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In brief: May 2007Calcium Uncoupling proteins 2 and 3 are fundamental for mitochondrial Ca2+ uniport Under physiological conditions, mitochondria take up Ca2+ from the cytosol; however, the mechanism of mitochondrial import is unknown. Trenker et al. show that uncoupling protein-2 (UCP2) and UCP3 are essential for mitochondrial Ca2+import. Overexpression of UCP2 or UCP3 enhanced mitochondrial Ca2+ uptake. Ca2+ uptake was inhibited by expression of dominant-negative mutants of UCP2 or UCP3, by depletion of UCP2 or UCP3 (mediated by small interfering RNAs) or in isolated Ucp2-/- mitochondria. By contrast, modulation of UCP1 expression had no effect on Ca2+ dynamics. It is unclear whether UCP2 and UCP3 form a channel in the inner mitochondrial membrane or whether they function indirectly to regulate the passage of Ca2+ ions. DNA repair The structural determinants of checkpoint activation The ATR (ATM and Rad3-mediated) kinase is a central sensor of many forms of genotoxic stress that activates a DNA-damage checkpoint through its downstream effector checkpoint kinase-1 (Chk1). Using Xenopus laevis egg extract, MacDougall et al. identified a minimal DNA structure that activated the ATR-mediated checkpoint response. The authors found that single-stranded (ss) DNA with short complementary DNA sequences annealed along it was sufficient for checkpoint activation by ATR. This primed ssDNA structure is similar to those that are often formed following many types of DNA damage. Importantly, the authors have generated a tool that can be used to probe ATR-dependent checkpoint signalling; for example, the length of ssDNA next to the primer influences the extent of Chk1 activation. In addition, this forms a starting molecule from which the structural determinants that are required for checkpoint activation can be refined. T-cell responses Limiting amounts of IL-7 do not control contraction of CD4+ T cell responses Following T-cell clonal expansion, only a few effector T cells escape apoptotic death and become memory T cells. How this happens is unclear, although it is thought that the selective expression of interleukin-7 receptor- Genomic instability HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability Simon Boulton and colleagues have shown that CDC-like kinase 2 (CLK2), homologous to the Caenorhabditis elegans biological clock protein CLK2, functions in the DNA damage response to replication stress. Cells deficient in CLK2 accumulate DNA damage and fail to activate the S-phase checkpoints as well as key members of the Fanconi anaemia and homologous recombination DNA repair pathways, which resulted from the defective regulation of CHK1 stability. This indicates that CLK2 mutations could account for a subset of patients with Fanconi anaemia and possibly other DNA-damage-response disorders, such as Seckel syndrome. Trial watch Surprise, surprise Ronald Simon and colleagues report that oestrogen receptor a (ESR1) is commonly amplified in the early stages of proliferative breast disease, and that the detection of ESR1 copy number could provide clinically relevant information about treatment strategies for patients with breast cancer. Using more than 2,000 breast cancer tissue samples to analyse ESR1 amplification by fluorescence in situ hybridization, they reported that ESR1 was amplified in 20.6% of these samples and that an additional 15.3% showed increases in ESR1 copy number. Low-grade mucinous cancers showed the highest rates of ESR1 amplifications (35.6%), and benign papillomas and ductal hyperplasias also showed ESR1 amplification (36% and 8.3%, respectively), indicating that ESR1 amplification is a very early event in tumorigenesis and might have prognostic value. Therefore, Simon and colleagues investigated the relationship between ESR1 amplification and the response to tamoxifen, which targets the oestrogen receptor. In a retrospective analysis of 175 breast cancer samples from patients who had undergone variable tamoxifen treatments, they showed that tamoxifen response correlated with and was dependent on ESR1 copy number. This is surprising for two reasons: first, this effect is the opposite to that of the androgen receptor in prostate cancer; and second, the importance of ESR1 amplification in breast cancer development and prognosis has not been detected until now. Technology Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies. Kittler et al. generated genome-wide libraries of endoribonuclease-prepared short interfering RNAs (esiRNAs) for highly specific and cost-effective screens. The authors identified esiRNAs targeting every protein-coding human gene using an in silico approach that predicts esiRNAs with