Carboxysomes are organelle-like polyhedral bodies found in cyanobacteria and many chemoautotrophic bacteria that are thought to facilitate carbon fixation. Carboxysomes are bounded by a proteinaceous outer shell and filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the first enzyme in the CO 2 fixation pathway, but exactly how they enhance carbon fixation is unclear. Here we report the three-dimensional structure of purified carboxysomes from Synechococcus species strain WH8102 as revealed by electron cryotomography. We found that while the sizes of individual carboxysomes in this organism varied from 114 to 137 nm, surprisingly, all were approximately icosahedral. There were on average ∼250 RuBisCOs per carboxysome, organized into 3-4 concentric layers. Some models of carboxysome function depend on specific contacts between individual RuBisCOs and the shell, but no evidence of such contacts was found: no systematic patterns of connecting densities or RuBisCO positions against the shell's presumed hexagonal lattice could be discerned, and simulations showed that packing forces alone could account for the layered organization of RuBisCOs.
Cell cycle arrest by FoxO transcription factors involves transcriptional repression of cyclin D, although the exact mechanism remains unclear. In this study, we used the BCR-ABL-expressing cell line BV173 as a model system to investigate the mechanisms whereby FoxO3a regulates cyclin D2 expression. Inhibition of BCR-ABL by STI571 results in down-regulation of cyclin D2 expression, activation of FoxO3a activity, and up-regulation of BCL6 expression. Using reporter gene assays, we demonstrate that STI571, FoxO3a, and BCL6 can repress cyclin D2 transcription through a STAT5/BCL6 site located within the cyclin D2 promoter. We propose that BCR-ABL inhibition leads to FoxO3a activation, which in turn induces the expression of BCL6, culminating in the repression of cyclin D2 transcription through this STAT5/BCL6 site. This process was verified by mobility shift and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to accumulation of BCL6 and down-regulation of cyclin D2 at protein and mRNA levels. Furthermore, silencing of FoxO3a and BCL6 in BCR-ABL-expressing cells abolishes STI571-mediated effects on cyclin D2. This report establishes the signaling events whereby BCR-ABL signals are relayed to cyclin D2 to mediate cell cycle progression and defines a potential mechanism by which FoxO proteins regulate cyclin D2 expression.In mammalian cells, the commitment to divide is made in the G 1 phase of the cell cycle in response to various stimuli, including growth factors. After passing the restriction point at mid-to late G 1 , cells become refractory to growth inhibition signals or do not require growth factors to progress into S phase (37). Progression of eukaryotic cells through the cell cycle is controlled by the two families of G 1 cyclins: (i) D-type cyclins (cyclins D1, D2, and D3) and cyclin E (cyclins E1 and E2) (29, 44) and (ii) the cyclin-dependent kinases (cdk's), their catalytic counterparts. The primary targets of the G 1 cyclin-cdk complexes are the retinoblastoma protein (pRb) family of pocket proteins, consisting of pRb, p107, and p130 (20,27,34). The phosphorylation state of pRb regulates the activity of the E2F family of transcription factors; in their hypophosphorylated forms, the pRb-related pocket proteins associate with members of the E2F family, negatively regulating transcription activity of E2F-regulated genes that are required for entry into the S phase of the cell cycle (15,35,40).In mammals, the phosphatidylinositol 3-kinase/protein kinase B (PI3-K/PKB) pathway is stimulated by a variety of growth factors and cytokines and by cell-matrix interactions, and it controls many biological functions, including cell proliferation, cell survival, and insulin responses (30). Importantly, constitutive activation of the PI3-K pathway facilitates tumor formation by two different mechanisms: it supports S-phase entry, and it confers resistance to apoptotic signals which normally restrict uncontrolled cell growth (49). Recently, it has been demonstrated that the member...
Supplementary data are available at Bioinformatics online.
The IL-2R promotes rapid expansion of activated T cells through signals mediated by the adaptor protein Shc and the transcription factor Stat5. The mechanisms that engage the cell cycle are not well defined. We report on the transcriptional regulation of the cell cycle gene cyclin D2 by the IL-2R. IL-2-responsive induction of a luciferase reporter gene containing 1624 bp of the cyclin D2 promoter/enhancer was studied in the murine CD8+ T cell line CTLL2. Reporter gene deletional analysis and EMSAs indicate an IL-2-regulated enhancer element flanks nucleotide −1204 and binds a complex of at least three proteins. The enhancer element is bound constitutively by Sp1 and an unknown factor(s) and inducibly by Stat5 in response to IL-2. The Stat5 binding site was essential for IL-2-mediated reporter gene activity, and maximum induction required the adjacent Sp1 binding site. Receptor mutagenesis studies in the pro-B cell line BA/FG (a derivative of the BA/F3 cell line) demonstrated a correlation between Stat5 activity and cyclin D2 mRNA levels when the Stat5 signal was isolated, disrupted, and then rescued. Further, a dominant-negative form of Stat5 lacking the trans-activation domain inhibited induction of cyclin D2 mRNA. We propose that the IL-2R regulates the cyclin D2 gene in part through formation of an enhancer complex containing Stat5 and Sp1.
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