CDK actively contributes to establishment of the stationary phase state in fission yeast.

Upon exhaustion of essential environmental nutrients, unicellular organisms cease cell division and enter stationary phase, a metabolically repressed state essential for cell survival in stressful environments. In the fission yeast Schizosaccharomyces pombe, cell size is reduced by cell division before entry into stationary phase; thus cyclin-dependent kinase (CDK) must actively contribute to stationary phase establishment. However, the contribution of CDK to stationary phase remains largely uncharacterized. Here, we examine the role of the sole S. pombe CDK, Cdc2, in the establishment of stationary phase. We show that in stationary phase, nuclear and chromosomal volumes and the nucleus-to-cell volume ratio are reduced, and sister chromatid separation and chromosome fluctuation are repressed. Furthermore, Cdc2 accumulates in the nucleolus. Most of these changes are induced by glucose depletion. Reduction in Cdc2 activity before and upon stationary phase entry alleviates the changes and shortens the survival time of stationary phase cells, whereas Cdc2 inhibition represses nucleolar Cdc2 accumulation and glucose depletion-induced nuclear volume reduction. These results demonstrate that CDK actively regulates stationary phase, both before and upon stationary phase entry.

Enzymological and structural characterization of Arabidopsis thaliana heme oxygenase-1.

Arabidopsis thaliana heme oxygenase-1 (AtHO-1), a metabolic enzyme in the heme degradation pathway, serves as a prototype for study of the bilin-related functions in plants. Past biological analyses revealed that AtHO-1 requires ferredoxin-NADP+ reductase (FNR) and ferredoxin for its enzymatic activity. Here, we characterized the binding and degradation of heme by AtHO-1, and found that ferredoxin is a dispensable component of the reducing system that provides electrons for heme oxidation. Furthermore, we reported the crystal structure of heme-bound AtHO-1, which demonstrates both conserved and previously undescribed features of plant heme oxygenases. Finally, the electron transfer pathway from FNR to AtHO-1 is suggested based on the known structural information.

Structural basis of bilin binding by the chlorophyll biosynthesis regulator GUN4.

The chlorophyll biosynthesis regulator GENOMES UNCOUPLED 4 (GUN4) is conserved in nearly all oxygenic photosynthetic organisms. Recently, GUN4 has been found to be able to bind the linear tetrapyrroles (bilins) and stimulate the magnesium chelatase activity in the unicellular green alga Chlamydomonas reinhardtii. Here, we characterize GUN4 proteins from Arabidopsis thaliana and the cyanobacterium Synechocystis sp. PCC 6803 for their ability to bind bilins, and present the crystal structures of Synechocystis GUN4 in biliverdin-bound, phycocyanobilin-bound, and phytochromobilin-bound forms at the resolutions of 1.05, 1.10, and 1.70 Å, respectively. These linear molecules adopt a cyclic-helical conformation, and bind more tightly than planar porphyrins to the tetrapyrrole-binding pocket of GUN4. Based on structural comparison, we propose a working model of GUN4 in regulation of tetrapyrrole biosynthetic pathway, and address the role of the bilin-bound GUN4 in retrograde signaling.

Amiodarone-induced thyroid dysfunction in Taiwan: a retrospective cohort study.

