP-bound states, and thereby act as a molecular switch in cells. Rab27a is a member of this family and exists in T-lymphocytes, melanocytes and pancreatic beta-cells. Rab27a regulates secretion of cytolytic granules from cytotoxic T-lymphocytes and intracellular transport of melanosomes in melanocytes. In pancreatic beta-cells, Rab27a controls pre-exocytotic stages of insulin secretion. A few GTP-dependent Rab27a effectors are known to mediate these cellular functions. We recently found that Rab27a also possesses the GDP-dependent effector coronin 3. Coronin 3 regulates endocytosis in pancreatic beta-cells through its interaction with GDP-Rab27a. These results imply that GTP- and GDP-Rab27a actively regulate distinct stages in the insulin secretory pathway. In this review, we provide an overview of the roles of both GTP- and GDP-Rab27a in pancreatic beta-cells.     

Expression,phosphorylation and function of the Rab-GTPase activating protein TBC1D1 in pancreatic beta-cells

The Rab-GTPase activating protein TBC1D1 is a paralog of AS160/TBC1D4. AS160/TBC1D4, a downstream effector of Akt, has been shown to play a central role in beta-cell function and survival. The two proteins have overlapping function in insulin signalling in muscle cells. However, the expression and the potential role of TBC1D1 in beta-cells remain unknown. Therefore, the aim of this study is to investigate whether TBC1D1 is expressed in beta-cells and whether it plays, as AS160/TBC1D4, a role in beta-cell function and survival. Using human and rat beta-cells, this study shows for the first time that TBC1D1 is expressed and phosphorylated in response to glucose in these cells. Knockdown of TBC1D1 in beta-cells resulted in increased basal and glucose-stimulated insulin release, decreased proliferation but no change in apoptosis.     

WFS1 is a novel component of the unfolded protein response and maintains homeostasis of the endoplasmic reticulum in pancreatic beta-cells

In Wolfram syndrome, a rare form of juvenile diabetes, pancreatic beta-cell death is not accompanied by an autoimmune response. Although it has been reported that mutations in the WFS1 gene are responsible for the development of this syndrome, the precise molecular mechanisms underlying beta-cell death caused by the WFS1 mutations remain unknown. Here we report that WFS1 is a novel component of the unfolded protein response and has an important function in maintaining homeostasis of the endoplasmic reticulum (ER) in pancreatic beta-cells. WFS1 encodes a transmembrane glyco-protein in the ER. WFS1 mRNA and protein are induced by ER stress. The expression of WFS1 is regulated by inositol requiring 1 and PKR-like ER kinase, central regulators of the unfolded protein response. WFS1 is normally up-regulated during insulin secretion, whereas inactivation of WFS1 in beta-cells causes ER stress and beta-cell dysfunction. These results indicate that the pathogenesis of Wolfram syndrome involves chronic ER stress in pancreatic beta-cells caused by the loss of function of WFS1.     

Characterization of a mutant Listeria monocytogenes strain expressing green fluorescent protein

To construct a recombinant strain of Listeria monocytogenes for the expression of heterologous genes, homologous recombination was utilized for insertional mutation, targeting its listeriolysin O gene (hly). The gene encoding green fluorescent protein (GFP) was used as the indicator of heterologous gene expression. The gene gfp was inserted into hly downstream from its promoter and signal sequence by an overlapping extension polymerase chain reaction, and was then cloned into the shuttle plasmid pKSV7 for allelic exchange with the L. monocytogenes chromosome. Homologous recombination was achieved by growing the electro-transformed L. monocytogenes cells on chloramphenicol plates at a non-permissive temperature. Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strain L. monocytogenes hly-gfp lost its hemolytic activity as visualized on the blood agar or when analyzed with the culture supernatant samples. Such insertional mutation resulted in a reduced virulence of about 2 logs less than its parent strain L. monocytogenes 10403s as shown by the 50%-lethal-dose assays in the mouse and embryonated chicken egg models. These results thus demonstrate that mutated L. monocytogeues could be a potential carrier for the expression of heterologous passenger genes or could act as an indicator organism in the food industry.     

