Senya Matsufuji

Epigenetic control of polyamines by the prion [PSI+]

Prion proteins are found in mammals and yeast, and can transmit diseases and encode heritable phenotypic traits. In Saccharomyces cerevisiae, eRF3, Rnq1, Ure2 and Swi1 are functional proteins with a soluble conformation that can switch to a non-functional, amyloid conformation denoted as [PSI+], [PIN+], [URE3] and [SWI+], respectively. The prion [PSI+] corresponds to an aggregated conformation of the translational release factor eRF3, which suppresses nonsense codons. [PSI+] modifies cellular fitness and induces several phenotypes according to the genetic background. An elegant series of studies has demonstrated that several [PSI+]-induced phenotypes occur as a consequence of decreased translational termination efficiency. However, the genes whose expression levels are controlled by [PSI+] remain largely unknown. Here, we show that [PSI+] enhances expression of antizyme, a negative regulator of cellular polyamines, by modulating the +1 frameshifting required for its expression. Our study also demonstrates that [PSI+] greatly affects cellular polyamines in yeast. We show that modification of the cellular content of polyamines by the prion accounts for half of the [PSI+]-induced phenotypes. Antizyme is the first protein to be described for which expression in its functional form is stimulated by [PSI+].

Conservation of polyamine regulation by translational frameshifting from yeast to mammals

Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals.

Identification of Nuclear Export Signals in Antizyme-1

Antizyme-1 (AZ1) is a protein that negatively regulates polyamine synthesis by inhibiting the key synthetic enzyme ornithine decarboxylase and targeting it for degradation by the 26 S proteasome. Recent work shows that antizyme protein translocates to the nucleus during mouse development (Gritli-Linde, A., Nilssom, J., Bohlooly, Y. M., Heby, O., and Linde, A. (2001) Dev. Dyn. 220, 259-275). However, the significance and mechanism of this phenomenon remain unclear. In this study, we expressed AZ1 fused with enhanced green fluorescent protein (EGFP) to study its localization in a living cell. We found that EGFP-AZ1 was predominantly localized in the cytoplasm and that treatment with leptomycin B, a specific inhibitor of chromosomal region maintenance 1 (CRM1) induced nuclear accumulation of EGFP-AZ1 in Chinese hamster ovary and NIH3T3 cells. Two independent nuclear export signal (NES) sequences, each containing essential hydrophobic residues, were identified in the 50 N-terminal amino acid residues and in the central part of AZ1. The activity of the second NES was inhibited by an N-terminal adjacent region and was only revealed in N-terminal truncated constructs. Both NESs were active when fused to an artificial nuclear protein SV40-NLS-EGFP-EGFP. The ability of AZ1 to shuttle between the nucleus and the cytoplasm suggests that it has a novel function in the nucleus.

Role of ornithine decarboxylase antizyme inhibitor in vivo.

Ornithine decarboxylase (ODC) antizyme inhibitor (AZI) has been shown to regulate ODC activity in cell cultures. However, its biological functions in an organism remain unknown. An embryonic stem (ES) cell clone was established, in which the Azin1 gene was disrupted by the gene trap technique. To identify the function of Azin1 gene in vivo, a mutant mouse line was generated using these trapped ES cells. Homozygous mutant mice died at P0 with abnormal liver morphology. Further analysis indicated that the deletion of Azin1 in homozygous mice resulted in the degradation of ODC, and reduced the biosynthesis of putrescine and spermidine. Our results thus show that AZI plays an important role in regulating the levels of ODC, putrescine and spermidine in mice, and is essential for the survival of mice.

Rho-kinase inhibition prevents the progression of diabetic nephropathy by downregulating hypoxia-inducible factor 1α

The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic nephropathy. Accumulating evidence shows that hypoxia-inducible factor-1α (HIF-1α) is a key regulator of renal sclerosis under diabetic conditions. However, the interactions of Rho-kinase and HIF-1α in the development of renal dysfunction have not been defined. Here, we assessed whether Rho-kinase blockade attenuates HIF-1α induction and the subsequent fibrotic response using type 2 diabetic mice and cultured mesangial cells. Fasudil, a Rho-kinase inhibitor, reduced urinary albumin excretion, mesangial matrix expansion, and the expression of fibrotic mediators in db/db mice. Mechanistically, HIF-1α accumulation and the expression of its target genes that contribute to diabetic glomerulosclerosis were also prevented by fasudil in the renal cortex. In mesangial cells, Rho/Rho-kinase signaling was activated under hypoxic conditions. Further in vitro studies showed that pharmacological and genetic inhibition of Rho-kinase promoted proteasomal HIF-1α degradation, which subsequently suppressed HIF-1-dependent profibrotic gene expression by upregulation of prolyl hydroxylase 2. Thus, we found a previously unrecognized renoprotective mechanism for the effects of Rho-kinase inhibition and this could be a potential therapeutic target for the treatment of diabetic nephropathy.

Cell culture analysis of the regulatory frameshift event required for the expression of mammalian antizymes

Background Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual +1 translational frameshift mechanism that responds to polyamine levels.