BACKGROUND: The incidence and risk factors of amiodarone-induced thyroid dysfunction are variable in the literature. OBJECTIVE: The aim of this study was to investigate the clinical and biochemical features and risk factors of amiodarone-induced thyroid dysfunction in Taiwan. SETTING: This study was conducted at a tertiary referral center for arrhythmia. METHOD: Retrospective analysis of patients treated with amiodarone during the years 2008-2009 was performed. MAIN OUTCOME MEASURE: Incidence and risk factors of amiodarone-induced thyrotoxicosis (AIT) and amiodarone-induced hypothyroidism (AIH) were assessed. RESULTS: Of the 527 patients, 437 (82.9 %) remained euthyroid, 21 (4.0 %) developed AIT, and 69 (13.1 %) were affected with AIH. In univariate analysis, AIT was associated with younger age, and the risk factors for AIH included older age, higher baseline thyroid stimulating hormone (TSH) titer, lower baseline free T4 level, lower cumulative amiodarone dosage, and shorter amiodarone treatment duration. Cox regression analysis was performed to determine the different risk categories in the elderly population of age 65-74 (young-old), 75-84 (old-old), and ≥85 years old (oldest-old). Additionally increased risk of AIH was found in the groups of old-old (HR 2.09, 95 % CI 1.11-3.96) and oldest-old (HR 2.57, 95 % CI 1.21-4.75). In the multivariate analysis of risk factors for AIH, baseline TSH level (HR 1.38, 95 % CI 1.12-1.70) and cumulative amiodarone dosage (HR 0.95, 95 % CI 0.93-0.97) remained statistically significant. CONCLUSION: AIH was much more common than AIT in Taiwan, an area with sufficient iodine intake. Higher baseline TSH level was the predominant independent risk factor for the development of AIH.

hnRNP-K is a nuclear target of TCR-activated ERK and required for T-cell late activation.

Sustained extracellular signal-regulated kinase (ERK)-signaling plays a critical role in T-cell-mediated IL-2 production. Although many downstream targets are known for ERK, details remain unknown about which molecules play functional roles in IL-2 production. Here, we addressed this question using proteomic analysis of nuclear proteins from TCR-activated T cells and identified hnRNP-K as one of the ERK targets essential for IL-2 production. hnRNP-K was previously shown by others to be a direct substrate of ERK and form complexes with multiple signaling proteins as well as DNA and RNA. Our data showed a clear ERK-dependent increase in one form of hnRNP-K after TCR stimulation. Small interfering RNA-mediated gene knockdown of hnRNP-K expression abrogated IL-2 production by T cells. Moreover, reduction of hnRNP-K expression caused a notable increase in proteolysis of Vav1, a binding target of hnRNP-K. Since Vav1 is an essential molecule for T-cell activation, the data suggest that ERK signaling is required for T-cell activation partly by inhibiting activation-induced proteolysis of Vav1.

Lck couples Shc to TCR signaling.

Recent genetic evidence demonstrated that Shc is a critical molecule for T cell activation and differentiation. However, how Shc is coupled to the T cell antigen receptor (TCR) has not been clearly characterized. Here we report that the tyrosine kinase Lck functions as a connecting molecule for TCR and Shc. Lck plays a critical role in TCR signal transduction by phosphorylating the immuno-receptor tyrosine based activation motif (ITAM). Our data shows that the PTB domain of Shc binds the SH2/3 domains of Lck in a phosphotyrosine-independent manner. Inhibition of the Lck/Shc interaction led to the loss of IL-2 promoter activation, confirming that the role of Shc in IL-2 production requires its interaction with Lck. Together, the data show that Shc is connected to the activated TCR via direct interaction with Lck.

A novel ERK-dependent signaling process that regulates interleukin-2 expression in a late phase of T cell activation.

Engagement of the T cell antigen receptor (TCR) rapidly induces multiple signal transduction pathways, including ERK activation. Here, we report a critical role for ERK at a late stage of T cell activation. Inhibition of the ERK pathway 2-6 h after the start of TCR stimulation significantly impaired interleukin-2 (IL-2) production, whereas the same treatment during the first 2 h had no effect. ERK inhibition significantly impaired nuclear translocation of c-Rel with a minimum reduction of NF-AT activity. Requirement for sustained ERK activation was also confirmed using primary T cells. To induce sustained activation of ERK, T cells required continuous engagement of TCR. Stimulation of T cells with soluble anti-TCR antibody resulted in activation of ERK lasting for 60 min, but failed to induce IL-2 production. In contrast, plate-bound anti-TCR antibody activated ERK over 4 h and induced IL-2. Furthermore, T cells treated with soluble anti-TCR antibody produced IL-2 when phorbol 12-myristate 13-acetate, which activates ERK, was present in the culture medium 2-6 h after the start of stimulation. Together, the data demonstrate the presence of a novel activation process following TCR stimulation that requires ERK-dependent regulation of c-Rel, a member of the NF-kappaB family.