Glutamate inhibits protein phosphatases and promotes insulin exocytosis in pancreatic beta-cells

In human type 2 diabetes mellitus, loss of glucose-sensitive insulin secretion from the pancreatic beta-cell is an early pathogenetic event, but the mechanisms involved in glucose sensing are poorly understood. A messenger role has been postulated for L-glutamate in linking glucose stimulation to sustained insulin exocytosis in the beta-cell, but the precise nature by which L-glutamate controls insulin secretion remains elusive. Effects of L-glutamate on the activities of ser/thr protein phosphatases (PPase) and Ca(2+)-regulated insulin exocytosis in INS-1E cells were investigated. Glucose increases L-glutamate contents and promotes insulin secretion from INS-1E cells. L-glutamate also dose-dependently inhibits PPase enzyme activities analogous to the specific PPase inhibitor, okadaic acid. L-glutamate and okadaic acid directly and non-additively promote insulin exocytosis from permeabilized INS-1E cells in a Ca(2+)-independent manner. Thus, an increase in phosphorylation state, through inhibition of protein dephosphorylation by glucose-derived L-glutamate, may be a novel regulatory mechanism linking glucose sensing to sustained insulin exocytosis.     

Proteomic analysis of oligodendrogliomas expressing a mutant isocitrate dehydrogenase-1

Gliomas are primary tumors of the human central nervous system with unknown mechanisms of progression. Isocitrate dehydrogenase-1 (IDH1) mutation is frequent in diffuse gliomas such as oligodendrogliomas. To gain insights into the physiopathology of oligodendrogliomas that have a better prognosis than other diffuse gliomas, we combined microdissection, 2-D DIGE and MS/MS focusing on proteome alterations associated with IDH1 mutation. We first compared tumor tissues (TT) and minimally infiltrated parenchymal tissues (MIT) of four IDH1-mutated oligodendrogliomas to verify whether proteins specific to oligodendroglioma tumor cells could be identified from one patient to another. This study resulted in identification of 68 differentially expressed proteins, with functions related to growth of tumor cells in a nervous parenchyma. We then looked for proteins distinctly expressed in TT harboring either mutant (oligodendrogliomas, n=4) or wild-type IDH1 (oligodendroglial component of malignant glio-neuronal tumors, n=4). This second analysis resulted in identification of distinct proteome patterns composed of 42 proteins. Oligodendrogliomas with a mutant IDH1 had noteworthy enhanced expression of enzymes controlling aerobic glycolysis and detoxification, and anti-apoptosis proteins. In addition, the mutant IDH1 migrated differently from the wild-type IDH1 form. Comparative proteomic analysis might thus be suitable to identify proteome alterations associated with a well-defined mutation.     

Imaging pancreatic beta-cells in the intact pancreas

We have developed a method to visualize fluorescent protein-labeled beta-cells in the intact pancreas through combined reflection and confocal imaging. This method provides a 3-D view of the beta-cells in situ. Imaging of the pancreas from mouse insulin I promoter (MIP)-green (GFP) and red fluorescent protein (RFP) transgenic mice shows that islets, beta-cell clusters, and single beta-cells are not evenly distributed but are aligned along the large blood vessels. We also observe the solitary beta-cells in both fetal and adult mice and along the pancreatic and common bile ducts. We have imaged the developing endocrine cells in the embryos using neurogenin-3 (Ngn3)-GFP mice crossed with MIP-RFP mice. The dual-color-coded pancreas from embryos (E15.5) shows a large number of green Ngn3-expressing proendocrine cells with a smaller number of red beta-cells. The imaging technique that we have developed, coupled with the transgenic mice in which beta-cells and beta-cell progenitors are labeled with different fluorescent proteins, will be useful for studying pancreatic development and function in normal and disease states.