Fus3 and Tpk2 protein kinases regulate the phosphorylation-dependent functions of RNA helicase Dhh1 in yeast mating and Ste12 protein expression.

Decapping of mRNA is a key regulatory step for mRNA decay and translation. The RNA helicase, Dhh1, is known as a decapping activator and translation repressor in yeast Saccharomyces cerevisiae. Dhh1 also functions as a gene-specific positive regulator in the expression of Ste12, a mating-specific transcription factor. A previous study showed that the N-erminal phosphorylation of Dhh1 regulates its association with the mRNA-binding protein, Puf6, to affect the protein translation of Ste12. Here, we investigated the roles of the phosphorylated residues of Dhh1 in yeast mating process and Ste12 expression. The phospho-deficient mutation, DHH1-T10A, was associated with decreased diploid formation during mating and decreased level of the Ste12 protein in response to α-mating pheromone. A kinase overexpression analysis revealed that Ste12 protein expression was affected by overexpression of Fus3 MAP kinase or Tpk2 kinase. Tpk2 was shown to be responsible for phosphorylation of Dhh1 at Thr10. Our study shows that overexpression of Fus3 or Tpk2 alters the Dhh1-Puf6 protein interaction and thereby affects Ste12 protein expression.

A photovoice study on the bereavement experience of mothers after the death of a child.

This study explored the bereavement experience of mothers after losing a child to cancer in Korea, using photovoice. The mothers took photos reflecting five subject areas they selected: (a) if I had one more day with my child, (b) memories with my child, (c) dreaming of my child's healthy future, (d) what gave me strength, and (e) fulfilling my child's wishes for the future. The findings show that mothers who lost a child to cancer need bereavement care to promote well-being. This study can help pediatric oncology providers develop bereavement interventions that address parental grief and improve quality of life.

Roles of Dhh1 RNA helicase in yeast filamentous growth: Analysis of N-terminal phosphorylation residues and ATPase domains.

In yeast Saccharomyces cerevisiae, the Dhh1 protein, a member of the DEAD-box RNA helicase, stimulates Dcp2/Dcp1-mediated mRNA decapping and functions as a general translation repressor. Dhh1 also positively regulates translation of a selected set of mRNAs, including Ste12, a transcription factor for yeast mating and pseudohyphal growth. Given the diverse functions of Dhh1, we investigated whether the putative phosphorylation sites or the conserved motifs for the DEAD-box RNA helicases were crucial in the regulatory roles of Dhh1 during pseudohyphal growth. Mutations in the ATPase A or B motif (DHH1-K96R or DHH1-D195A) showed significant defects in pseudohyphal colony morphology and agar invasive phenotypes. The N-terminal phospho-mimetic mutation, DHH1-T16E, showed defects in pseudohyphal phenotypes. Decreased levels of Ste12 protein were also observed in these pseudohyphal-defective mutant cells under filamentous-inducing low nitrogen conditions. We suggest that the ATPase motifs and the Thr16 phosphorylation site of Dhh1 are crucial to its regulatory roles in pseudohyphal growth under low nitrogen conditions.

Mutational analysis of the RNA helicase Dhh1 in Ste12 expression and yeast mating.

Dhh1 and Dhh1 homologues (RCK/p54/DDX6) are members of the DEAD-box protein family of RNA helicases. These proteins display conserved sequence motifs for ATPase and RNA binding activities. Dhh1 is a component of the P-bodies (processing bodies) of mRNA granules and functions as an mRNA decapping activator in Saccharomyces cerevisiae. Dhh1 also contributes to gene-specific regulation during yeast mating. The dhh1 deletion mutation results in a significant decrease in the expression of Ste12, a mating-specific transcription factor, showing severe mating defects. Here, we introduced amino-acid substitution mutations in the ATPase and RNA binding domains of Dhh1 and also constructed a deletion of 79 amino acids at the Q/P-rich C-terminal region. The mutations in ATPase A and B motif (K96R, D195A) and C-terminus deletion showed reduced levels of mating efficiency as well as Ste12 protein expression. The Q/P-rich C-terminal region of Dhh1 was dispensable for growth at nonpermissive temperature 37°C but appeared to play an important role in regulating the Ste12 protein expression and mating processes. The P-body accumulation induced by treatment with α-mating factor required ATPase, RNA-binding and the Q/P-rich C-terminal domains of Dhh1.