optimal specificity.They then generated more than 16,000 esiRNAs that showed higher in vivo silencing efficacy and reduced off-target specificity compared with chemically synthesized siRNAs. The authors have provided their database (RiDDLE) as an open resource for retrieving optimized esiRNA and primer sequences to generate esiRNAs targeting the human, mouse and rat transcriptomes. Human disease Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Systemic lupus erythematosus is an autoimmune disease in which antibodies are made against 'autoantigens' such as DNA and RNA- and DNA-associated proteins. Tian et al. reveal a new mechanism by which dentritic cells and B cells are activated in response to DNA. They show that high-mobility group box 1 (HMGB1), which is a nuclear DNA-binding protein, is essential for the stimulation of cytokine production through a pathway that involves Toll-like receptor 9 and the receptor RAGE, ultimately leading to autoimmune disease. Polarity LRK-1, a C. elegans PARK8-related kinase, regulates axonal-dendritic polarity of SV proteins. lrk-1 deletion in Caenorhabditis elegans causes defects in the polarization of synaptic vesicle proteins: instead of localizing strictly to axons, these proteins were found at both axonal and dendritic terminals. Surprisingly, this effect was dependent on unc-101, a gene encoding a subunit of the AP-1 clathrin adaptor complex that mediates the transport of odorant receptors in sensory neurons. The authors provide evidence that LRK-1 might function in the Golgi to exclude synaptic vesicle proteins from a UNC-101-dependent vesicle-sorting pathway that results in dendrite-specific transport. Psychiatric disorders Association and linkage of allelic variants of the dopamine transporter gene in ADHD. A genome-wide scan previously identified an attention deficit/hyperactivity disorder (ADHD) linkage peak on the distal part of the short arm of chromosome 5, which includes the dopamine transporter gene (DAT1). Friedel et al. investigated the DAT1 gene and the adjacent loci on chromosome 5 for association and linkage by genotyping 30 single nucleotide polymorphisms (SNPs) in 329 families with 523 ADHD-affected offspring. One SNP located in intron 4 of the DAT1 gene was found to be strongly associated with ADHD among the tested patients, confirming a role for DAT1 in the aetiology of this childhood disorder. Microglia UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. ATP released from injured neurons has been shown to trigger microglial chemotaxis. Now, researchers show that leakage of endogenous UDP from damaged hippocampal neurons is required to stimulate microglial phagocytosis of neuronal debris both in vitro and in vivo. UTP activates metabotropic P2Y6 receptors in microglia that may function as sensors of neuronal damage and could be a potential target for pharmacological modulation of microglia activity. Imaging and visualization Fluorescent reporter proteins without oxygen Fluorescent proteins such as GFP are extremely useful, genetically encodable probes for biological imaging. All proteins derived from the GFP family, however, require molecular oxygen as a cofactor for chromophore fluorescence and therefore cannot be used in anaerobic systems. Drepper et al. now describe the engineering of blue-light flavin mononucleotide-based fluorescent proteins that do not require oxygen for formation of the chromophore. These proteins should find application for imaging in anaerobic microbes or hypoxic solid tumors. Gene transfer Guiding Sleeping Beauty Transposons are mobile genetic elements that integrate into the genome at random. Although the efficiency of their integration makes them suitable as gene delivery vectors, their unspecific insertion can be problematic when it disrupts genes or regulatory elements. Yant et al. now show that by fusing the Sleeping Beauty transposon to a DNA-binding domain, they can direct the integration to specific sequences in human cells. Mast cells The sphingosine kinase–sphingosine-1-phosphate axis is a determinant of mast cell function and anaphylaxis The stimulation of mast cells by allergens induces two mammalian sphingosine kinases (SPHK1 and SPHK2) to produce sphingosine 1-phosphate (S1P), but little is known about their specific role in regulating immune-cell function. Here, Olivera et al. used mice in which either one or both of the Sphk1 and Sphk2 genes were deleted to investigate their role in mast-cell function. They showed that, unexpectedly, SPHK2, but not SPHK1, is the main regulator of S1P production, mediating several mast-cell signals and functions. An in vivo anaphylaxis challenge showed that the responsiveness of mast cells is determined by circulating amounts