Eukaryotic translation initiation factor 5A inhibition alters physiopathology and immune responses in a "humanized" transgenic mouse model of type 1 diabetes.

Therapeutic options for treatment of type 1 diabetes (T1D) are still missing. New avenues for immune modulation need to be developed. Here we attempted at altering the diabetes outcome of our humanized model of T1D by inhibiting translation-initiation factor eIF5A hypusination in vivo. Double-transgenic (DQ8-GAD65) mice were immunized with adenoviral vectors carrying GAD65 for diabetes induction. Animals were subsequently treated with deoxyhypusine synthase (DHS) inhibitor GC7 and monitored for diabetes development over time. On one hand, helper CD4(+) T cells were clearly affected by the downregulation of the eIF5A not just at the pancreas level but overall. On the other hand, the T regulatory cell component of CD4 responded with activation and proliferation significantly higher than in the non-GC7-treated controls. Female mice seemed to be more susceptible to these effects. All together, our results show for the first time that downregulation of eIF5A through inhibition of DHS altered the physiopathology and observed immune outcome of diabetes in an animal model that closely resembles human T1D. Although the development of diabetes could not be abrogated by DHS inhibition, the immunomodulatory capacity of this approach may supplement other interventions directed at increasing regulation of autoreactive T cells in T1D.

Synergistic effect of inhibiting translation initiation in combination with cytotoxic agents in acute myelogenous leukemia cells.

We have previously shown that inhibition of translation initiation, using the small molecule inhibitor silvestrol, induces apoptosis in a pre-clinical murine lymphoma model when combined with daunorubicin. Silvestrol blocks ribosome recruitment by targeting the RNA helicase, eIF4A, which is required for this process. Here we investigate the sensitivity of acute myelogenous leukemia (AML) cell lines to protein synthesis inhibition in combination with the standard cytotoxic agents daunorubicin, etoposide, and cytarabine. Silvestrol shows synergy with standard-of-care agents in AML cell lines and synergizes with ABT-737, a small molecule inhibitor of Bcl-X(L) and Bcl-2. The in vitro synergy between silvestrol and the cytotoxic drugs used in AML therapy provides a basis for in vivo evaluation of these combinations.

Translational control in heat-shocked Drosophila embryos. Evidence for the inactivation of initiation factor(s) involved in the recognition of mRNA cap structure.

Lysates from normally growing (25 degrees C) or heat shocked (37 degrees C, 45 min) Drosophila melanogaster embryos were obtained and the effect of analogues of the mRNA 5'-terminal cap, m7G(5')ppp(5')N structure and of potassium ions on their endogenous protein synthesis activity was studied. At optimal concentration of KCH3COO (75-80 mM), protein synthesis in normal lysates is strongly inhibited by cap analogues (m7GpppG, m7GDP, and m7GMP). At the same ionic conditions, heat shock lysates translate preferentially the heat shock messengers, and this translation is almost unaffected by the cap analogues. In contrast, residual synthesis of normal proteins in heat shock lysates was reduced by these compounds. By lowering the concentration of potassium ions it was possible to gradually reverse the inhibitory effect of the cap analogues in normal lysates and also to increase specifically the translation of normal mRNAs in heat shock lysates. Translation of normal mRNAs is also partial but specifically rescued by supplementing heat shock lysates with polypeptide chain initiation factors partially purified from rabbit reticulocytes. These data are consistent with the notion that the failure of normal mRNAs to be translated under heat shock conditions might be due, at least to some extent, to the inactivation of polypeptide chain initiation factor(s) involved in the recognition of the mRNA 5'-terminal cap structure.

Indirect inactivation of eukaryotic initiation factor 2 in reticulocyte lysate by selenite.

Addition of selenite to rabbit reticulocyte lysate produces a biphasic pattern of translational inhibition. Sucrose density gradient shows that the onset of translational inhibition is accompanied by decreased Met-tRNAf binding to 43 SN ribosomal subunits and loss of polysomes. Control rates of translation are restored by the addition of exogenous eukaryotic initiation factor 2 (eIF-2). Selenite also directly inhibits Met-tRNAf binding activity of eIF-2. While selenite could react directly with unpaired cysteine residues of eIF-2 to inhibit protein synthesis initiation, a more complex mechanism than a direct inactivation of eIF-2 is suggested by the following observations: 1) translational inhibition produced by selenite is accompanied by an apparent increase in the phosphorylation state of eIF-2alpha; and 2) the extent of translational inhibiton is not proportional to steady-state level of phosphorylation. Rather, the time required for the onset of translational inhibition decreases as the level of eIF-2alpha phosphorylation is increased. This suggests a multistep sequence for eIF-2 inactivation, dependent upon an initial activation of eIF-2alpha kinase and followed by additional eIF-2 modification(s).