Host CD25+CD4+Foxp3+ regulatory T cells primed by anti-CD137 mAbs inhibit graft-versus-host disease.

CD25(+)CD4(+)Foxp3(+) regulatory T cells (Tregs) play a pivotal role in the maintenance of self-tolerance and regulation of immune responses. Previous studies have demonstrated that CD137 signals can promote proliferation and survival of Tregs in vitro. Here, we show that in vivo CD137-induced expansion of Tregs in naive mice was dependent upon IL-2 secreted by memory T cells. Tregs primed by anti-CD137 mAbs had a higher immunosuppressive capacity. Preconditioning with anti-CD137 mAbs significantly inhibited graft-versus-host disease (GVHD) in the C57BL/6 → (C57BL/6 × DBA/2) F1 acute GVHD model. In this disease model, a high proportion of host Tregs remained long-term in the recipient spleen, whereas donor hematopoietic cells replaced other host bone marrow-derived cells. Transient depletion of Tregs before transfer of donor cells completely abrogated the inhibitory effect of anti-CD137 mAbs on GVHD. In addition, adoptive transfer of anti-CD137-primed Tregs ameliorated GVHD. Our results demonstrate that it is possible to enhance the survival and/or the immunosuppressive activity of host Tregs in nonmyeloablative GVHD, and that 1 way of accomplishing this is through the prophylactic use of anti-CD137 mAbs in nonmyeloablative GVHD.

Induction of lethal graft-versus-host disease by anti-CD137 monoclonal antibody in mice prone to chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is an increasingly frequent complication of allogeneic stem cell transplantation. We previously showed that anti-CD137 monoclonal antibody (mAb) can cure advanced cGVHD by inducing activation-induced cell death of donor T cells. In this study, we examined whether administration of anti-CD137 mAb can prevent the development of cGVHD after bone marrow transplantation (BMT) in mice conditioned with total body irradiation (TBI). We used the B10.D2-->Balb/c (H-2(d)) minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. A single injection of anti-CD137 mAb was administered immediately after BMT. In contrast to the results obtained from the curing model of cGVHD, anti-CD137 given simultaneously with BMT resulted in lethal GVHD. Histopathologic evaluation revealed inflammation and damage of target organs from acute GVHD (aGVHD) in anti-CD137-treated mice. Anti-CD137-induced lethal aGVHD required host cells, as well as irradiation and mature donor T cells. Apparently, anti-CD137 mAb rapidly induced activation of donor T cells and sustained their activation status under the inflammatory condition triggered by irradiation. When given on day 12 after irradiation and BMT, anti-CD137 mAb could still exacerbate GVHD, but when given on day 30, it could not. Our data demonstrate that anti-CD137 mAb can amplify inflammation induced by host preconditioning, subsequently resulting in lethal aGVHD; thus, alleviating irradiation-induced toxicity is critical to allow the use of anti-CD137 mAb as GVHD prophylaxis.

Breaking of CD8+ T cell tolerance through in vivo ligation of CD40 results in inhibition of chronic graft-versus-host disease and complete donor cell engraftment.

In the DBA/2 --> unirradiated (C57BL/6 x DBA/2)F(1) model of chronic graft-vs-host disease (cGVHD), donor CD4(+) T cells play a critical role in breaking host B cell tolerance, while donor CD8(+) T cells are rapidly removed and the remaining cells fall into anergy. Previously we have demonstrated that in vivo ligation of GITR (glucocorticoid-induced TNF receptor-related gene) can activate donor CD8(+) T cells, subsequently converting the disease pattern from cGVHD to an acute form. In this study, we investigated the effect of an agonistic mAb against CD40 on cGVHD. Treatment of anti-CD40 mAb inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis. The inhibition of cGVHD occurred because anti-CD40 mAb prevented donor CD8(+) T cell anergy such that subsequently activated donor CD8(+) T cells deleted host CD4(+) T cells and host B cells involved in autoantibody production. Additionally, functionally activated donor CD8(+) T cells induced full engraftment of donor hematopoietic cells and exhibited an increased graft-vs-leukemia effect. However, induction of acute GVHD by donor CD8(+) T cells seemed to be not so apparent. Further CTL analysis indicated that there were lower levels of donor CTL activity against host cells in mice that received anti-CD40 mAb, compared with mice that received anti-GITR mAb. Taken together, our results suggest that a different intensity of donor CTL activity is required for removal of host hematopoietic cells, including leukemia vs induction of acute GVHD.