Maintenance of skeletal muscle mass relies on the dynamic balance between anabolic and catabolic processes and is important for motility, systemic energy homeostasis, and viability. We identified direct target genes of the glucocorticoid receptor (GR) in skeletal muscle, i.e., REDD1 and KLF15. As well as REDD1, KLF15 inhibits mTOR activity, but via a distinct mechanism involving BCAT2 gene activation. Moreover, KLF15 upregulates the expression of the E3 ubiquitin ligases atrogin-1 and MuRF1 genes and negatively modulates myofiber size. Thus, GR is a liaison involving a variety of downstream molecular cascades toward muscle atrophy. Notably, mTOR activation inhibits GR transcription function and efficiently counteracts the catabolic processes provoked by glucocorticoids. This mutually exclusive crosstalk between GR and mTOR, a highly coordinated interaction between the catabolic hormone signal and the anabolic machinery, may be a rational mechanism for fine-tuning of muscle volume and a potential therapeutic target for muscle wasting.
We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.
Because of its very high affinity for DNA, NF-jB is believed to make long-lasting contacts with cognate sites and to be essential for the nucleation of very stable enhanceosomes. However, the kinetic properties of NF-jB interaction with cognate sites in vivo are unknown. Here, we show that in living cells NF-jB is immobilized onto high-affinity binding sites only transiently, and that complete NF-jB turnover on active chromatin occurs in less than 30 s. Therefore, promoter-bound NF-jB is in dynamic equilibrium with nucleoplasmic dimers; promoter occupancy and transcriptional activity oscillate synchronously with nucleoplasmic NF-jB and independently of promoter occupancy by other sequence-specific transcription factors. These data indicate that changes in the nuclear concentration of NF-jB directly impact on promoter function and that promoters sample nucleoplasmic levels of NF-jB over a timescale of seconds, thus rapidly re-tuning their activity. We propose a revision of the enhanceosome concept in this dynamic framework.
Acentric, autonomously replicating extrachromosomal structures called double-minute chromosomes (DMs) frequently mediate oncogene amplification in human tumors. We show that DMs can be removed from the nucleus by a novel micronucleation mechanism that is initiated by budding of the nuclear membrane during S phase. DMs containing c-myc oncogenes in a colon cancer cell line localized to and replicated at the nuclear periphery. Replication inhibitors increased micronucleation; cell synchronization and bromodeoxyuridine–pulse labeling demonstrated de novo formation of buds and micronuclei during S phase. The frequencies of S-phase nuclear budding and micronucleation were increased dramatically in normal human cells by inactivating p53, suggesting that an S-phase function of p53 minimizes the probability of producing the broken chromosome fragments that induce budding and micronucleation. These data have implications for understanding the behavior of acentric DNA in interphase nuclei and for developing chemotherapeutic strategies based on this new mechanism for DM elimination.
Reduction-oxidation (redox) regulation has been implicated in the activation of the transcription factor NF-B. However, the significance and mechanism of the redox regulation remain elusive, mainly due to the technical limitations caused by rapid proton transfer in redox reactions and by the presence of many redox molecules within cells. Here we establish versatile methods for measuring redox states of proteins and their individual cysteine residues in vitro and in vivo, involving thiolmodifying reagents and LC-MS analysis. Using these methods, we demonstrate that the redox state of NF-B is spatially regulated by its subcellular localization. While the p65 subunit and most cysteine residues of the p50 subunit are reduced similarly in the cytoplasm and in the nucleus, Cys-62 of p50 is highly oxidized in the cytoplasm and strongly reduced in the nucleus. The reduced form of Cys-62 is essential for the DNA binding activity of NF-B. Several lines of evidence suggest that the redox factor Ref-1 is involved in Cys-62 reduction in the nucleus. We propose that the Ref-1-dependent reduction of p50 in the nucleus is a necessary step for NF-B activation. This study also provides the first example of a drug that inhibits the redox reaction between two specific proteins.The redox states of cysteine residues, which can change reversibly within cells, often greatly influence the various properties of proteins, such as protein stability, chaperone activity, enzymatic activity, and protein structure (1-5). It has also been suggested that several transcription factors bind to their cognate sites in a redox-regulated manner. Well characterized cases include the prokaryotic transcription factors SoxR and OxyR, which function as oxidative stress sensors, their DNA binding activated through oxidation of critical cysteine residues (6 -7). In most cases, however, the roles and mechanisms of redox regulation are not fully defined because it is difficult to monitor the alteration of redox states of proteins mainly due to the rapid proton transfer in redox reactions. A few have directly quantified the redox state of cysteine clustered with iron or amounts of oxidized cysteines using physicochemical or biochemical techniques (3, 8 -9), but these methods cannot describe the whole picture of redox states of a protein and are not widely applicable to other proteins. Therefore, most researchers have chosen an indirect way of using cysteine-substitution mutant proteins (3-5, 7).NF-B 1 is a eukaryotic transcription factor that regulates a wide variety of genes involved in immune function and development (10). NF-B is composed of two subunits, p50 and p65, both of which are members of the Rel family of transcription factors. NF-B normally exists in the cytoplasm, forming an inactive ternary complex with the inhibitor protein IB␣. Following the application of appropriate stimuli, NF-B is released from IB␣ and translocates into the nucleus, where it binds DNA and activates transcription of target genes. Mechanisms of NF-B activation have been exten...
