Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Direct interaction of the Rab3 effector RIM with Ca2+ channels, SNAP-25, and synaptotagmin.

To define the role of the Rab3-interacting molecule RIM in exocytosis we searched for additional binding partners of the protein. We found that the two C(2) domains of RIM display properties analogous to those of the C(2)B domain of synaptotagmin-I. Thus, RIM-C(2)A and RIM-C(2)B bind in a Ca(2+)-independent manner to alpha1B, the pore-forming subunit of N-type Ca(2+) channels (EC(50) = approximately 20 nm). They also weakly interact with the alpha1C but not the alpha1D subunit of L-type Ca(2+) channels. In addition, the C(2) domains of RIM associate with SNAP-25 and synaptotagmin-I. The binding affinities for these two proteins are 203 and 24 nm, respectively, for RIM-C(2)A and 224 and 16 nm for RIM-C(2)B. The interactions of the C(2) domains of RIM with SNAP-25 and synaptotagmin-I are modulated by Ca(2+). Thus, in the presence of Ca(2+) (EC(50) = approximately 75 microm) the interaction with synaptotagmin-I is increased, whereas SNAP-25 binding is reduced. Synaptotagmin-I binding is abolished by mutations in two positively charged amino acids in the C(2) domains of RIM and by the addition of inositol polyphosphates. We propose that the Rab3 effector RIM is a scaffold protein that participates through its multiple binding partners in the docking and fusion of secretory vesicles at the release sites.

Rabphilin dissociated from Rab3 promotes endocytosis through interaction with Rabaptin-5.

Rabphilin is a secretory vesicle protein that interacts with the GTP-bound form of the small GTPase Rab3. We investigated the involvement of Rabphilin in endocytosis using different point mutants of the protein. Overexpression of wild-type Rabphilin in the insulin-secreting cell line HIT-T15 did not affect receptor-mediated transferrin endocytosis. By contrast, Rabphilin V61A, a mutant that is unable to interact with Rab3, enhanced the rate of transferrin internalization. The effect of Rabphilin V61A was not mimicked by Rabphilin L83A, another mutant with impaired Rab3 binding. Careful analysis of the properties of the two mutants revealed that Rabphilin V61A and Rabphilin L83A are both targeted to secretory vesicles, have stimulatory activity on exocytosis, and bind equally well to alpha-actinin. However, Rabphilin L83A fails to interact with Rabaptin-5, an important component of the endocytotic machinery. These results indicate that Rabphilin promotes receptor-mediated endocytosis and that its action is negatively modulated by Rab3. We propose that the hydrolysis of GTP that is coupled to the exocytotic event disrupts the Rabphilin-Rab3 complex and permits the recruitment of Rabaptin-5 at the fusion site. Our data show that immediately after internalization the transferrin receptor and VAMP-2 colocalize on the same vesicular structures, suggesting that Rabphilin favors the rapid recycling of the components of the secretory vesicle.

Anaphylactoid purpura like vasculitis following fibrinolytic therapy: role of the immune response to streptokinase.

We report the case of a patient who presented with a clinical picture of serum sickness with some characteristics of anaphylactoid purpura (Henoch-Schönlein purpura) five days after receiving streptokinase as a treatment for myocardial infarction. The appearance of Henoch-Schönlein like vasculitis after streptokinase treatment was particularly intriguing in view of the common association of streptococcus infection with this type of vasculitis. The production of streptokinase specific IgG and IgA antibodies was studied in the patient and compared with six controls treated with identical doses of streptokinase without adverse effects. A more rapid increase of streptokinase specific IgA was detected in the patient, with a significant higher amount of streptokinase specific IgA and IgG after six days of treatment. These results suggest that different kinetics could induce a precipitation of the immune complexes responsible for vasculitis. However, we cannot exclude that the IgA found in the vessel walls was only an "innocent bystander" deposited as a secondary event.

Murine Thy-1+ dendritic epidermal T cell lines express granule-associated perforin and a family of granzyme molecules.

Two T cell receptor gamma/delta + murine dendritic epidermal T cell (DETC) lines with cytotoxic potential towards various tumor cell lines are shown to express perforin and granzyme A both at the mRNA and protein levels. Furthermore, mRNA transcripts for granzyme B and at least one of the other granzymes D, E, F and G are detected in amounts equivalent to a murine IL-2-dependent alpha/beta + cytotoxic T lymphocyte cell line. Hemolytic granules containing serine-esterase (granzyme A) activity are isolated from a DETC line. Thus, cytolytically-active Thy-1+ DETC lines contain the granule-associated pore-forming protein, perforin, and at least one member of each of the three subgroups of granzyme serine esterases (granzyme A, B and D/E/F/G). These data support the proposed role of gamma/delta + DETC in immune surveillance, possibly exerting cytolytic functions against virus- or parasite-infected, transformed or stressed cells.