of S1P and by a partnership between SPHK1 (as an extrinsic regulator) and SPHK2 (as an intrinsic regulator). So, SPHKs and S1P have a key role in mast-cell responsiveness. Signalling c-Src activates endonuclease-mediated mRNA decay Peng and Schoenberg have now shown that the Drosophila melanogaster mRNA endonuclease, polysomal ribonuclease 1 (PMR1), is activated by phosphorylation and that activated SRC is the kinase responsible. SRC is commonly overactive in cancer cells, and the authors showed that the epidermal growth factor-mediated activation of SRC induced the phosphorylation of exogenously expressed PMR1, and therefore increased mRNA decay. This potentially provides a new arm to the oncogenic receptor tyrosine kinase signalling networks and requires further investigation to identify the target mRNAs. Tumorigenesis 14-3-3σ controls mitotic translation to facilitate cytokinesis The σ isoform of the 14-3-3 proteins, which have numerous roles in cell-cycle regulation and apoptosis, is expressed in response to p53 activation, and so is frequently downregulated in cancer cells and early in tumorigenesis. Michael Yaffe and colleagues now show that the tumour-suppressor function of 14-3-3σ might derive from its ability to regulate translation factors in mitosis. The loss of 14-3-3σ impaired mitotic exit and led to the accumulation of binucleated cells, indicating a route by which tumorigenesis might occur. Oxygen sensing HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells Hypoxia-inducing factor-1 (HIF1) is activated under low-oxygen conditions to promote pathways of anaerobic metabolism. The authors show that, in mammalian cells, HIF1 also modulates aerobic metabolism by regulating the production of a catalytic subunit in the mitochondrial electron-transport chain — cytochrome c oxidase-4 (COX4). Under normal conditions, cells predominantly produce COX4-1, an isoform that generates moderate amounts of harmful reactive oxygen species (ROS). Under hypoxic conditions, HIF1 induces the production of COX4-2 and activates a mitochondrial LON protease that degrades COX4-1. COX4-2 is a more efficient isoform that enhances aerobic respiration during periods of hypoxic stress and generates lower levels of ROS. However, forced overproduction of COX4-2 under non-hypoxic conditions was more harmful to cells than was forced overproduction of COX4-1, which may explain why COX4-2 production is usually kept to low levels. The swapping of cytochrome oxidase subunits also occurs in yeast. Yeast lack a HIF1 homologue and regulate subunit production by a different mechanism, which provides a clear example of convergent evolution in eukaryotes. Tolerance Tracing the action of IL-2 in tolerance to islet-specific antigen The σ isoform of the 14-3-3 proteins, which have numerous roles in cell-cycle regulation and apoptosis, is expressed in response to p53 activation, and so is frequently downregulated in cancer cells and early in tumorigenesis. Michael Yaffe and colleagues now show that the tumour-suppressor function of 14-3-3σ might derive from its ability to regulate translation factors in mitosis. The loss of 14-3-3σ impaired mitotic exit and led to the accumulation of binucleated cells, indicating a route by which tumorigenesis might occur. Regulatory T cells Foxp3 controls regulatory T-cell function by interacting with AML1/Runx1 Forkhead box P3 (FOXP3) is a crucial regulator of the development and function of naturally occurring CD4 Microarrays Making protein arrays with AFM Atomic force microscopy (AFM) is a useful technology for manipulating molecules on surfaces. Though it showed potential for the creation of protein arrays, this application was limited to very stable proteins, as it required relatively harsh conditions unfriendly to delicate proteins. Tinazli et al. now demonstrate the fabrication of 'rewritable' protein nanoarrays under physiological conditions using AFM-based nanolithography. Virology Structure of a herpesvirus-encoded cysteine protease reveals a unique class of deubiquitinating enzymes The host ubiquitination pathway is a key point of regulation for various cellular processes, and so is an obvious target for subversion by both bacterial and viral pathogens. Previous work using a functional proteomics approach had identified a deubiquitinating activity in cells infected with herpes simplex virus 1 (HSV-1), which was mapped to the N-terminal region of the UL36 protein, a constituent of the HSV-1 tegument. This activity was confirmed in the UL36 homologues in many other herpesviruses. The crystal structure of the ubiquitin-specific protease domain of the murine cytomegalovirus US36 homologue (M48 | |
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