CD26 is a T cell costimulatory molecule with dipeptidyl peptidase IV activity in its extracellular region. We previously reported that recombinant soluble CD26 enhanced T cell proliferation induced by the recall antigen tetanus toxoid (TT). However, the mechanism involved in this enhancement is not yet elucidated. We now demonstrate that CD26 binds Caveolin-1 on antigen-presenting cells, and that residues 201-211 of CD26 along with the serine catalytic site at residue 630 contribute to binding to caveolin-1 scaffolding domain. In addition, after CD26 -caveolin-1 interaction on TT-loaded monocytes, caveolin-1 is phosphorylated, which links to activate NF-B, followed by up-regulation of CD86. Finally, reduced caveolin-1 expression on monocytes inhibits CD26-mediated CD86 up-regulation and abrogates CD26 effect on TT-induced T cell proliferation. Taken together, these results strongly suggest that CD26 -caveolin-1 interaction plays a role in the up-regulation of CD86 on TT-loaded monocytes and subsequent engagement with CD28 on T cells, leading to antigen-specific T cell activation. C D26 is a widely distributed, 110-kDa cell-surface glycoprotein with known dipeptidyl peptidase IV (DPPIV) (EC 3.4.14.5) activity in its extracellular domain (1, 2), capable of cleaving amino-terminal dipeptides with either L-proline or L-alanine at the penultimate position. The CD4 ϩ CD26 high T cells respond maximally to recall antigens, such as tetanus toxoid (TT) (3). Crosslinking of CD26 and CD3 with solid-phase immobilized mAbs induces T cell costimulation and IL-2 production by either human CD4 ϩ T cells or CD26 Jurkat transfectants (4, 5, 6). Importantly, DPPIV enzyme activity is required for CD26-mediated T cell costimulation (7). More recently, we have shown that internalization of CD26 after crosslinking is mediated in part by the mannose-6-phosphate͞ insulin-like growth factor II receptor (M6P͞IGF-IIR), and that CD26-M6P͞IGFIIR interaction plays a role in CD26-induced T cell costimulation (8).Caveolin-1 was first identified as a major tyrosine phosphorylated protein in v-Src-transformed chicken embryo fibroblasts (9). Multiple lines of evidence suggest that caveolin-1 acts as a scaffolding protein capable of directly interacting with and modulating the activity of caveolin-bound signaling molecules. In support of this hypothesis, caveolin-1 binding can functionally modulate the activity of G protein-coupled proteins, membrane proteins, nonreceptor tyrosine and serine͞threonine kinases, protein kinase C isoforms, epidermal growth factor receptor, and endothelial nitric oxide synthetase (10, 11). In immune cells, caveolin-1 on monocytes͞ macrophages regulates metabolism of scavenged lipids (12). However, it is unknown whether caveolin-1 also plays a role in signal transduction in antigen-presenting cells (APC).Maximal T cell activation requires both an antigen-specific stimulus provided by an MHC peptide complex and a costimulatory signal (13). Engagement of CD28 on T cell surface by B7-1 (CD80) or B7-2 (CD86) expressed on AP...
The presence of micronuclei in mammalian cells is related to several mutagenetic stresses. In order to understand how micronuclei emerge, behave in cells, and affect cell fate, we performed extensive time-lapse microscopy of HeLa H2B-GFP cells in the presence of hydroxyurea at low concentration. Micronuclei formed after mitosis from lagging chromatids or chromatin bridges between anaphase chromosomes and were stably maintained in the cells for up to one cell cycle. Nuclear buds also formed from chromatin bridges or during interphase. If the micronuclei-bearing cells entered mitosis, they either produced daughter cells without micronuclei or, more frequently, produced cells with additional micronuclei. Low concentrations of hydroxyurea efficiently induced multipolar mitosis, which generated lagging chromatids or chromatin bridges, and also generated multinuclear cells that were tightly linked to apoptosis. We found that the presence of micronuclei is related to apoptosis but not to multipolar mitosis. Furthermore, the structural heterogeneity among micronuclei, with respect to chromatin condensation or the presence of lamin B, derived from the mechanism of micronuclei formation. Our study reinforces the notion that micronucleation has important implications in the genomic plasticity of tumor cells.
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