Regulation of ornithine decarboxylase by antizymes and antizyme inhibitor in zebrafish (Danio rerio)

Mammalian polyamine synthesis is regulated by a unique feedback mechanism. When cellular polyamine levels increase, antizyme, an ornithine decarboxylase (ODC) inhibitory protein, is induced by polyamine-dependent translational frameshifting. Antizyme not only inhibits ODC, a key enzyme in polyamine synthesis, it also targets the enzyme degradation by the 26S proteasome. Furthermore, it suppresses cellular uptake of polyamines. Previously, we isolated two zebrafish antizymes with different expressions and activities. This suggested that a common feedback mechanism of polyamine metabolism might operate in mammals and zebrafish (Danio rerio). In the present study, cDNAs of zebrafish ODC and antizyme inhibitor, another regulatory protein that inhibits antizyme action, were cloned. The presence of ODC and antizyme inhibitor mRNAs was confirmed by Northern blotting in embryos and adult fish, as well as in a zebrafish-derived cell line (BRF41). The activity of the ODC cDNA expression product was inhibited by short and long zebrafish antizymes, and recombinant zebrafish antizyme inhibitor reversed this inhibition. In the BRF41 cells, the ODC half-life was considerably longer than that of mammalian ODC but shorter than that of Schizosaccharomyces pombe. Spermidine elicited a rapid decay of ODC activity and ODC protein in a protein synthesis-dependent manner.

The change of antizyme inhibitor expression and its possible role during mammalian cell cycle

Antizyme inhibitor (AIn), a homolog of ODC, binds to antizyme and inactivates it. We report here that AIn increased at the G1 phase of the cell cycle, preceding the peak of ODC activity in HTC cells in culture. During interphase AIn was present mainly in the cytoplasm and turned over rapidly with the half-life of 10 to 20 min, while antizyme was localized in the nucleus. The level of AIn increased again at the G2/M phase along with ODC, and the rate of turn-over of AIn in mitotic cells decreased with the half-life of approximately 40 min. AIn was colocalized with antizyme at centrosomes during the period from prophase through late anaphase and at the midzone/midbody during telophase. Thereafter, AIn and antizyme were separated and present at different regions on the midbody at late telophase. AIn disappeared at late cytokinesis, whereas antizyme remained at the cytokinesis remnant. Reduction of AIn by RNA interference caused the increase in the number of binucleated cells in HTC cells in culture. These findings suggested that AIn contributed to a rapid increase in ODC at the G1 phase and also played a role in facilitating cells to complete mitosis during the cell cycle.

Subcellular Localization and Phosphorylation of Antizyme 2

Antizymes (AZs) are polyamine‐induced proteins that negatively regulate cellular polyamine synthesis and uptake. Three antizyme isoforms are conserved among mammals. AZ1 and AZ2 have a broad tissue distribution, while AZ3 is testis specific. Both AZ1 and AZ2 inhibit ornithine decarboxylase (ODC) activity by binding to ODC monomer and target it to the 26S proteasome at least in vivo. Both also inhibit extra‐cellular polyamine uptake. Despite their being indistinguishable by these criteria, we show here using enhanced green fluorescent protein (EGFP)‐AZ2 fusion protein that in mammalian cells, the subcellular location of AZ2 is mainly in the nucleus, and is different from that of AZ1. The C‐terminal part of AZ2 is necessary for the nuclear distribution. Within a few hours, a shift in the distribution of EGFP‐AZ2 fusion protein from cytoplasm to the nucleus or from nucleus to cytoplasm is observable in NIH3T3 cells. In addition, we found that in cells a majority of AZ2, but not AZ1, is phosphorylated at Ser‐186, likely by protein kinase CK2. There may be a specific function of AZ2 in the nucleus. J. Cell. Biochem. 108: 1012–1021, 2009.

Rho-kinase regulation of TNF-α-induced nuclear translocation of NF-κB RelA/p65 and M-CSF expression via p38 MAPK in mesangial cells

The small GTPase Rho and its downstream effector, Rho-associated coiled-coil containing protein kinase (Rho-kinase), regulate a number of cellular processes, including organization of the actin cytoskeleton, cell adhesion, and migration. While pharmacological inhibitors of Rho-kinase signaling are known to block renal inflammation, the molecular basis for this effect is unclear. Here, we provide evidence that proinflammatory TNF-α promotes mesangial expression of macrophage colony-stimulating factor (M-CSF), a key regulator for the growth and differentiation of mononuclear phagocytes, in a Rho-kinase-dependent manner. Consistent with this observation, TNF-α-mediated renal expression of M-CSF in insulin-resistant db/db mice was downregulated by Rho-kinase inhibition. Small interfering RNA-facilitated knockdown of Rho-kinase isoforms ROCK1 and ROCK2 indicated that both isoforms make comparable contributions to regulation of M-CSF expression in mesangial cells. From a mechanistic standpoint, Western blotting and EMSA showed that Rho-kinase and its downstream target p38 MAPK regulate nuclear translocation of NF-κB RelA/p65 and subsequent DNA binding activity, with no significant effects on IκBα degradation and RelA/p65 phosphorylation. Moreover, we showed that Rho-kinase-mediated cytoskeletal organization is required for the nuclear uptake of RelA/p65. Collectively, these findings identify Rho-kinase as a critical regulator of chemokine expression and macrophage proliferation.